WO2023041435A1

WO2023041435A1 - Bacteriocin for applications against mycobacterium

Info

Publication number: WO2023041435A1
Application number: PCT/EP2022/075118
Authority: WO
Inventors: Anandi MARTIN; Philippe Gabant
Original assignee: Syngulon S.A.
Priority date: 2021-09-14
Filing date: 2022-09-09
Publication date: 2023-03-23

Abstract

Described herein are bacteriocin peptides, peptidomimetics, compositions, and pharmaceutical compositions comprising same. Aspects and embodiments described herein may be used in medical treatment and disinfection of surfaces.

Description

Bacteriocin for applications against Mycobacterium

Field

Aspects herein generally pertain to the field of antimicrobial compounds, more particularly to bacteriocin peptides, peptidomimetics and/or compositions comprising same, and their uses in medical treatment and disinfection of surfaces.

Background

Extensive and widespread use of antimicrobial compounds to reduce or eliminate or neutralize undesired microbial organisms has led to the emergence of resistant strains of microbial organisms. As a result, these resistant microbial organisms are no longer susceptible to the currently available antimicrobial compounds or disinfection methods. Accordingly, there is a need for improved antimicrobial compounds and disinfection methods compared to the ones currently available in the art.

Bacteria of the genus Mycobacterium, such as the ones belonging to the Mycobacterium tuberculosis complex (MTB or MTBC), and in particular Mycobacterium tuberculosis, as well as nontuberculous mycobacteria, and in particular Mycobacterium abscessus, are pathogenic bacteria which are the main causes of tuberculosis (TB) and other infections and/or diseases such as pulmonary disease resembling tuberculosis, skin and soft tissue infections (SSTIs), leprosy, central nervous system infections, bacteremia, ocular infections, and lymphadenopathies in animals and humans worldwide. Accordingly, there is a need for improved treatments against infections and/or diseases caused by bacteria of the genus Mycobacterium, for example by Mycobacterium tuberculosis or Mycobacterium abscessus. There is further a need for disinfection methods of surfaces contaminated by bacteria of the genus Mycobacterium.

Tuberculosis (TB) remains one of the deadliest diseases in the world despite the availability of existing tuberculosis treatments, which is focused on administration of antibiotics. Accordingly, there is a need for improved tuberculosis treatments.

The emergence of multiple-drug resistant and extensively-drug resistant strains, such as strains causing multipledrug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have decreased the efficacy of existing treatments (Skrahina et al., 2013). Accordingly, there is a need for improved treatments against diseases caused by such strains, and especially against multiple-drug resistant tuberculosis and extensively drug resistant tuberculosis. There is further a need for disinfection methods of surfaces contaminated by such strains.

Summary

An aspect of the invention relates to a bacteriocin peptide or peptidomimetic, wherein said peptide or peptidomimetic comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or by an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 for use in the treatment, prevention and/or delaying of an infection and/or a disease in a subject, wherein said infection and/or disease is caused by a bacterium of the genus Mycobacterium. In some embodiments, said bacterium is selected from a species belonging to the Mycobacterium tuberculosis complex or a nontuberculous Mycobacterium. In some embodiments, said bacterium is Mycobacterium tuberculosis. In some embodiments, said bacterium is Mycobacterium abscessus. In some embodiments, said infection and/or disease is tuberculosis. In some embodiments, said bacterium is antibiotic-resistant. In some embodiments, the antibiotic resistance is caused by a mutation in a gene selected from rpoB, gyrA, or gyrB. In some embodiments, said antibiotic is a polyketide, preferably is rifampicin. In some embodiments, said antibiotic is a fluoroquinolone, preferably is ofloxacin or moxifloxacin. In some embodiments, said antibiotic is a macrolide, preferably is clarithromycin. In some embodiments, said antibiotic is a tetracycline or a glycylcycline, preferably is tigecycline. In some embodiments, said antibiotic is a [3-lactam or a carbapenem, preferably is imipenem. In some embodiments, said antibiotic is an aminoglycoside, preferably is amikacin.

In some embodiments, an antibiotic is further used, preferably selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a p-lactam, a carbapenem, an aminoglycoside, or a combination thereof, more preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

Another aspect of the invention relates to a composition comprising a bacteriocin peptide or peptidomimetic according to the invention. In some embodiments, the composition further comprises an antibiotic, preferably selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a p-lactam, a carbapenem, an aminoglycoside, or a combination thereof, more preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

In some embodiments, the composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients and is for use according to the invention. In some embodiments, a bacteriocin peptide, peptidomimetic, or pharmaceutical composition for use according to the invention is such that administration of said peptide, peptidomimetic, or composition is selected from intrapulmonary, oral, or parenteral administration.

In some embodiments, the composition according to the invention is such that said composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, optionally further comprising one or more antimicrobial compounds and/or a solvent.

Another aspect of the invention relates to an ex-vivo method of disinfecting a surface comprising contacting said surface with a bacteriocin peptide or peptidomimetic, or a composition, according to the invention. In some embodiments, said surface is contaminated with a bacterium of the genus Mycobacterium. In some embodiments, the bacterium is selected from a species belonging to the Mycobacterium tuberculosis complex or a nontuberculous Mycobacterium. In some embodiments, said bacterium is Mycobacterium tuberculosis. In some embodiments, said bacterium is Mycobacterium abscessus.

Description

The present inventors have surprisingly found that bacteriocins described herein are able to effectively inhibit bacteria of the genus Mycobacterium. Particularly, and as elaborated in the experimental part, the present inventors have surprisingly found that said bacteriocins are able to inhibit strains that are resistant to antibiotics. Further, the bacteriocins described herein demonstrate a strong synergistic inhibition effect when combined with antibiotics. Accordingly, the aspects and embodiments described herein solve at least some of the problems and needs discussed herein.

Bacteriocin

Bacteriocins are antimicrobial compounds. A “bacteriocin” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to proteinaceous (peptidic) toxins produced by bacteria. The biological activity of bacteriocins is the inhibition of microbial cells other than the host cell by which the peptide is made. Said biological activity may also be referred to as antimicrobial activity. A bacteriocin may inhibit at least one cell and/or strain other than the host cell and/or strain in which the peptide is made, including cells and/or strains clonally related to the host cell and other microbial cells. Detailed descriptions of bacteriocins, including methods and compositions for using bacteriocins to control the growth of microbial cells can be found, for example, in U.S. Patent No. 9,333,227, which is hereby incorporated by reference in its entirety. Bacteriocins are typically produced by both Gram-positive and Gram-negative bacteria. Examples of Gram-positive bacteriocin-producing bacteria are Lactococcus lactis, Staphylococcus aureus, and Staphylococcus epidermidis. Bacteriocin production may be strain-specific, e.g. it may be produced predominantly by a specific strain such as a Lactococcus lactis, Staphylococcus aureus, or Staphylococcus epidermidis strain. Novel bacteriocins may be identified using modern bioinformatics tools according to standard methods available in the art, such as the BAGEL4 software (van Heel et al. Nucleic Acids Res 46(W1): W278-W281 , incorporated by reference herein in its entirety), available at http://bagel4.molgenrug.nl/, which enable researchers to mine bacterial (meta-)genomic DNA for bacteriocin-encoding genes.

Within the context of the invention, a bacteriocin may be a peptide or a peptidomimetic. A "peptide” as described herein also encompasses polypeptides, as well as variants of peptides and polypeptides as described later herein. A definition of "peptidomimetic” is provided later herein. A "peptidomimetic” as described herein also encompasses variants of peptidomimetics.as described later herein. A bacteriocin peptide or peptidomimetic as described herein may be comprised in a composition, such as a pharmaceutical composition, optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients. A description of compositions, such as pharmaceutical compositions, according to the invention is given in the section titled "compositions”.

The present inventors have surprisingly found that a bacteriocin peptide which comprises the following sequence: MAG FLKVVQLLAKYGSKAVQWAWANKGKI LDWLNAGQAI DWVVSKI KQI LG I K (NCBI Accession number: WP_058206662.1 , SEQ ID NO: 1) exhibits advantageous antimicrobial properties, as it inhibits bacteria of the genus Mycobacterium, particularly strains that are resistant to antibiotics. Further, said bacteriocin demonstrates a strong synergistic inhibition effect when combined with antibiotics.

The present inventors have further surprisingly found that the same advantageous antimicrobial properties are exhibited by a bacteriocin peptide which comprises the following sequence:

MAGFLKVVQILAKYGSKAVQWAWANKGKILDWINAGQAIDWVVEKIKQILGIK

(NCBI Accession number: BAF75975.1 , SEQ ID NO: 2)

MSWLNFLKYIAKYGKKAVSAAWKYKGKVLEWLNVGPTLEWVWQKLKKIAGL

(NCBI Accession number: WP_032072954.1 , SEQ ID NO: 3)

MAAFMKLIQFLATKGQKYVSLAWKHKGTILKWINAGQSFEWIYKQIKKLWA

(NCBI Accession number: 6SIF_A, SEQ ID NO: 4)

Accordingly, in an aspect, the invention provides a bacteriocin peptide, peptidomimetic, preferably peptide, or a pharmaceutical composition comprising a bacteriocin peptide or peptidomimetic, preferably peptide, optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients, wherein said peptide or peptidomimetic comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, for use in the treatment, prevention and/or delaying of an infection and/or a disease in a subject, wherein said infection and/or disease is caused by a bacterium of the genus Mycobacterium.

In an aspect, the invention provides a method of treatment, prevention, and/or delaying of an infection and/or disease, wherein said infection and/or disease is caused by a bacterium of the genus Mycobacterium, comprising administering a bacteriocin peptide, peptidomimetic, preferably peptide, or pharmaceutical composition comprising a bacteriocin peptide or peptidomimetic, preferably peptide, optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients, to a subject such as a subject in need thereof, wherein said peptide or peptidomimetic comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In an aspect, the invention provides the use of a bacteriocin peptide, peptidomimetic, preferably peptide, or pharmaceutical composition comprising a bacteriocin or peptidomimetic, preferably peptide, optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients, for the manufacture of a medicament forthe treatment, prevention, and/or delaying of an infection and/or disease, wherein said infection and/or disease is caused by a bacterium of the genus Mycobacterium, wherein said peptide or peptidomimetic comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 1 , preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 2, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 2, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 2.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 3, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 3, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 3.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 4, or by an amino acid sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with SED ID NO: 4, preferably having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 4.

"Inhibition”, "neutralization”, and variations of the terms as used herein have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure. They include any form of inhibition or arrest of microbial growth and/or division (bacteriostatic effect), as well as any cytotoxic or bactericidal effect (killing). Inhibition and/or neutralization may be full or partial, meaning a whole microbial cell population, such as a target microbial population, or only a part thereof may be growth-inhibited or killed. Partial inhibition may mean that at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 99% of an initial microbial population, such as a target microbial population, is not growth-inhibited or killed.

The ability of an antimicrobial compound such as a bacteriocin peptide or peptidomimetic to inhibit and/or neutralize a microbial cell (i.e. its biological activity) such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, may be determined using standard methods in the art, for example utilizing standard commercial in vitro tests such as ASTM E2149-20 or ASTM E1054-08 (ASTM, PA, USA), and the like, as well as methods such as the resazurin microtiter assay (REMA) described in Palomino et al. (2002) and Martin et al. (2003), both of which are incorporated herein by reference in their entireties. A REMA assay typically includes the incubation of a liquid microbial cell culture, to which resazurin and an antimicrobial compound such as a bacteriocin peptide or peptidomimetic have been added, under conditions suitable for the growth of said microbial cell, which will depend on the microbial cell and will be known to the skilled person. Resazurin typically has a blue colour, which changes to pink when active microbial growth occurs. Antimicrobial activity of the added compound can thus be determined by naked eye observation or any colorimetric method known in the art, by observing the lack of change of colour from blue to pink in the incubated culture. This method is compatible with serial dilutions, i.e. progressively lower concentrations of the antimicrobial compound may be applied in order to accurately pinpoint the relevant concentrations wherein antimicrobial activity is present. An exemplary application of REMA for the determination of biological activity of a bacteriocin is further provided in the experimental section herein. The same methods can be used for determination of biological activity of antibiotics, such as, but not limited to rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, and the like, as well as combinations of a bacteriocin with antibiotics. As a nonlimiting example, a bacteriocin peptide or peptidomimetic as described herein may be considered biologically active against a target microbial cell when application of a concentration of 1000 pg/ml or lower of said bacteriocin in a resazurin microtiter assay (REMA) as described above does not result in a resazurin change of colour from blue to pink. Non-limiting exemplary concentration values may be from 0.01 to 1000 pg/ml, from 0.1 to 1000 pg/ml, from 1 to 1000 |jg/ml , from 2 to 500 pg/ml, from 3 to 400 pg/ml, from 4 to 300 pg/ml, from 5 to 200 pg/ml, from 10 to 150 |jg/ml, from 25 to 125 pg/ml, from 25 to 100 pg/ml, or from 50 to 100 pg/ml.

Biological activity of a bacteriocin peptide or peptidomimetic as described herein may also be assessed using its minimal inhibitory concentration (MIC) value against a target microbial cell such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis. "Minimal inhibitory concentration” as used herein refers to the lowest concentration of a bacteriocin peptide or peptidomimetic which inhibits the growth of the target cell such that a change of resazurin colour from blue to pink in a liquid microbial cell culture is prevented, when the resazurin microtiter assay method as described above is used for determination of said concentration. Alternatively, or in addition, the minimum bactericidal concentration (MBC), also known as minimum lethal concentration (MLC) may be used to assess biological activity, which refers to the lowest concentration of a bacteriocin peptide or peptidomimetic which is able to kill 99.9% of the cells present in a liquid culture of a bacterium such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, after incubation for at least 21 days under conditions suitable for the growth of said bacterium, as compared to a reference culture which has not been exposed to said peptide or peptidomimetic. Determination of MIC and MBC may be combined in successive assays, for example cultures that are used for MIC determination may be later used for MBC determination. Exemplary methods of MIC and MBC determination are further provided in the experimental section herein.

The skilled person understands that a bacteriocin peptide or peptidomimetic exhibiting a lower MIC and/or MBC value relative to another bacteriocin peptide or peptidomimetic may be considered to have an increased biological activity. The skilled person understands that the MIC and MBC values of a bacteriocin peptide or peptidomimetic as described herein will vary depending on the target microbial cell, for example different species or strains of the genus Mycobacterium. The MIC and/or MBC value of a bacteriocin peptide or peptidomimetic may be determined against a bacterium of the genus Mycobacterium, preferably against Mycobacterium tuberculosis, more preferably against Mycobacterium tuberculosis H37Rv (ATCC 27294) or another multi-drug resistant (MDR-TB) strain, for example a clinical isolate or a strain isolated from a hospital environment, such as described in the experimental section herein. The MIC and/or MBC value of a bacteriocin peptide or peptidomimetic may be determined against Mycobacterium abscessus, more preferably against Mycobacterium abscessus CCUG 41449 or any other strain, for example a clinical isolate or a strain isolated from a hospital environment, in some cases being multi-drug resistant. A description of multi-drug resistant Mycobacterium strains is provided later herein. Said values may be the same or may differ. The MIC and/or MBC values for different target microbial cells may also be determined according to methods commonly used in the art, such as discussed in standard handbooks such as Schwalbe R. et al., Antimicrobial susceptibility testing protocols, Boca Raton: CRC Press (2007) (incorporated herein by reference in its entirety), and/or protocols (CLSI Clinical and Laboratory Standards Institute, 2018; NCCLS, 1999; both of which incorporated herein by reference in their entireties) and/or commercially available kits such as ETEST® (Biomerieux, NC, USA). The MBC/MIC ratio may also be determined, once MIC and MBC values are known. The MIC value, MBC value, and MBC/MIC value of an antimicrobial compound such as an antibiotic, or a combination of a bacteriocin peptide or peptidomimetic with an antimicrobial compound such as an antibiotic, against a target microbial cell such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, may also be determined using the methods described above.

In some embodiments, the MIC and/or MBC value of a bacteriocin peptide or peptidomimetic, preferably peptide, as described herein ranges from 0.01 to 1000 pg/ml, from 0.1 to 1000 pg/ml, from 1 to 1000 pg/ml, from 2 to 500 |jg/ml , from 3 to 400 pg/ml, from 4 to 300 pg/ml, from 5 to 200 pg/ml, from 10 to 150 pg/ml, from 25 to 125 pg/ml, from 25 to 100 pg/ml, or from 50 to 100 pg/ml.

In some embodiments, the MIC value of a bacteriocin peptide or peptidomimetic, preferably peptide, is 0.01 pg/ml or lower, 0.1 pg/ml or lower, 1 pg/ml or lower, 2 pg/ml or lower, 3 pg/ml or lower, 4 pg/ml or lower, 5 pg/ml or lower, 6 pg/ml or lower, 7 pg/ml or lower, 8 pg/ml or lower, 9 pg/ml or lower, 10 pg/ml or lower, 11 pg/ml or lower, 12 pg/ml or lower, 13 pg/ml or lower, 14 pg/ml or lower, 15 pg/ml or lower, 16 pg/ml or lower, 17 pg/ml or lower, 18 pg/ml or lower, 19 pg/ml or lower, 20 pg/ml or lower, 21 pg/ml or lower, 22 pg/ml or lower, 23 pg/ml or lower, 24 pg/ml or lower, 25 pg/ml or lower, 26 pg/ml or lower, 27 pg/ml or lower, 28 pg/ml or lower, 29 pg/ml or lower, 30 pg/ml or lower, 31 pg/ml or lower, 32 pg/ml or lower, 33 pg/ml or lower, 34 pg/ml or lower, 35 pg/ml or lower, 36 pg/ml or lower, 37 pg/ml or lower, 38 pg/ml or lower, 39 pg/ml or lower, 40 pg/ml or lower, 41 pg/ml or lower, 42 pg/ml or lower, 43 pg/ml or lower, 44 pg/ml or lower, 45 pg/ml or lower, 46 pg/ml or lower, 47 pg/ml or lower, 48 pg/ml or lower, 49 pg/ml or lower, 50 pg/ml or lower, 51 pg/ml or lower, 52 pg/ml or lower, 53 pg/ml or lower, 54 pg/ml or lower, 55 pg/ml or lower, 56 pg/ml or lower, 57 pg/ml or lower, 58 pg/ml or lower, 59 pg/ml or lower, 60 pg/ml or lower, 61 pg/ml or lower, 62 pg/ml or lower, 63 pg/ml or lower, 64 pg/ml or lower, 65 pg/ml or lower, 66 pg/ml or lower, 67 pg/ml or lower, 68 pg/ml or lower, 69 pg/ml or lower, 70 pg/ml or lower, 71 pg/ml or lower, 72 pg/ml or lower, 73 pg/ml or lower, 74 pg/ml or lower, 75 pg/ml or lower, 76 pg/ml or lower, 77 pg/ml or lower, 78 pg/ml or lower, 79 pg/ml or lower, 80 pg/ml or lower, 81 pg/ml or lower, 82 pg/ml or lower, 83 pg/ml or lower, 84 pg/ml or lower, 85 pg/ml or lower, 86 pg/ml or lower, 87 pg/ml or lower, 88 pg/ml or lower, 89 pg/ml or lower, 90 pg/ml or lower, 91 pg/ml or lower, 92 pg/ml or lower, 93 pg/ml or lower, 94 pg/ml or lower, 95 pg/ml or lower, 96 pg/ml or lower, 97 pg/ml or lower, 98 pg/ml or lower, 99 pg/ml or lower, 100 pg/ml or lower, 110 pg/ml or lower, 120 pg/ml or lower, 130 pg/ml or lower, 140 pg/ml or lower, 150 pg/ml or lower, 160 pg/ml or lower, 170 pg/ml or lower, 180 pg/ml or lower, 190 pg/ml or lower, 200 pg/ml or lower, 250 pg/ml or lower, 300 pg/ml or lower, 400 pg/ml or lower, 500 pg/ml or lower, 550 pg/ml or lower, 600 pg/ml or lower, 650 pg/ml or lower, 700 pg/ml or lower, 750 pg/ml or lower, 800 pg/ml or lower, 850 pg/ml or lower, 900 pg/ml or lower, 950 pg/ml or lower, 1000 pg/ml or lower, preferably it is 50 pg/ml or lower or 100 pg/ml or lower.

In some embodiments, the MBC value of a bacteriocin peptide or peptidomimetic, preferably peptide, is 0.01 pg/ml or lower, 0.1 pg/ml or lower, 1 pg/ml or lower, 2 pg/ml or lower, 3 pg/ml or lower, 4 pg/ml or lower, 5 pg/ml or lower, 6 pg/ml or lower, 7 pg/ml or lower, 8 pg/ml or lower, 9 pg/ml or lower, 10 pg/ml or lower, 11 pg/ml or lower, 12 pg/ml or lower, 13 pg/ml or lower, 14 pg/ml or lower, 15 pg/ml or lower, 16 pg/ml or lower, 17 pg/ml or lower, 18 pg/ml or lower, 19 pg/ml or lower, 20 pg/ml or lower, 21 pg/ml or lower, 22 pg/ml or lower, 23 pg/ml or lower, 24 pg/ml or lower, 25 pg/ml or lower, 26 pg/ml or lower, 27 pg/ml or lower, 28 pg/ml or lower, 29 pg/ml or lower, 30 pg/ml or lower, 31 pg/ml or lower, 32 pg/ml or lower, 33 pg/ml or lower, 34 pg/ml or lower, 35 pg/ml or lower, 36 pg/ml or lower, 37 pg/ml or lower, 38 pg/ml or lower, 39 pg/ml or lower, 40 pg/ml or lower, 41 pg/ml or lower, 42 pg/ml or lower, 43 pg/ml or lower, 44 pg/ml or lower, 45 pg/ml or lower, 46 pg/ml or lower, 47 pg/ml or lower, 48 pg/ml or lower, 49 pg/ml or lower, 50 pg/ml or lower, 51 pg/ml or lower, 52 pg/ml or lower, 53 pg/ml or lower, 54 pg/ml or lower, 55 pg/ml or lower, 56 pg/ml or lower, 57 pg/ml or lower, 58 pg/ml or lower, 59 pg/ml or lower, 60 pg/ml or lower, 61 pg/ml or lower, 62 pg/ml or lower, 63 pg/ml or lower, 64 pg/ml or lower, 65 pg/ml or lower, 66 pg/ml or lower, 67 pg/ml or lower, 68 pg/ml or lower, 69 pg/ml or lower, 70 pg/ml or lower, 71 pg/ml or lower, 72 pg/ml or lower, 73 pg/ml or lower, 74 pg/ml or lower, 75 pg/ml or lower, 76 pg/ml or lower, 77 pg/ml or lower, 78 pg/ml or lower, 79 pg/ml or lower, 80 pg/ml or lower, 81 pg/ml or lower, 82 pg/ml or lower, 83 pg/ml or lower, 84 pg/ml or lower, 85 pg/ml or lower, 86 pg/ml or lower, 87 pg/ml or lower, 88 pg/ml or lower, 89 pg/ml or lower, 90 pg/ml or lower, 91 pg/ml or lower, 92 pg/ml or lower, 93 pg/ml or lower, 94 pg/ml or lower, 95 pg/ml or lower, 96 pg/ml or lower, 97 pg/ml or lower, 98 pg/ml or lower, 99 pg/ml or lower, 100 pg/ml or lower, 110 pg/ml or lower, 120 pg/ml or lower, 130 pg/ml or lower, 140 pg/ml or lower, 150 pg/ml or lower, 160 pg/ml or lower, 170 pg/ml or lower, 180 |jg/ml or lower, 190 pg/ml or lower, 200 pg/ml or lower, 250 pg/ml or lower, 300 pg/ml or lower, 400 pg/ml or lower, 500 |jg/ml or lower, 550 pg/ml or lower, 600 pg/ml or lower, 650 pg/ml or lower, 700 pg/ml or lower, 750 pg/ml or lower, 800 pg/ml or lower, 850 pg/ml or lower, 900 pg/ml or lower, 950 pg/ml or lower, 1000 pg/ml or lower, preferably it is 100 pg/ml or lower.

Within the context of bacteriocin peptides, peptidomimetics, or pharmaceutical compositions for use, methods, and uses of the invention, one or more antimicrobial compounds such as an antifungal agent, antiviral agent, essential oil, another bacteriocins, and/or an antibiotic, preferably an antibiotic, may optionally be used in combination with the bacteriocin peptide or peptidomimetic. The skilled person understands that the choice of a particular antimicrobial compound may depend on the target microbial cell. A bacteriocin may be any class I or class II bacteriocin, including any corresponding subgroup bacteriocin, as summarized in Cotter, P.D. et al. , Nature Reviews Microbiology 2012;11 (2): 95-105, incorporated by reference herein in its entirety. Non-limiting examples of suitable groups of bacteriocins are shown in Table 1 .

Table 1 . Examples of suitable groups of bacteriocins

The further use of an antibiotic may be particularly suitable. An antibiotic may be any compound selected from, but not limited to, the group of polyketides (e.g. the rifamycin group such as rifampicin, rifapentine, rifabutin), penicillins (0-lactams), glycylcyclines, aminonucleosides, nucleoside analogues, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulphonamides, polypeptides, glycopeptides, lipoglycopeptides, aminoglycosides (e.g. kanamycin, amikacin, capreomycin), fluoroquinolones (e.g. ofloxacin, moxifloxacin, levofloxacin), monobactams, oxazolidinones, streptogramins, rifamycins, carbapenems, chloramphenicol, clindamycin, daptomycin, fosfomycin, lefamulin, metronidazole, mupirocin, nitrofurantoin, tigecycline, puromycin, hygromycin B (hygrovetine), geneticin (G418), bleomycin, zeocin, isoniazid, amikacin, cefoxitine, meropenem, streptomycin (SM), pyrazinamide, clarithromycin, imipenem, and blasticidin. Preferred antibiotics are polyketides and fluoroquinolones, such as, but not limited to, antibiotics of the rifamycin group (e.g. rifampicin, rifapentine, rifabutin), ofloxacin, levofloxacin, and moxifloxacin, with rifampicin, moxifloxacin, and ofloxacin being further preferred, and with rifampicin and ofloxacin being even further preferred. Additional preferred antibiotics are macrolides, tetracyclines, glycylcyclines, 0- lactams, carbapenems, and aminoglycosides, of which clarithromycin, tigecycline, imipenem, and amikacin are further preferred A combination of multiple different antibiotics may also be optionally used.

Accordingly, in some embodiments, an antibiotic is further used. In some embodiments, a polyketide antibiotic, preferably selected from the rifamycin group, more preferably selected from rifampicin, rifapentine, rifabutin, or a combination thereof, is further used. In some embodiments, a fluoroquinolone antibiotic, preferably selected from ofloxacin, levofloxacin, moxifloxacin , or a combination thereof, is further used. In some embodiments, a macrolide antibiotic is further used. In some embodiments, a tetracycline antibiotic is further used. In some embodiments, a glycylcycline antibiotic is further used. In some embodiments, a 0-lactam antibiotic is further used. In some embodiments, a carbapenem antibiotic is further used. In some embodiments, an amikacin antibiotic is further used. In preferred embodiments, the further used antibiotic is selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a 0-lactam, a carbapenem, an aminoglycoside, or a combination thereof, preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof, more preferably selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

In other preferred embodiments, the further used antibiotic is selected from rifampicin, ofloxacin or a combination thereof. In some embodiments, the further used antibiotic is rifampicin. In some embodiments, the further used antibiotic is ofloxacin. In some embodiments, the further used antibiotic is moxifloxacin. In some embodiments, the further used antibiotic is clarithromycin. In some embodiments, the further used antibiotic is tigecycline. In some embodiments, the further used antibiotic is imipenem. In some embodiments, the further used antibiotic is amikacin.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , or by an amino acid sequence having at least 70%, 80%, 90%, or 95% identity or similarity with SED ID NO: 1 , and the further used antibiotic is selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, or a combination thereof, preferably selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, or a combination thereof. In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 2, or by an amino acid sequence having at least 70%, 80%, 90%, or 95% identity or similarity with SED ID NO: 2, and the further used antibiotic is selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, or a combination thereof, preferably selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, or a combination thereof.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 3, or by an amino acid sequence having at least 70%, 80%, 90%, or 95% identity or similarity with SED ID NO: 3, and the further used antibiotic is selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof, preferably selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

In some embodiments, the bacteriocin peptide or peptidomimetic, preferably peptide, comprises, consists essentially of, or consists of, preferably comprises, a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 4, or by an amino acid sequence having at least 70%, 80%, 90%, or 95% identity or similarity with SED ID NO: 4, and the further used antibiotic is selected from rifampicin, clarithromycin, tigecycline, or a combination thereof.

As discussed above, and further demonstrated in the experimental section herein, the bacteriocin peptides or peptidomimetics described herein demonstrate a strong synergistic inhibition effect against target microbial cells such as bacteria of the genus Mycobacterium and particularly against antibiotic-resistant target microbial cells such as, but not limited to, antibiotic-resistant strains of Mycobacterium tuberculosis or Mycobacterium abscessus, when combined with antibiotics. A "synergistic inhibition effect” or "synergistic effect” or "synergistic biological activity” as used herein refers to the biological activity of the combination of the bacteriocin peptide or peptidomimetic with the antibiotic being greater than the sum (the additive biological activity) of the individual biological activities of said bacteriocin and antibiotic. Said activity may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, or at least 100- fold higher than the sum of the individual biological activities of said bacteriocin and antibiotic. Assessment of synergistic biological activity may be performed using the methods described above, some of which are exemplified in the experimental section herein.

Alternatively, synergistic biological activity may be assessed by determination of the fractional inhibitory concentration index (FIC index). The term "fractional inhibitory concentration index” as used herein refers to a dimensionless number which arises from determination of the presence of synergistic biological activity by using a checkerboard assay, as described in standard publications such as Hsieh et al., 1993, which is incorporated herein by reference in its entirety. The checkerboard assay may be combined with a resazurin microtiter assay as described earlier herein, as described in standard publications such as Santos et al., 2018, which is incorporated herein by reference in its entirety. Determination of the FIC index generally involves the determination of the MIC value of an antimicrobial compound, such as a bacteriocin peptide or peptidomimetic as described herein, individually and in combination with another antimicrobial compound, such as an antibiotic. The FIC index may then be calculated using Formula I as follows: ... (Formula I), with:

“A, combination” corresponding to the MIC value of another antimicrobial compound, such as an antibiotic, which is used in combination with the bacteriocin peptide or peptidomimetic as described herein;

"A, alone” corresponding to the MIC value of another antimicrobial compound, such as an antibiotic, when determined individually;

”B, combination” corresponding to the MIC value of a bacteriocin peptide or peptidomimetic as described herein, which is used in combination with another antimicrobial compound such as an antibiotic, and;

”B, alone” corresponding to the MIC value of a bacteriocin peptide or peptidomimetic as described herein when determined individually.

A FIC index of 0.5 or lower (< 0.5) demonstrates synergistic biological activity (i.e. a synergistic inhibition effect). A FIC index of higher than 0.5 (> 0.5) but lower than or equal to 1 (< 1) demonstrates additive biological activity (no synergy). Values higher than 1 (> 1) but lower than or equal to 4 (< 4) demonstrate an indifferent effect, with values higher than >4 demonstrating antagonism between tested compounds. An exemplary method of FIC index determination is further provided in the experimental section herein.

In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and an antibiotic, preferably polyketide, fluoroquinolone, macrolide, tetracycline, glycylcycline, -lactam, carbapenem, or aminoglycoside, more preferably rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, or amikacin, has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and an antibiotic, preferably rifampicin, ofloxacin, or moxifloxacin, more preferably rifampicin or ofloxacin, has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and rifampicin has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and ofloxacin has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and moxifloxacin has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and clarithromycin has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and tigecycline has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and imipenem has a FIC index of 0.5 or lower, preferably of 0.4 or lower. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and amikacin has a FIC index of 0.5 or lower, preferably of 0.4 or lower.

The FIC index is preferably determined against a bacterium of the genus Mycobacterium, preferably against Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably against an antibiotic-resistant strain of Mycobacterium tuberculosis or Mycobacterium abscessus.

A combination of a bacteriocin peptide or peptidomimetic, preferably peptide, with an antibiotic, preferably rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, or amikacin, which demonstrates synergistic biological activity will have a MIC and/or MBC value against a target microbial cell such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis, which is lower than the MIC value against said target microbial cell which is demonstrated by the bacteriocin peptide or peptidomimetic alone. The MIC and/or MBC value of the combination may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (2-fold), at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, or at least 100-fold lower relative to the MIC value of the bacteriocin peptide or peptidomimetic alone. In some embodiments, the combination of a bacteriocin or peptidomimetic, preferably peptide, and an antibiotic, preferably rifampicin, ofloxacin, or moxifloxacin, more preferably rifampicin or ofloxacin, has a MIC value against a bacterium such as a bacterium of the genus Mycobacterium, preferably against Mycobacterium tuberculosis, more preferably against an antibiotic-resistant strain of Mycobacterium tuberculosis, that is at least 4-fold, 8-fold, or 16- fold lower, preferably at least 4-fold lower or at least 8-fold lower, relative to the MIC value of the bacteriocin peptide or peptidomimetic alone.

Within the context of the invention, a bacteriocin peptide may be produced by a cell or be synthetic. Production of a peptide by a cell can be endogenous or exogenous. Endogenous production refers to production of a peptide by a cell that is natively able to produce it (i.e. a cell that comprises the required genetic information for its production). In some embodiments, endogenous production refers to production by Lactococcus lactis, for example production of a bacteriocin peptide represented by SEQ ID NO: 1 or SEQ ID NO: 2 by Lactococcus lactis. In some embodiments, endogenous production refers to production by Staphylococcus aureus, for example production of a bacteriocin peptide represented by SEQ ID NO: 3 by Staphylococcus aureus. In some embodiments, endogenous production refers to Staphylococcus epidermidis, for example production of a bacteriocin peptide represented by SEQ ID NO: 4 by Staphylococcus epidermidis. Exogenous production typically refers to production of the peptide by a different organism and/or cell (i.e. a host), by which said peptide is not natively produced, the capability of which having been introduced via means of recombinant DNA technology. Within the context of the invention, the term exogenous production also encompasses cases wherein the native production of a peptide, preferably by Lactococcus lactis, Staphylococcus aureus, or Staphylococcus epidermidis, is increased via means of recombinant DNA technology using standard molecular toolbox techniques as compared to the corresponding endogenous production. It also encompasses cases wherein a variant peptide is produced, as described later herein. Said increase may be achieved by modification of any of the steps of bacteriocin production, including transcription, post- transcriptional modification, translation, post-translational modification, and secretion. Said increase may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% compared to the corresponding endogenous production. A definition of peptide production, alternatively referred to herein as peptide expression, is provided in the section titled "general information”.

Exogenous production can be achieved by introduction of a nucleotide sequence comprising a bacteriocin encoding sequence (ORF) to a host organism and/or cell. Recombinant DNA techniques, suitable host organisms and/or cells for exogenous protein production, and culturing methods are well-known in the art and are described in standard handbooks such as Ausubel et al., Current Protocols in Molecular Biology, 3^rd edition, John Wley & Sons Inc (2003) and in Sambrook and Green, Molecular Cloning. A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press (2012); both of which are incorporated herein by reference in their entireties. To achieve exogenous protein production, a bacteriocin ORF, operably linked to (i. e. in a functional relationship with) a suitable transcription initiation sequence such as a promoter, will typically be introduced to a suitable host cell according to standard techniques. A promoter may be constitutive i.e. allowing constant expression of a bacteriocin peptide, or inducible i.e. only allowing expression of a bacteriocin peptide under specific culture conditions or upon induction with chemical compounds. Optionally, other regulatory sequences such as transcription terminators, enhancers, kozak sequences, polyA sequences and the like may be operably linked to the bacteriocin ORF. The choice of a particular regulatory sequence will depend on the choice of the host cell and is well within the capabilities of the skilled person. As a non-limiting example, a nucleotide sequence comprising, consisting essentially of, or consisting of, preferably comprising, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a nucleotide sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, may be introduced in a suitable host cell.

The bacteriocin ORF may be stably integrated in a suitable cell’s genome or may be introduced in a self-replicating vector. The bacteriocin ORF may be codon-optimized for expression in a particular host cell, e.g. Escherichia coli, using commonly used computer algorithms. A definition of codon optimization is given in the section titled "general information”. Suitable host cells may be selected from mammalian, insect, plant, or microbial cells, preferably are selected from microbial cells. Examples of suitable microbial cells include eukaryotes such as yeasts, filamentous fungi, and algae, and prokaryotes such as bacteria and archaea, of which bacteria is preferred. Bacterial host cells include both Gram-negative and Gram-positive bacteria and can be selected from suitable groups known in the art such as Bacillus species (for example Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis) , Paenibacillus species, Streptomyces species, Staphylococcus species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species, Pseudomonas species, Lactobacillus species, Lactococcus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus, Pseudomonas stutzeri, Clostridium thermocellus, Escherichia coli, including strains thereof, of which Lactococcus species and Staphylococcus species are preferred, with Lactococcus lactis, Staphylococcus aureus, and Staphylococcus epidermidis being more preferred. Algae host cells may be selected from suitable groups known in the art such as Botryococcus braunii, Chlorella species, Dunaliella tertiolecta, Gracilaria species, Pleurochrysis carterae, and Sargassum species. Yeast host cells may be selected from suitable groups known in the art such as Saccharomyces species (for example, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (for example, Candida utilis, Candida krusei), Schizosaccharomyces species (for example Schizosaccharomyces pombe, Schizosaccharomyces japonicus), Pichia or Hansenula species (for example, Pichia pastoris or Hansenula polymorpha) species, and Brettanomyces species (for example, Brettanomyces claussenii). Filamentous fungal host cells may be selected from suitable groups known in the art such as Acremonium, Agaricus, Altemaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocaffimastix, Neurospora, Paecilomyces, Peniciffium, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria. Species include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenaturn, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Penicillium chrysogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

A bacteriocin peptide may be isolated and/or purified from its producing cell. Suitable downstream processing methods for isolation and/or purification of products from cell cultures are well-known in the art and are described in standard handbooks such as Wesselingh, J.A and Krijgsman, J., 1^st edition, Downstream Processing in Biotechnology, Delft Academic Press (2013), incorporated herein by reference in its entirety. Examples of suitable isolation and/or purification techniques are chromatographic methods such as high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, immunoprecipitation via the use of tags, and the like. Accordingly, in some embodiments the bacteriocin peptide is an isolated and/or purified peptide.

Within the context of the invention, a bacteriocin peptide may be produced in vitro, using isolated and/or purified cellular components (cell-free extracts) comprising the necessary transcription and translation machinery. In vitro protein production typically comprises transcription and translation of isolated circular or linear DNA, or only translation when isolated mRNA is used as a template, said DNA or mRNA comprising a bacteriocin encoding sequence optionally operably linked to regulatory sequences as discussed elsewhere herein. The corresponding cellular components may be isolated/purified and the reaction conditions can be chosen according to standard methods, such as for example described in Gregorio et al., Methods Protoc 2(1):24 (2019), incorporated herein by reference in its entirety. Alternatively, commercial in vitro protein synthesis kits such as PURExpress® (New England Biolabs, MA, USA) may be used. Depending the on the cell used for isolation/purification of the cellular components, e.g. Escherichia coli, and/or the commercial kit used, the bacteriocin ORF may be codon optimized for expression in that particular cell and/or commercial kit. As a non-limiting example, a nucleotide sequence comprising, consisting essentially of, or consisting of, preferably comprising, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a nucleotide sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, may be used in conjunction with PURExpress® according to the manufacturer’s protocol to produce a bacteriocin. Accordingly, in some embodiments the bacteriocin peptide is an in vitro produced peptide.

Within the context of the invention, a bacteriocin peptide may be synthetic. The term “synthetic peptide’’ has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to a peptide which is generated by means of chemical peptide synthesis. A synthetic bacteriocin peptide according to the invention may be prepared or synthesized using conventional methods that are well-known in the art. For instance, peptides can be synthesized by commonly used solid-phase synthesis methods such as those that involve a tert-butyloxycarbonyl-protecting group (t-BOC) or fluorenylmethyloxycarbonyl-protecting group (FMOC) for protection of alpha-amino groups. In such methods, amino acids are added sequentially to a growing amino acid chain. Such methods are, for instance, described in Merrifield, J. Am. Chem. Soc. 85(14):2149-2154 (1963), and Atherton & Sheppard, Solid Phase Peptide Synthesis: A practical Approach, IRL Press, Oxford, UK (1999), both of which are incorporated herein by reference in their entireties. Accordingly, in some embodiments, the bacteriocin peptide is a synthetic peptide.

Within the context of the invention, a bacteriocin may be a peptidomimetic. As used herein, a “peptidomimetic” (alternatively referred to as "mimetic”) is understood to encompass all compounds whose essential elements mimic a natural peptide and which retain the ability to interact with the biological target and exert the natural peptide’s biological activity. The biological activity of a bacteriocin peptidomimetic may be the same, decreased, or increased as compared to a bacteriocin peptide. Decreased biological activity of a bacteriocin peptidomimetic may mean that the peptidomimetic exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the biological activity of the corresponding bacteriocin peptide. Increased biological activity of a bacteriocin peptidomimetic may mean that the peptidomimetic exhibits an increase of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in biological activity as compared to the corresponding bacteriocin peptide. A definition of "biological activity” and measurement methods thereof are provided elsewhere herein.

In some embodiments, the peptidomimetic comprises, consists essentially of, or consists of, preferably comprises, a non-naturally occurring amino acid sequence. In some embodiments, the peptidomimetic does not occur in nature and is considered to be man-made. Peptidomimetics typically arise either from modification of an existing peptide, or by designing similar systems that mimic peptides, such as peptoids and -peptides. Structures and synthesis of peptidomimetics are for instance described in William D. Lubell (ed.), Peptidomimetics I and II, Topics in Heterocyclic Chemistry (Book 48), Springer 1^st ed., XVI, 310 p (2017); Trabocchi A. Chapter 6 - Principles and applications of small molecule peptidomimetics, Small Molecule Drug Discovery Methods, Molecules and Applications, pp 163-195, Elsevier (2020); Vagner et al., Curr Opin Chem Biol. 12(3): 292-296 (2008), all of which incorporated herein by reference in their entireties. A bacteriocin peptidomimetic may be a structural mimetic of a bacteriocin peptide described herein. Structural mimetics, also known as type I mimetics, have analogous structural features to the bacteriocin peptide they mimic. A bacteriocin peptidomimetic may be a functional mimetic of a bacteriocin peptide described herein. Functional mimetics, also known as type II mimetics, retain the ability to interact with the biological target and exert the natural peptide’s biological activity without apparent structural analogy to the peptide. A bacteriocin peptidomimetic may be a functional-structural mimetic of a bacteriocin peptide described herein. Functional-structural mimetics, also known as type III mimetics, generally comprise a scaffold having a structure different from the bacteriocin peptide that they mimic, in which all the functional groups needed for the biological activity are mounted in a well-defined spatial orientation.

In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which a modification has been introduced, for example to the backbone and/orthe side chains.

In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which a non-natural amino acid has been introduced. Examples of non-natural amino acids are provided later herein. In some embodiments, a natural amino acid is substituted by a non-natural amino acid or a D-amino acid, which may, for example, be corresponding as described later herein.

In some embodiments, a bacteriocin peptidomimetic corresponds to a bacteriocin peptide in which the peptide backbone has been replaced completely, for example by a heterocycle, a sugar, or other scaffold. Examples of suitable scaffolds are known to the skilled person and discussed, for example, in Pelay-Gimeno et al., Angew Chem Int Ed Engl; 54(31): 8896-8927 (2015), incorporated herein by reference in its entirety. In some embodiments, a bacteriocin peptidomimetic corresponds to a peptoid. In some embodiments, a bacteriocin peptidomimetic corresponds to a -peptide.

Modification of an existing peptide may be the result of natural processes, such as post-translational processing, or chemical modification techniques. In some embodiments, a peptidomimetic refers to a compound containing non- peptidic structural elements. Typical but non-limiting examples of non-peptidic structural elements are modifications of one or more existing amino acids, conformational restraints, cyclization of the polypeptide, isosteric replacement or other modifications. In some embodiments, a peptidomimetic may contain one or more or all substitutions of an amino acid by the corresponding D-amino acid. As used herein, “corresponding D-amino acid’’ denotes the D-amino acid counterpart of an L-amino acid. A peptidomimetic may also optionally contain non-natural amino acids. As used herein, “non-natural amino acid’’ has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to non-genetically encoded amino acids, irrespective of whether they appear in nature or not. Non-natural amino acids that can be present in a peptidomimetic as described herein include: 0- amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl-L-tyrosine; 2-aminoadipic acid; 3-aminoadipic acid; beta-alanine; 4-tert-butyl hydrogen 2-azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid; 2,4-diamino butyric acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2- aminopimelic acid; p-aminophenylalanine; 2,3-diaminobutyric acid; 2,3-diamino propionic acid; 2,2’-diaminopimelic acid; p-amino-L-phenylalanine; p-azido-L- phenylalanine; D-allyl glycine; p-benzoyl-L- phenylalanine; 3-benzothienyl alanine p-bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosine; epsilon-amino hexanoic acid; N- ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3-fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxy lysine; alio-hydroxy lysine; 3-(3-methyl-4-nitrobenzyl)-L- histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3- methyl-phenylalanine; N- methylglycine; N-methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; 16e sulfoxide; 2- napthylalanine; L-3-(2-naphthyl)alanine; isoserine; 3-phenylserine; norvaline; norleucine; 5,5,5-trifluoro-DL-leucine; ornithine; 3-chloro-tyrosine; N5-carbamoylornithine; penicillamine; phenylglycine; piperidinic acid; pyridylalanine; 1 ,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid; beta-2-thienylalanine; y-carboxy-DL-glutamic acid; 4-fluoro-DL- glutamic acid; D-thyroxine; allo-threonine; 5-hydroxy-tryptophan; 5-methoxy-tryptophan; 5-fluoro-tryptophan; 3- fluoro-valine. In some embodiments, a natural amino acid of a bacteriocin peptide or peptidomimetic according to the invention is substituted by a corresponding non-natural amino acid. As used herein, a “corresponding nonnatural amino acid’’ refers to a non-natural amino acid that is a derivative of the reference natural amino acid. For instance, a natural amino acid can be substituted by the corresponding 0-amino acid, which has its amino group bonded to the 0-carbon rather than the a-carbon. In some embodiments, a peptide or peptidomimetic of the invention may further be provided with a targeting moiety. It is known that peptidomimetics are able to circumvent some of the disadvantages associated with natural peptides: e.g. stability against proteolysis (duration of activity) and poor bioavailability. Certain other properties, such as receptor selectivity or potency, often can be substantially improved.

Within the context of the invention, a bacteriocin peptide or peptidomimetic may further be modified by natural processes, such as post-translational processing, or by chemical modification techniques. Such modifications may be inserted in the peptide at any location, including in the backbone, amino acid side-chains and at the N- or C- terminus. Multiple types of modifications may occur in a single peptide, or a peptide may comprise several modifications of a single type. Types of modifications and modification techniques are well-known in the art and described in standard handbooks such as Peptide Modifications to Increase Metabolic Stability and Activity, 1^st edition, Ed. Predrag Cudic, Humana Press (2013), incorporated herein by reference in its entirety. Accordingly, in some embodiments, the bacteriocin peptide or peptidomimetic comprises at least one amino acid modification selected from the group consisting of alkylation, acetylation, amidation, acylation, phosphorylation, methylation, demethylation, ADP-ribosylation, disulfide bond formation, ubiquitination, gamma-carboxylation, glycosylation, hydroxylation, iodination, oxidation, pegylation, succinylation, and sulfation, preferably selected from methylation or glycosylation.

Within the context of the invention, a bacteriocin peptide or peptidomimetic may comprise one or more modifications in its sequence, resulting in bacteriocin peptide or peptidomimetic variants (alternatively referred to herein as mutants). Said sequence modifications may include amino acid substitutions, deletions and/or insertions. Variant peptides or peptidomimetics can, for example, be synthetically made or made by cellular (or in vitro) production as described elsewhere herein, after modifying the nucleotide sequence encoding forsaid peptides using mutagenesis techniques known to the skilled person, such as, random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, CRISPR/Cas-mediated mutagenesis and the like, so that the resulting nucleotide sequence encodes a peptide that differs by at least one amino acid from the non-modified peptide or peptidomimetic, i.e. wherein at least one amino acid is substituted by a different amino acid and/or at least one amino acid is deleted and/or at least one amino acid is inserted. Amino acid substitutions may be conservative. A definition of a "conservative” substitution is provided in the section titled "general information”. Variant peptides and/or peptidomimetics according to the invention may retain decreased, but still detectable, or increased biological activity as compared to the corresponding non-modified peptide or peptidomimetic. Biological activity (i.e. antimicrobial activity) against a target microbial cell such as a bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis, may be assessed as described elsewhere herein. Decreased biological activity of a variant may mean that the variant exhibits at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the biological activity of the corresponding non-modified bacteriocin peptide or peptidomimetic. Increased biological activity of a variant may mean that the variant exhibits an increase of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, or at least 200% in biological activity as compared to the corresponding non-modified bacteriocin peptide or peptidomimetic.

Within the context of the invention, the physicochemical properties of a bacteriocin peptide or peptidomimetic may be the same or differ as compared to a corresponding naturally-occurring (wild-type) peptide. The skilled person is aware of such properties, non-limiting examples of which include susceptibility to enzymatic degradation (e.g. by proteinases, peptidases, aminopeptidases, carboxypeptidases, Rnases, phospholipases, amylases, and the like), susceptibility to degradation by organic solvents (e.g. to acetone, chloroform, acetonitrile, ethanol, 2-propanol, butanol, methanol, and the like), susceptibility to degradation by surfactants (e.g. anionic, non-ionic, cationic, amphoteric, silicon-based, fluorinated, polymeric, and the like), susceptibility to reducing agents (e.g. DTT, 0- mercaptoethanol, and the like), susceptibility to heat degradation, pH optimum, and the like. In some embodiments, a bacteriocin peptide or peptidomimetic exhibits at least one improved physicochemical property as compared to a corresponding naturally-occurring (wild-type) peptide. Physicochemical properties of peptides or peptidomimetics may be assessed by commonly used methods in the art, such as discussed in standard handbooks like Hansen, P. R., Antimicrobial Peptides: Methods and Protocols, 1^st Edition, Humana Press, US, (2017) and Remington: The Science and Practice of Pharmacy, 23^rd ed., Ed. Adejare A., Academic Press, US (2021), both of which are incorporated herein by reference in their entireties.

Within the context of the invention, a bacteriocin peptide or peptidomimetic, preferably peptide, as described herein may comprise, essentially consist of, or consist of, preferably comprise, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one amino acid of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 has been substituted by any amino acid.

"Any amino acid” as used herein includes any of the natural (L- and D- configuration) amino acids, non-natural aminoacids, as well as modified versions of natural and/or non-natural aminoacids, as described elsewhere herein. Said substitution may be conservative. Said substitution may correspond to specific amino acid positions of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. When multiple amino acids are substituted, they may correspond to consecutive positions or may be spatially apart in the peptide sequence. Determination of amino acids to be substituted in a peptide sequence corresponding to specific positions of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 may be performed by routine sequence alignment methods, further elaborated upon in the section titled "general information” herein. The skilled person understands that the methionine (M) residue at the N-terminus end of SEQ ID NO: 1 corresponds to position 1 , that the lysine (K) at the C-terminus end of SEQ ID NO: 1 corresponds to position 53, and that the amino acids in between the two ends of SEQ ID NO: 1 correspond to positions 2-52, respectively. The skilled person further understands that the methionine (M) residue at the N-terminus end of SEQ ID NO: 2 corresponds to position 1 , that the lysine (K) at the C-terminus end of SEQ ID NO: 2 corresponds to position 53, and that the amino acids in between the two ends of SEQ ID NO: 2 correspond to positions 2-52, respectively. The skilled person further understands that the methionine (M) residue at the N- terminus end of SEQ ID NO: 3 corresponds to position 1 , that the leucine (L) at the C-terminus end of SEQ ID NO:

3 corresponds to position 51 , and that the amino acids in between the two ends of SEQ ID NO: 3 correspond to positions 2-50, respectively. The skilled person further understands that the methionine (M) residue at the N- terminus end of SEQ ID NO: 4 corresponds to position 1 , that the alanine (A) at the C-terminus end of SEQ ID NO:

4 corresponds to position 51 , and that the amino acids in between the two ends of SEQ ID NO: 4 correspond to positions 2-50, respectively.

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one methionine (M) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by a cysteine (C) or proline (P).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one alanine (A) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by an amino acid selected from leucine (L), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glycine (G) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one phenylalanine (F) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by a tyrosine (Y) or tryptophan (W).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one leucine (L) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one lysine (K) of SEQ ID NO: 1 has been substituted by any amino acid, preferably an amino acid selected from arginine (R) or histidine (H).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one valine (V) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamine (Q) of SEQ ID NO: 1 has been substituted by any amino acid, preferably an amino acid selected from serine (S), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tyrosine (Y) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by a tryptophan (W) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one serine (S) of SEQ ID NO: 1 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tryptophan (W) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by a tyrosine (Y) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one asparagine (N) of SEQ ID NO: 1 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and serine (S).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one isoleucine (I) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and valine (V).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one aspartic acid (D) of SEQ ID NO: 1 has been substituted by any amino acid, preferably by a glutamic acid (E).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein the amino acid sequence of SEQ ID NO: 1 , has been modified in length.

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one methionine (M) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by a cysteine (C) or proline (P).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one alanine (A) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an amino acid selected from leucine (L), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glycine (G) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one phenylalanine (F) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by a tyrosine (Y) or tryptophan (W).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one leucine (L) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one lysine (K) of SEQ ID NO: 2 has been substituted by any amino acid, preferably an amino acid selected from arginine (R) or histidine (H).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one valine (V) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamine (Q) of SEQ ID NO: 2 has been substituted by any amino acid, preferably an amino acid selected from serine (S), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one isoleucine (I) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and valine (V).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tyrosine (Y) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by a tryptophan (W) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one serine (S) of SEQ ID NO: 2 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tryptophan (W) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by a tyrosine (Y) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one asparagine (N) of SEQ ID NO: 2 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and serine (S).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one aspartic acid (D) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by a glutamic acid (E).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamic acid (E) of SEQ ID NO: 2 has been substituted by any amino acid, preferably by an aspartic acid (D).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein the amino acid sequence of SEQ ID NO: 2, has been modified in length. In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one methionine (M) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by a cysteine (C) or proline (P).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one serine (S) of SEQ ID NO: 3 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tryptophan (W) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by a tyrosine (Y) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one leucine (L) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one asparagine (N) of SEQ ID NO: 3 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and serine (S).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one phenylalanine (F) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by a tyrosine (Y) or tryptophan (W).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one lysine (K) of SEQ ID NO: 3 has been substituted by any amino acid, preferably an amino acid selected from arginine (R) or histidine (H).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tyrosine (Y) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by a tryptophan (W) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one isoleucine (I) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and valine (V).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one alanine (A) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an amino acid selected from leucine (L), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glycine (G) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), leucine (L), and isoleucine (I). In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one valine (V) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamic acid (E) of SEQ ID NO: 3 has been substituted by any amino acid, preferably by an aspartic acid (D).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one proline (P) of SEQ ID NO: 3 has been substituted by any amino acid, preferably an amino acid selected from cysteine (C) or methionine (M).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one threonine (T) of SEQ ID NO: 3 has been substituted by any amino acid, preferably an amino acid selected from serine (S), glutamine (Q), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein the amino acid sequence of SEQ ID NO: 3, has been modified in length.

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one methionine (M) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by a cysteine (C) or proline (P).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one alanine (A) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an amino acid selected from leucine (L), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one phenylalanine (F) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by a tyrosine (Y) or tryptophan (W).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one lysine (K) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from arginine (R) or histidine (H).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one leucine (L) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), glycine (G), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one isoleucine (I) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and valine (V). In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamine (Q) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from serine (S), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one threonine (T) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from serine (S), glutamine (Q), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glycine (G) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), valine (V), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tyrosine (Y) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by a tryptophan (W) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one valine (V) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an amino acid selected from alanine (A), glycine (G), leucine (L), and isoleucine (I).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one serine (S) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and asparagine (N).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one tryptophan (W) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by a tyrosine (Y) or phenylalanine (F).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one histidine (H) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from arginine (R) or lysine (K).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one asparagine (N) of SEQ ID NO: 4 has been substituted by any amino acid, preferably an amino acid selected from glutamine (Q), threonine (T), and serine (S).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein at least one glutamic acid (E) of SEQ ID NO: 4 has been substituted by any amino acid, preferably by an aspartic acid (D).

In some embodiments, a bacteriocin peptide or peptidomimetic, preferably peptide, comprises, essentially consists of, or consists of, preferably comprises, a peptide or peptidomimetic represented by an amino acid sequence wherein the amino acid sequence of SEQ ID NO: 4, has been modified in length. A length modification may arise from the deletion of amino acids (shortening) and/or insertion of amino acids (lengthening). An inserted amino acid may be any amino acid as described earlier herein. The skilled person understands that deletion and/or insertion of amino acids may occur at any position of the peptide chain, including the end points and any position in between.

Within the context of the invention, a bacteriocin peptide or peptidomimetic may be in salt form. Suitable salts forms of peptides and peptidomimetics and their preparation are known in the art and discussed in standard handbooks, such as Remington: The Science and Practice of Pharmacy (supra) and Koutsopoulos, Peptide Applications in Biomedicine, Biotechnology and Bioengineering’ 1st Edition, Woodhead Publishing, UK (2017), incorporated herein by reference in their entireties. Preparation of peptide salts generally involves mixing of the peptide or peptidomimetic with an acid or base, for instance, by reacting the free acid or free base forms of the peptide or peptidomimetic with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water, which is then removed by vacuum or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.

The terms "infection” and "disease” as used herein have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure. An infection refers to the invasion and growth of a microbial cell in a subject’s (host’s) body and/ortissues, which may be accompanied by spreading throughout the body and/orto other tissues. A disease refers to an abnormal condition that negatively affects the structure or function of all or part of an organism due to the unwanted growth of a microbial cell in a subject’s body and/or tissues. A disease may be an infectious disease. An infection and/or disease may result in injury to the affected body and/or tissue. The microbial cell may come into contact with the subject and/or tissue via the environment, such as physical contact with a contaminated surface, or may already be present in the subject and/or tissue as part of the microbiome of said tissue and/or subject. An infection and/or disease may be caused by a pathogenic or potentially pathogenic microbial cell. An infection and/or disease may be associated with a pathogenic or potentially pathogenic microbial cell. As used herein, "associated with” means that a pathogenic or potentially pathogenic microbial cell may commonly appear in patients suffering from an infection and/or disease that is not directly caused by it, increasing the risk of further infections and/or diseases in these patients. As a non-limiting example, Mycobacterium abscessus is associated with cystic fibrosis patients. "Pathogenicity” as used herein has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It refers to the infection- and/or disease-causing capacity of a microbial cell. A potentially pathogenic microbial cell will cause an infection and/or disease under certain conditions, for example in cases wherein the immune system of the host is compromised or a bodily wound allows for entry of said cell in the body.

An infection and/or disease may be caused by a bacterium of the genus Mycobacterium. An infection and/or disease may be associated with a bacterium of the genus Mycobacterium. The genus Mycobacterium belongs to the family Mycobacteriaceae of the order Actinomycetales of the class Actino my cetes. The mycobacteria that form this genus are typically shaped like slightly curved rods, measuring from 1 to 10 mm in length and 0.2 to 0.6 mm in diameter. These bacteria typically cannot be classified as either Gram-positive or Gram- negative. Said bacteria have a complex cellular envelope, containing a relatively high percentage of lipids, which include the long chains of mycolic acid. This envelope confers strong hydrophobicity, making it resistant to lysis and relatively impermeable to antibiotics and other chemical agents. Bacteria of this genus are typically labelled acid-alcohol resistant, i.e., they are resistant to discoloration caused by weak acids after staining with fuchsine or similar dyes. The genomic DNA contains a high content of guanosine/cytosine, between 58-79%. These aspects are typically considered basic characteristics for identifying a bacterium as a member of the genus Mycobacterium (Orme, I. (1995) Medical Intelligent Unit: Immunity to Mycobacteria. Austin: R.G. Lands Company, p. 5; Jawetz et al., (1998) Medical Microbiology. 21^st Ed. Appleton & Lange, Stamford, Connecticut; Shinnick, T .M. & Good, R. C. (1994) Mycobacterial taxonomy. Eur J Clin Microbiol Infect MDis 11 : 884-901 ; all of which are incorporated herein by reference in their entireties). Out of the bacteria of the genus Mycobacterium, Mycobacterium tuberculosis is the most wide-spread and is the main cause of tuberculosis world-wide. It is a pathogenic bacterium which infects mammalian hosts through the lungs.

A bacterium of the genus Mycobacterium may be a nontuberculous Mycobacterium (NTM). The term "nontuberculous mycobacteria” as used herein comprises all of Mycobacteria species that do not cause tuberculosis. Non-limiting examples of such bacteria are Mycobacterium abscessus and subspecies thereof (alternately referred to as the Mycobacterium abscessus complex (MABSC), such as Mycobacterium abscessus subsp. abscessus, Mycobacterium abscessus subsp. massiliense, and Mycobacterium abscessus subsp. bolletii), Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracell ulare, Mycobacterium leprae, Mycobacterium chimaera, and Mycobacterium chelonae.

Nontuberculous mycobacteria cause, or are associated with, multiple infections and/or diseases, including, but not limited to, pulmonary infections, pulmonary disease resembling tuberculosis, chronic lung infections, skin and soft tissue infections (SSTIs), leprosy, central nervous system infections, bacteremia, ocular infections, lymphadenopathy, cystic fibrosis, and the like.

Accordingly, in some embodiments, the bacterium of the genus Mycobacterium is a nontuberculous Mycobacterium, preferably it is Mycobacterium abscessus. In some embodiments, the bacterium of the genus Mycobacterium is selected from Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracell ulare, Mycobacterium leprae, Mycobacterium chimaera, or Mycobacterium chelonae, preferably it is Mycobacterium abscessus. In some embodiments, the infection and/or disease is caused by a nontuberculous Mycobacterium, preferably it is caused by Mycobacterium abscessus. In some embodiments, the infection and/or disease is associated with a nontuberculous Mycobacterium, preferably with Mycobacterium abscessus. In preferred embodiments, the infection is an infection caused by Mycobacterium abscessus in a subject, preferably a human subject, who is a cystic fibrosis patient.

A bacterium of the genus Mycobacterium may cause tuberculosis. Non-limiting examples of such bacteria are species belonging to the Mycobacterium tuberculosis complex (MTBC). The Mycobacterium tuberculosis complex is a group of Mycobacterium species that can cause tuberculosis, including, but not limited to, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Mycobacterium dassie, and Mycobacterium oryx. Tuberculosis (TB) is an infectious disease occurring in humans and animals arising from infection by a tuberculosis-causing bacterium of the genus Mycobacterium, with Mycobacterium tuberculosis being the most wide spread. It may occur in any part of the body, but typically occurs in the lungs (known as pulmonary tuberculosis). Extrapulmonary TB occurs when tuberculosis develops outside of the lungs, although extrapulmonary TB may coexist with pulmonary TB. Non-limiting examples of affected areas in extrapulmonary TB include the pleura (e.g. in tuberculous pleurisy), the central nervous system (e.g. in tuberculous meningitis, intracranial tuberculomas, and spinal tuberculous arachnoiditis), the lymphatic system (e.g. in scrofula of the neck), the lymph nodes (e.g. in tubercular lymphadenitis), the genitourinary system (e.g. in urogenital tuberculosis), the Gl tract (e.g. in gastrointestinal tuberculosis and tuberculous peritonitis), the bones (e.g. in skeletal tuberculosis) and joints (e.g. in Pott disease of the spine), and the like. Another example of extrapulmonary TB is disseminated tuberculosis (military tuberculosis).

TB may be asymptomatic (latent TB) or symptomatic (active). Active tuberculosis typically occurs in the lungs. In some cases, the infection spreads outside the lungs, causing extrapulmonary TB. Extrapulmonary TB occurs more commonly in people with a weakened immune system and young children. In those with HIV, this occurs in more than 50% of cases.

Symptoms of active tuberculosis include, but are not limited to, fever, coughing, chills, night sweats, shortness of breath, loss of appetite, weight loss, nail clubbing, inflammation, and fatigue. Tuberculosis is potentially fatal if left untreated or if not treated successfully.

Accordingly, in some embodiments, the bacterium of the genus Mycobacterium is a tuberculosis-causing Mycobacterium. In some embodiments, the bacterium of the genus Mycobacterium is selected from a species belonging to the Mycobacterium tuberculosis complex (MTBC), preferably it /s Mycobacterium tuberculosis. In some embodiments, the bacterium of the genus Mycobacterium is selected from Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Mycobacterium dassie, and Mycobacterium oryx, preferably it is Mycobacterium tuberculosis.

In some embodiments, the infection and/or disease is tuberculosis. In some embodiments, the infection and/or disease is pulmonary tuberculosis. In some embodiments, the infection and/or disease is extrapulmonary tuberculosis. In some embodiments, the infection and/or disease is latent tuberculosis. In some embodiments, the infection and/or disease is active tuberculosis. In some embodiments, the infection and/or disease is a pulmonary infection. In some embodiments, the infection and/or diseases is a pulmonary infection in a subject, preferably a human subject, who is a cystic fibrosis patient. In some embodiments, the infection and/or disease is a skin and soft tissue infection (SSTI).

Typically, treatment of an infection and/or disease, such as, but not limited to, tuberculosis, caused by a bacterium of the genus Mycobacterium, such as, but not limited to, Mycobacterium tuberculosis, is done by administration of antibiotics, examples of which being isoniazid, rifampicin and/or a fluoroquinolone. An additional example of antibiotic administration is the administration of macrolides, for example clarithromycin, against infections and/or diseases caused by Mycobacterium abscessus. However, multiple Mycobacterium strains, and in particular Mycobacterium tuberculosis strains, have developed resistance to conventional antibiotic treatments. Mycobacterium strains are typically characterized as multiple-drug resistant (MDR strains), when they are simultaneously resistant to at least two antibiotics such as, but not limited to, isoniazid and rifampicin. Mycobacterium strains are characterized as extensively drug-resistant tuberculosis (XDR strains) when they are simultaneously resistant to additional antibiotics, such as, but not limited to, polyketides (e.g. other antibiotics belonging to the rifamycin group such as rifapentine, rifabutin), penicillins ( -lactams), glycylcy clines, aminonucleosides, nucleoside analogues, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulphonamides, polypeptides, glycopeptides, lipoglycopeptides, aminoglycosides (such as kanamycin, amikacin, capreomycin), fluoroquinolones (e.g. ofloxacin, moxifloxacin, levofloxacin), monobactams, oxazolidinones, streptogramins, rifamycins, carbapenems, chloramphenicol, clindamycin, daptomycin, fosfomycin, lefamulin, metronidazole, mupirocin, nitrofurantoin, tigecycline, puromycin, hygromycin B (hygrovetine), geneticin (G418), bleomycin, zeocin, amikacin, cefoxitine, meropenem, streptomycin (SM), pyrazinamide, clarithromycin, imipenem, and blasticidin.

In the case of Mycobacterium tuberculosis, MDR strains are the cause of multiple-drug resistant tuberculosis (MDR- TB) and XDR strains are the cause of extensively drug-resistant tuberculosis (XDR-TB). MDR-TB and XDR-TB are described in standard publications such as the Technical Report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis (World Health Organization, 2018; WHO/CDS/TB/2018.5). In bacteria of the genus Mycobacterium, mutations in the rpoB gene (DNA-directed RNA polymerase), such as Ser450Leu, Ser531 Leu, and the like, have typically been shown to be responsible for resistance to polyketides (e.g. rifampicin) (described in e.g. Shea et al. (2021) J Clin Microbiol 59:e01885-20 and Sinha et al. (2020), BMC Biotechnology 20: 284, incorporated herein by reference in their entireties). Additional mutations in rpoB associated with rifampicin resistance include Asp435Val, His445Tyr, His445Asp, Asp435Tyr, Ser450Trp, Leu452Pro, His445Leu, Ser450Phe, His445Arg, Val170Phe, lle491 Phe, Asp435Phe, His445Cys, Gln432Lys, Gln432Pro, Ser441 Leu, Gln432Leu, His445Ser, Ser441 Gln, Ser450Gln, rpoB_1296_ins_3_a_attc, and rpoB_1328_ins_3_t_tgac, described in the World Health Association’s Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance (version of 25 June 2021 , ISBN 9789240028173, incorporated herein by reference in its entirety).

Isoniazid resistance is coupled with a variety of mutations affecting one or more than one gene such as katG (catalase-peroxidase, such as Ser315Thr, Gly944Cys, and the like, described in e.g. Riska et al, (2000) Int J Tuberc Lung Dis 4(2):S4-S10 and Ando et al. (2010), ASM Antimicrobial Agents and Chemotherapy 54(5): 1793-1799, incorporated herein by reference in their entireties). Isoniazid resistance may further arise from mutations in inhA (enoyl-[acyl-carrier-protein] reductase). Additional mutations in katG associated with isoniazid resistance include Ser315Asn and Trp328Leu, Mutations in inhA associated with isoniazid resistance include inhA_c777t (fabG1_c15t), and inhA_g154a (fabG1_L203L), These mutations are described in the World Health Association’s Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance (supra). Fluoroquinolone resistance (e.g. to ofloxacin or moxifloxacin) is typically caused by mutations in gyrA (DNA gyrase subunit A; such as Ala90Val, Asp94Gly, Asp94Asn, and the like) and gyrB (DNA gyrase subunit B; such as Gly512Arg, and the like) genes, described in e.g. Maruri et al. (2012) J Antimicrob Chemother 67(4): 819-831 and Chien et al. (2016) ASM Antimicrobial Agents and Chemotherapy 60(4): 2090-2096, incorporated herein by reference in their entireties). Additional mutations in gyrA associated with fluoroquinolone resistance include Asp94Ala, Ser91 Pro, Asp94Tyr, Asp94His, and Gly88Cys. An additional mutation in gyrB associated with fluoroquinolone resistance is Asp461Asn. These mutations are described in the World Health Association’s Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance (supra). Clarithromycin resistance in M. abscessus is seen through the inducible erm(41) gene or via acquired mutations in the rrl (23S rRNA) gene. Strains harboring the erm(41) T28 mutation demonstrate inducible clarithromycin resistance, while those harboring C28 mutation are susceptible. Exemplary mutations in the rrl gene are A2058C, A2058G, A2059C, A2059G, delta wt A2058T, and delta wt A2059T. Amikacin resistance is coupled with a variety of mutations affecting the rrs gene. Exemplary rrs mutations are A1408G, delta wt T1406A, and delta wt C1409T.

Determination of antibiotic resistance may be performed using the methods described earlier herein, some of which are further exemplified in the experimental section herein. Alternatively, antibiotic resistance may be determined by molecular phenotyping, for example by identifying mutations in rpoB, katG, gyrA, or gyrB, or in another relevant gene for a respective antibiotic resistance (such as erm(41), rrl, and rrs), using standard molecular toolbox techniques known in the art. Alternatively, antibiotic resistance may be determined using other commercially available methods, such as analyses offered by laboratories such as the Belgian National Reference Center for tuberculosis and Mycobacteria (Sciensano, BE), the GenoType NTM-DR test offered by Hain Lifescience GmbH (DE) and the GenoType LepraeDR test offered by Hain Lifescience GmbH (DE), the latter of which being particularly suitable for Mycobacterium leprae.

An exemplary antibiotic-resistant Mycobacterium strain is M. tuberculosis H37Rv (ATCC 27294). Another exemplary antibiotic-resistant Mycobacterium strain is a clinical MDR-TB strain comprising a mutation in katG (Ser315Thr), in rpoB (Ser450Leu) and in gyrA (Ala90Val), resistant to rifampicin, isoniazid, and ofloxacin, as described above, which is used in the experimental section herein.

Typically, a Mycobacterium tuberculosis strain may be classified as polyketide-antibiotic resistant when said polyketide displays a MIC value of equal to or higher than (>) 0.5 pg/ml against that strain. The polyketide may preferably be rifampicin.

Typically, a Mycobacterium tuberculosis strain may be classified as fluoroquinolone-antibiotic resistant when said fluoroquinolone displays a MIC value of equal to or higherthan (>) 4 pg/ml against that strain. The fluoroquinolone may preferably be ofloxacin or moxifloxacin.

Typically, a Mycobacterium tuberculosis strain may be classified as isoniazid resistant when said antibiotic displays a MIC value of equal to or higher than (>) 0.25 pg/ml against that strain.

Typically, a Mycobacterium abscessus strain may be classified as clarithromycin resistant when said antibiotic displays a MIC value of equal to or higher than (>) 8 pg/ml against that strain.

Typically, a Mycobacterium abscessus strain may be classified as imipenem resistant when said antibiotic displays a MIC value of equal to or higher than (>) 32 pg/ml against that strain.

Typically, a Mycobacterium abscessus strain may be classified as amikacin resistant when said antibiotic displays a MIC value of equal to or higher than (>) 64 pg/ml against that strain.

Fortigecycline resistance upto today there is insufficient data to establish breakpoints; only MIC should be reported. We considered a strain to be tigecycline resistant when said antibiotic displays a MIC value of equal to or higher than (>) 4 pg/ml against that strain.

Accordingly, in some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, is antibiotic-resistant. In some embodiments, the antibiotic resistance is caused by a mutation in a gene selected from rpoB, katG, gyrA, or gyrB, preferably selected from rpoB, gyrA, or gyrB, more preferably selected from rpoB and gyrA.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, is resistant to a polyketide antibiotic, such as, but not limited to, antibiotics belonging to the rifamycin group (e.g. rifampicin, rifapentine, rifabutin), preferably to rifampicin. In some embodiments, the resistance to the polyketide antibiotic is caused by a mutation in the rpoB gene, preferably by a mutation in the rpoB gene selected from Ser450Leu, Ser531 Leu, Asp435Val, His445Tyr, His445Asp, Asp435Tyr, Ser450Trp, Leu452Pro, His445Leu, Ser450Phe, His445Arg, Val170Phe, lle491 Phe, Asp435Phe, His445Cys, Gln432Lys, Gln432Pro, Ser441 Leu, Gln432Leu, His445Ser, Ser441 Gln, Ser450Gln, rpoB_1296_ins_3_a_attc, and rpoB_1328_ins_3_t_tgac, more preferably it is caused by a Ser450Leu mutation in the rpoB gene.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, is resistant to a fluoroquinolone antibiotic, such as, but not limited to, ofloxacin, levofloxacin, or moxifloxacin, preferably to ofloxacin or moxifloxacin, more preferably to ofloxacin. In some embodiments, the resistance to the fluoroquinolone antibiotic is caused by a mutation in the gyrA or gyrB genes, preferably by a mutation in the gyrA gene selected from Ala90Val, Asp94Gly, Asp94Asn, Asp94Ala, Ser91 Pro, Asp94Tyr, Asp94His, and Gly88Cys, or by a mutation in the gyrB gene selected from Gly512Arg and Asp461Asn, more preferably it is caused by an Ala90Val mutation in the gyrA gene.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium tuberculosis, is resistant to isoniazid. In some embodiments, the resistance to isoniazid is caused by a mutation in the katG or inhA genes, preferably by a mutation in the katG gene selected from Ser315Thr, Gly944Cys, Ser315Asn, and Trp328Leu, or by a mutation in the inhA gene selected from inhA_c777t (fabG1_c15t), and inhA_g154a (fabG1_L203L), more preferably it is caused by a Ser315Thr mutation in the katG gene.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium abscessus, is resistant to a macrolide antibiotic, preferably to clarithromycin. In some embodiments, the resistance to clarithromycin is caused by a mutation in the erm(41) or rrl gene.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium abscessus, is resistant to a tetracycline or glycylcycline antibiotic, preferably to tigecycline.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium abscessus, is resistant to a -lactam or carbapenem antibiotic, preferably to imipenem.

In some embodiments, the bacterium of the genus Mycobacterium, preferably Mycobacterium tuberculosis or Mycobacterium abscessus, more preferably Mycobacterium abscessus, is resistant to an aminoglycoside antibiotic, preferably to amikacin. In some embodiments, the resistance to amikacin is caused by a mutation in the rrs gene. The skilled person understands that a bacterium of the genus Mycobacterium may simultaneously be resistant to multiple antibiotics.

Within the context of bacteriocin peptides, peptidomimetics, or pharmaceutical compositions for use, methods, and uses of the invention, the subject treated may be a vertebrate, preferably a mammal such as a cat, a mouse, a rat, a dog, or a human. In preferred embodiments, the subject treated is a human.

Within the context of bacteriocin peptides, peptidomimetics, or pharmaceutical compositions for use, methods, and uses of the invention, administration of a bacteriocin peptide, peptidomimetic, or pharmaceutical composition may be performed to an individual, a cell, tissue, and/or an organ of an individual affected and/or at risk of developing an infection and/or disease as discussed herein. Administration may be performed directly or indirectly in vivo, ex vivo or in vitro, using suitable means known in the art. When administering a bacteriocin peptide, peptidomimetic, or pharmaceutical composition as described herein, it is preferred that it is dissolved in a solution that is compatible with the delivery method. Such solutions are generally known in the art, see for example Remington: The Science and Practice of Pharmacy (supra). Improvements in means for providing an individual or a cell, tissue, and/or organ of said individual with the bacteriocin peptide, peptidomimetic and/or pharmaceutical composition are anticipated, considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the invention. The skilled person understands that the type and frequency of administration will vary depending on the infection and/or disease. Administration may be one-time (single) or may involve multiple administrations overtwo, three, four, five, six, seven, eight, nine, ten days or more. Administration may be once daily or multiple times daily. Administration modes are generally known in the art. An administration mode may be topical, transdermal, intradermal, parenteral, intravenous, intramuscular, intraperitoneal, via inhalation, intraparenchymal, subcutaneous, intraarticular, intra-adipose tissue, oral, intrahepatic, intrapulmonary, intrasplanchnic, intra-ear, intrathoracic, intracardial, or intratracheal administration.

A preferred administration mode for treatment, prevention and/or delaying of an infection and/or disease, preferably tuberculosis, as described herein is intrapulmonary, oral, or parenteral administration. Intrapulmonary administration refers to administration within the lungs, for example by means of injection. Oral administration refers to administration via the mouth. Parenteral administration refers to administration that bypasses the Gl tract. Accordingly, in some embodiments, administration of the bacteriocin peptide, peptidomimetic, or pharmaceutical composition is selected from intrapulmonary, oral, or parenteral administration. Another preferred administration mode is topical (i.e. at the site of infection), transdermal, or intradermal administration.

In some embodiments, a bacteriocin peptide, peptidomimetic, or pharmaceutical composition for use, methods, and uses according to the invention result in the alleviation of at least one symptom and/or the improvement of at least one parameter associated with an infection and/or disease discussed herein, preferably tuberculosis. Alleviating a symptom of a disease and/or infection, preferably tuberculosis, as discussed herein may mean that said symptom is improved or decreased or that the progression of a typical symptom has been slowed down in an individual, in a cell, tissue or organ of said individual as assessed by a physician. A decrease or improvement of a typical symptom may mean a slowdown in progression of symptom development or a complete disappearance of symptoms. Symptoms, and thus also a decrease in symptoms, can be assessed using a variety of methods, to a large extent the same methods as used in diagnosis of the relevant infection and/or disease, including clinical examination and routine laboratory tests. Laboratory tests may include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays orCT-scans, biochemical methods, immunohistochemical methods, culturing methods, and others. In this context, “decrease” (respectively “improvement”) means at least a detectable decrease (respectively a detectable improvement) using an assay known to a person of skill in the art. The decrease may be a decrease of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. The decrease may be seen after at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days or more of treatment using a bacteriocin peptide, peptidomimetic, and/or pharmaceutical composition as described herein. Symptoms of infections and/or diseases discussed herein are known in the art. In some embodiments, administration of the bacteriocin peptide, peptidomimetic, or pharmaceutical composition results in the alleviation of at least one symptom associated with tuberculosis, preferably of at least one symptom selected from fever, coughing, chills, night sweats, shortness of breath, loss of appetite, weight loss, nail clubbing, inflammation, and fatigue.

Administration may involve a therapeutically effect amount of a peptide, peptidomimetic, or pharmaceutical composition as described herein. As used herein, an “effective amount” is an amount sufficient to exert beneficial or desired results. Accordingly, a “therapeutically effective amount” is an amount that, when administered to a subject such as a subject in need thereof, is sufficient to exert some therapeutic effect as described herein, such as, but not limited to, a reduction in the magnitude of at least one symptom and/or the improvement of at least one parameter associated with an infection and/or disease, preferably tuberculosis, as described earlier herein. An amount that is "therapeutically effective" will vary from subject to subject, depending on the age, the infection and/or disease type and its progression, and overall general condition of the individual. An appropriate "therapeutically effective" amount in any individual case may be determined by the skilled person using routine experimentation, such as discussed elsewhere herein. A "subject in need” may be any individual affected by, and/or at risk of developing an infection and/or disease, preferably tuberculosis.

Within the context of a bacteriocin peptide, peptidomimetic, pharmaceutical composition for use, methods, and uses described herein, the target microbial cell such as a bacterium of the genus Mycobacterium, may be comprised in a biofilm. A "biofilm” comprises any syntrophic consortium of microbial cells, preferably bacteria of the genus Mycobacterium, more preferably Mycobacterium tuberculosis or Mycobacterium abscessus, most preferably Mycobacterium tuberculosis, in which cells stick to each other and often also to a surface. Typically, biofilms are resistant to commonly used antimicrobials, such as antibiotics. Compositions

A bacteriocin peptide or peptidomimetic as described herein exhibits a number of activities that can be advantageously used in a wide range of applications, including therapeutic applications and applications in disinfection of surfaces (including surfaces of chemically fragile medical devices), biotechnology, biofermentation processes, and food preservation.

Provided herein, therefore, are compositions comprising a bacteriocin peptide or peptidomimetic, preferably peptide, as described earlier herein. Optionally, the compositions will further comprise an acceptable ingredient, such as a carrier, diluent, and/or excipient as discussed elsewhere herein. The skilled person is aware that each of the acceptable ingredients will be suitable for the intended use or application, for example pharmaceutical or application in the disinfection of surfaces. In embodiments wherein the compositions comprise bacteriocin peptide or peptidomimetic salts, preferably peptide salts, as described earlier herein, the skilled person is aware that said salts will be suitable for the intended use or application, for example pharmaceutical, or application in the disinfection of surfaces. Non-limiting examples of pharmaceutically acceptable acids or bases suitable for the preparation of a bacteriocin or peptidomimetic salt as described earlier herein include organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid, and thiocyanic acid, which form ammonium salts with free amino groups of polypeptides, and bases that form carboxylate salts with free carboxylic groups of polypeptides, such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other mono-, di-and trialkylamines, and arylamines.

Accordingly, in a further aspect, the invention provides a composition comprising a bacteriocin peptide or peptidomimetic, preferably peptide, as defined herein. Said composition may optionally further comprise another antimicrobial compound. Said antimicrobial compound may be selected from any antimicrobial compound such as, but not limited to, antifungal agents, antiviral agents, essential oils, other bacteriocins, and/or antibiotics, preferably antibiotics, as described earlier herein.

In some embodiments, the composition further comprises an antibiotic. In some embodiments, the further comprised antibiotic is a polyketide antibiotic, preferably selected from the rifamycin group, more preferably selected from rifampicin, rifapentine, rifabutin, or a combination thereof. In some embodiments, the further comprised antibiotic is a fluoroquinolone antibiotic, preferably selected from ofloxacin, levofloxacin, moxifloxacin, or a combination thereof, more preferably from ofloxacin or moxifloxacin, most preferably is ofloxacin. In some embodiments, the further comprised antibiotic is a macrolide antibiotic, preferably is clarithromycin. In some embodiments, the further comprised antibiotic is a tetracycline or glycylcycline antibiotic, preferably is tigecycline. In some embodiments, the further comprised antibiotic is a p-lactam or a carbapenem antibiotic, preferably is imipenem. In some embodiments, the further comprised antibiotic is an aminoglycoside antibiotic, preferably is amikacin.

In preferred embodiments, the further comprised antibiotic is selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a p-lactam, a carbapenem, an aminoglycoside, or a combination thereof, preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof, more preferably selected from rifampicin, ofloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof. In other preferred embodiments, the further comprised antibiotic is selected from rifampicin, ofloxacin, or a combination thereof. In some embodiments, the composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients. Said compositions may exhibit synergistic biological activity (i.e. inhibition effect), as described earlier herein.

In some embodiments, the composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, and optionally further comprises one or more antimicrobial compounds and/or a solvent. The skilled person understands that "suitable for disinfecting” in the context of the invention refers to the composition being capable of inhibiting a bacterium of the genus Mycobacterium, such as, but not limited to Mycobacterium tuberculosis and Mycobacterium abscessus, as defined earlier herein, when said composition is applied on a surface contaminated with said bacterium. A definition of a "contaminated surface” is provided later herein.

Typically, compositions and pharmaceutical compositions as described herein comprise a bacteriocin peptide or peptidomimetic, preferably peptide, as described earlier herein at a concentration value from 0.01 to 1000 pg/ml, from 0.1 to 1000 pg/ml, from 1 to 1000 pg/ml, from 2 to 500 pg/ml, from 3 to 400 pg/ml, from 4 to 300 pg/ml, from 5 to 200 pg/ml, from 10 to 150 pg/ml, from 25 to 125 pg/ml, from 25 to 100 pg/ml, or from 50 to 100 pg/ml.

Thus, a concentration value of a bacteriocin peptide or peptidomimetic may be at least 0.01 pg/ml, at least 0.1 pg/ml, at least 1 pg/ml, at least 2 pg/ml, at least 3 pg/ml, at least 4 pg/ml, at least 5 pg/ml, at least 6 pg/ml, at least 7 pg/ml, at least 8 pg/ml, at least 9 pg/ml, at least 10 pg/ml, at least 11 pg/ml, at least 12 pg/ml, at least 13 pg/ml, at least 14 pg/ml, at least 15 pg/ml, at least 16 pg/ml, at least 17 pg/ml, at least 18 pg/ml, at least 19 pg/ml, at least 20 pg/ml, at least 21 pg/ml, at least 22 pg/ml, at least 23 pg/ml, at least 24 pg/ml, at least 25 pg/ml, at least 26 pg/ml, at least 27 pg/ml, at least 28 pg/ml, at least 29 pg/ml, at least 30 pg/ml, at least 31 pg/ml, at least 32 pg/ml, at least 33 pg/ml, at least 34 pg/ml, at least 35 pg/ml, at least 36 pg/ml, at least 37 pg/ml, at least 38 pg/ml, at least 39 pg/ml, at least 40 pg/ml, at least 41 pg/ml, at least 42 pg/ml, at least 43 pg/ml, at least 44 pg/ml, at least 45 pg/ml, at least 46 pg/ml, at least 47 pg/ml, at least 48 pg/ml, at least 49 pg/ml, at least 50 pg/ml, at least 51 pg/ml, at least 52 pg/ml, at least 53 pg/ml, at least 54 pg/ml, at least 55 pg/ml, at least 56 pg/ml, at least 57 pg/ml, at least 58 pg/ml, at least 59 pg/ml, at least 60 pg/ml, at least 61 pg/ml, at least 62 pg/ml, at least 63 pg/ml, at least 64 pg/ml, at least 65 pg/ml, at least 66 pg/ml, at least 67 pg/ml, at least 68 pg/ml, at least 69 pg/ml, at least 70 pg/ml, at least 71 pg/ml, at least 72 pg/ml, at least 73 pg/ml, at least 74 pg/ml, at least 75 pg/ml, at least 76 pg/ml, at least 77 pg/ml, at least 78 pg/ml, at least 79 pg/ml, at least 80 pg/ml, at least 81 pg/ml, at least 82 pg/ml, at least 83 pg/ml, at least 84 pg/ml, at least 85 pg/ml, at least 86 pg/ml, at least 87 pg/ml, at least 88 pg/ml, at least 89 pg/ml, at least 90 pg/ml, at least 91 pg/ml, at least 92 pg/ml, at least 93 pg/ml, at least 94 pg/ml, at least 95 pg/ml, at least 96 pg/ml, at least 97 pg/ml, at least 98 pg/ml, at least 99 pg/ml, at least 100 pg/ml, at least 110 pg/ml, at least 120 pg/ml, at least 130 pg/ml, at least 140 pg/ml, at least 150 pg/ml, at least 160 pg/ml, at least 170 pg/ml, at least 180 pg/ml, at least 190 pg/ml, at least 200 pg/ml, at least 250 pg/ml, at least 300 pg/ml, at least 400 pg/ml, at least 500 pg/ml, at least 550 pg/ml, at least 600 pg/ml, at least 650 pg/ml, at least 700 pg/ml, at least 750 pg/ml, at least 800 pg/ml, at least 850 pg/ml, at least 900 pg/ml, at least 950 pg/ml, or at least 1000 pg/ml.

Compositions and pharmaceutical compositions as described herein may be in any form as commonly used in the art. The skilled person is aware that the form of the respective composition will be suitable for the intended use or application, for example pharmaceutical or application in the disinfection of surfaces. Non-limiting examples of suitable forms include tablets, capsules, pills, lyophilized, liquids, creams, ointments, gels, pastes, powders, emulsions, lotions, suspensions, sticks, aerosols (i.e. sprays), and the like.

As used herein, a "solvent” includes any solvent or mixture of solvents in which a bacteriocin peptide or peptidomimetic as described herein can be dissolved at a suitable concentration. Generally, the number and types of ionic charges in the peptide determine its solubility in aqueous solutions. Generally, the more charged residues the peptide possesses, the more soluble it is in aqueous solutions. In addition, peptides generally have more charges at pH 6-8 than at pH 2-6. It is for this reason that peptides are generally better dissolved at near neutral pH. Among the many exceptions to the rule are peptide sequences that are very hydrophobic and those that tend to aggregate. While the hydrophobicity of the sequence is the primary cause of aggregation, peptides can also aggregate or "gel" through extensive hydrogen bonding network. Non-limiting examples of solvents that can be used in the context of the invention are water, ethanol, ammoniumhydroxide, dimethylsulfoxide (DMSO), acetic acid, acetonitrile and dimethylformamide (DMF). Dissolution can be enhanced by sonication.

A "pharmaceutical composition” is a composition which is suitable for use in therapy. As used herein, ‘‘pharmaceutically acceptable ingredients’’ include pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and/or excipients. Accordingly, the one or more pharmaceutically acceptable ingredients may be selected from the group consisting of pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and/or excipients. Such pharmaceutically acceptable carriers, fillers, preservatives, solubilizers, vehicles, diluents and/or excipients may be found in standard handbooks such as in Remington: The Science and Practice of Pharmacy (supra).

Compositions and pharmaceutical compositions as described herein may optionally comprise additional compounds. Said compounds may help in delivery of the compositions. Suitable compounds in this context are: compounds capable of forming complexes, nanoparticles, micelles and/or liposomes that deliver each constituent as described herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these compounds are known in the art. Suitable compounds comprise polyethylenimine (PEI), orsimilarcationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives; synthetic amphiphiles (SAINT-18); lipofectinTM, DOTAP. The skilled person will know which type of formulation is the most appropriate for a composition as described herein.

Non-therapeutic methods/uses

In an aspect, the invention provides an ex-vivo method of disinfecting a surface comprising contacting said surface with a bacteriocin peptide, peptidomimetic, preferably peptide, or a composition, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, optionally further comprising one or more antimicrobial compounds and/or a solvent, as defined herein. In some embodiments, a surface is contaminated with a bacterium of the genus Mycobacterium. In some embodiments, a surface is contaminated with a nontuberculous Mycobacterium (NTM), preferably by Mycobacterium abscessus. In some embodiments, the bacterium of the genus Mycobacterium is selected from Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracell ulare, Mycobacterium leprae, Mycobacterium chimaera, or Mycobacterium chelonae, preferably it is Mycobacterium abscessus.

In some embodiments, a surface is contaminated with a tuberculosis-causing Mycobacterium. In some embodiments, a surface is contaminated with a bacterium of the genus Mycobacterium selected from a species belonging to the Mycobacterium tuberculosis complex, preferably by Mycobacterium tuberculosis. In some embodiments, the bacterium of the genus Mycobacterium is selected from Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Mycobacterium dassie, or Mycobacterium oryx, preferably it is Mycobacterium tuberculosis.

A "surface” as used herein refers to any non-living, preferably solid, surface which may serve as scaffold (i.e. provide physical support) for microbial growth. The method is further applicable both to the disinfection of instruments, such as medical instruments, and articles placed in small disinfection chambers, biological safety cabinets, isolators, glove boxes, incubators, materials airlocks, and the like. The method is also applicable for disinfection of food containers, industrial equipment and the like. In some embodiments, the surface is a surface of a medical instrument. Non-limiting examples of medical instruments include bedpans, cannulas, cardioverters, defibrillators, catheters, dialysers, electrocardiograph machines, enema equipment, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control equipment such as masks, gowns, face shields, and goggles, instrument sterilizers, kidney dishes, nasogastric tubes, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiographers, sphygmomanometers, thermometers, tongue depressors, transfusion kits, tuning forks, ventilators, watches, and the like. In some embodiments, the surface is the surface of industrial equipment. Non-limiting examples of industrial equipment include fermentation equipment, such as fermenters, tubing, feeding vessels, spargers, mixers, compressors, and the like, freezers, fridges, cargo vehicles, storage vessels, rotor blades, mills, and the like. A surface is "contaminated” with a bacterium, when its presence can be detected on said surface, using standard methods in the art such as swab tests.

"Disinfection”, otherwise known as "decontamination” has its customary and ordinary meaning as understood by one of skill in the art in view of this disclosure. It generally refers to the inhibition and/or killing of microbial cells on inert surfaces. Disinfection may be partial, i.e. a part of the target microbial cell population may not be neutralized and/or killed. Partial disinfection may mean that at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95%, or at most 99% of the targeted population is not inhibited and/or killed.

The ex-vivo method may result in at least a 2 log (a factor of 100), at least a 3 log (a factor of 1000), at least a 4 log (a factor of 10000), at least a 5 log (a factor of 100000), or at least a 6 log (a factor of 1000000) reduction of the non-inhibited and/or alive target microbial cell population, preferably a population of a bacterium of the genus Mycobacterium, more preferably a population of a bacterium selected from Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Mycobacterium dassie, or Mycobacterium oryx, most preferably a population of Mycobacterium tuberculosis.

The ex-vivo method may result in at least a 2 log (a factor of 100), at least a 3 log (a factor of 1000), at least a 4 log (a factor of 10000), at least a 5 log (a factor of 100000), or at least a 6 log (a factor of 1000000) reduction of the non-inhibited and/or alive target microbial cell population of a bacterium selected from Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracell ulare, Mycobacterium leprae, Mycobacterium chimaera, or Mycobacterium chelonae, preferably a population of Mycobacterium abscessus.

Contacting with a surface may be followed by a waiting period, wherein the bacteriocin peptide, peptidomimetic, or composition is left in contact with the surface. Said period may last at least 15 seconds, at least 30 seconds, at least 45 seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 25 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, or more. After contacting with a surface or after the waiting period, the bacteriocin peptide, peptidomimetic or composition may be removed from the surface, e.g. by rinsing with water or by wiping said surface with a clean cloth.

Accordingly, in some embodiments, the invention provides an ex-vivo method of disinfecting a surface, preferably a surface contaminated with a population of a bacterium of the genus Mycobacterium, more preferably a population of a bacterium selected from Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Mycobacterium dassie, or Mycobacterium oryx, most preferably a population of Mycobacterium tuberculosis, comprising contacting said surface with a bacteriocin peptide, peptidomimetic, or a composition, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, optionally further comprising one or more antimicrobial compounds and/or a solvent as defined herein, followed by a waiting period of at least 15 seconds.

In some embodiments, the invention provides an ex-vivo method of disinfecting a surface contaminated with a population of a bacterium selected from Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium chimaera, or Mycobacterium chelonae, preferably a population of Mycobacterium abscessus, comprising contacting said surface with a bacteriocin peptide, peptidomimetic, or a composition, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, optionally further comprising one or more antimicrobial compounds and/or a solvent as defined herein, followed by a waiting period of at least 15 seconds.

The antimicrobial effect of an ex-vivo method according to the invention may be assessed by standard methods in the art, such as commercial in vitro laboratory tests such as ASTM E2149-20 or ASTM E1054-08 (ASTM, PA, USA), and the like, or alternative methods discussed elsewhere herein. In some embodiments, the ex-vivo method results in a reduction of the target microbial cell population by at least 2-fold as assessed using the ASTM E1054-08 test in vitro.

Within the ex-vivo methods described herein, the target microbial cell such as a bacterium of the genus Mycobacterium, may be comprised in a biofilm.

General information

Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs, and read in view of this disclosure.

Sequence identity

In the context of the invention, a nucleic acid encoding a bacteriocin peptide is represented by a nucleotide sequence. In the context of the invention, a bacteriocin peptide or peptidomimetic is represented by an amino acid sequence. It is to be understood that each nucleic acid molecule or peptide or peptidomimetic as identified herein by a given sequence identity number (SEQ ID NO) is not limited to said specific sequence as disclosed..

Throughout this application, each time one refers to a specific nucleotide sequence SEQ ID NO (take SEQ ID NO: X as example) encoding a given protein fragment or polypeptide or peptide or derived peptide, one may replace it by: i. a nucleotide sequence comprising a nucleotide sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity with SEQ ID NO: X; ii. a nucleotide sequence the sequence of which differs from the sequence of a nucleic acid molecule of (i) due to the degeneracy of the genetic code; or iii. a nucleotide sequence that encodes an amino acid sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% amino acid identity or similarity with an amino acid sequence encoded by a nucleotide sequence SEQ ID NO: X. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

Throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 99%.

Each nucleotide sequence or amino acid sequence described herein by virtue of its identity or similarity percentage with a given nucleotide sequence or amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given nucleotide or amino acid sequence, respectively.

The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (peptide, polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length of two given sequences (for example as represented by a SEQ ID NO herein) or on a part thereof, preferably based on the full length of two given sequences. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Bioinformatics and the Cell: Modern Computational Approaches in Genomics, Proteomics and transcriptomics, Xia X., Springer International Publishing, New York, 2018; and Bioinformatics: Sequence and Genome Analysis, Mount D., Cold Spring Harbor Laboratory Press, New York, 2004, each incorporated by reference herein in its entirety.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman-Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith-Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the program EMBOSS needle or EMBOSS water using default parameters) share at least a certain minimal percentage of sequence identity (as described below).

A global alignment is suitably used to determine sequence similarity or identity when the two sequences have similar lengths. When sequences have a substantially different overall length, local alignments, such as those using the Smith-Waterman algorithm, are preferred. EMBOSS needle uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. EMBOSS water uses the Smith-Waterman local alignment algorithm. Generally, the EMBOSS needle and EMBOSS water default parameters are used, with a gap open penalty = 10 (nucleotide sequences) 1 10 (proteins) and gap extension penalty = 0.5 (nucleotide sequences) 1 0.5 (proteins). For nucleotide sequences the default scoring matrix used is DNAfull and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919, incorporated herein by reference in its entirety).

Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of some embodiments of the present invention can further be used as a “query sequence’’ to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al., J. Mol. Biol. 215:403-10 (1990), incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402 (1997), incorporated herein by reference in its entirety. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information accessible on the world wide web at www.ncbi.nlm.nih.gov/. The above algorithms may also be used to determine corresponding nucleotide or amino acid residue positions between sequences being aligned. For example, an amino acid residue in sequence Y which corresponds to position 1 (or any other position) of sequence X may be determined.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so- called conservative amino acid substitutions. As used herein, “conservative’’ amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

Alternative conservative amino acid residue substitution classes :

Alternative physical and functional classifications of amino acid residues:

For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.

Codon optimization

“Codon optimization’’, as used herein, refers to the processes employed to modify an existing coding sequence, or to design a coding sequence, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host cell. For example, to suit the codon preference of mammalian, insect, plant, or microbial cells, preferably microbial cells. Examples of microbial cells include eukaryotes such as yeasts, filamentous fungi, and algae, and prokaryotes such as bacteria and archaea. Codon optimization also eliminates elements that potentially impact negatively RNA stability and/or translation (e. g. termination sequences, TATA boxes, splice sites, ribosomal entry sites, repetitive and/or GC rich sequences and RNA secondary structures or instability motifs).

Proteins and amino acids

The terms "protein" or "peptide" or “amino acid sequence’’ are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. In amino acid sequences as described herein, amino acids or “residues" are denoted by three-letter symbols. These three-letter symbols as well as the corresponding one-letter symbols are well known to a person of skill in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, Y (Tyr) is tyrosine. A residue may be any proteinogenic amino acid, but also any non-proteinogenic amino acid such as D-amino acids and modified amino acids formed by post-translational modifications, and also any non-natural amino acid.

Peptide expression

Peptide "expression” or "production” by a cell may be assessed by any method known to a person of skill in the art. For example, expression may be assessed by measuring the levels of gene expression on the level of the mRNA or the peptide by standard assays known to a person of skill in the art, such as qPCR, RNA sequencing, Northern blot analysis, Western blot analysis, mass spectrometry analysis of protein-derived peptides or ELISA.

General terms

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb ‘‘to consist’’ may be replaced by ‘‘to consist essentially of or "to essentially consist of”, meaning that a composition as described herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention. In addition, the verb ‘‘to consist’’ may be replaced by ‘‘to consist essentially of meaning that a method or use as described herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention. In addition, the verb ‘‘to consist’’ may be replaced by ‘‘to consist essentially of meaning that a nucleotide or amino acid sequence as described herein may comprise additional nucleotides or amino acids than the ones specifically identified, said additional nucleotides or amino acids not altering the unique characteristics of the invention.

Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

As used herein, with "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 etc.

The word ‘‘about’’ or ‘‘approximately’’ when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 1 % of the value.

As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

Various embodiments are described herein. Each embodiment as identified herein may be combined together unless otherwise indicated.

All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. Table 2. Sequences

Examples

General procedures M. tuberculosis strains

M. tuberculosis H37Rv reference strain (ATCC 27294), and a clinical MDR-TB strain resistant to isoniazid (INH), rifampicin (RIF), moxifloxacin (MFX), and ofloxacin (OFLO) having a mutation in genes katG (Ser315Thr), in rpoB (Ser450Leu) and in gyrA (Ala90Val) respectively, were used in Examples 1 and 2. M. abscessus CCUG 41449 reference strain was used in Examples 3 and 4. Bacteriocins synthesis

Bacteriocin peptides (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) were synthesized in vitro (Gabant et al., (2019) Front. Bioeng. Biotechnol 7:213). Their amino acid sequences were reverse-translated and codon optimized for Escherichia coli (Scilico LLC, PA, USA, tool available at www.bioinformatics.org/ sms2/rev_trans.html). The codon optimized nucleotide sequences were cloned in a pUC57 vector backbone (Thermo-Scientific, MA, USA), comprising the T7 promoter, a start codon (ATG) and stop codon (TAA), and the T7 terminator region. Recombinant vectors were cloned in the E. coli DH10B according to standard methods. The vectors were used as templates for cell-free protein synthesis using PURExpress® in vitro Protein Synthesis Kit (New England Biolabs, MA, USA) following the manufacturer’s protocol.

Preparation of antibiotic solutions

Rifampicin (RIF), ofloxacin (OFLO), moxifloxacin (MFX), clarithromycin (CLA), tigecycline (TGC), imipenem (I Ml), and amikacin (AMK) (Sigma-Aldrich, MO, USA) solutions were prepared at concentrations of 10 mg/ml in methanol (RIF), and of 1 mg/ml in 0.1 M NaOH (OFLO, MFX), DMSO (CLA), and sterile distilled water (TGC, IMI, AMK) respectively, filter sterilized, and aliquots were kept at -20°C until used.

Screening for presence of bacteriocin antimicrobial activity

The screening of the bacteriocins (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) was performed using the well-established high-throughput-method of the resazurin microtiter assay (REMA) (Palomino et al. (2002); Martin et al. (2003), against M. tuberculosis H37Rv and the MDR-TB strain. The bacteriocins were diluted in sterile distilled water to have a concentration of 100 pg/ml in middlebrook broth 7H9 medium (Sig ma-Ald rich, MO, USA) containing 0.5% glycerol and supplemented with 10% OADC (oleic acid, albumin, dextrose, and catalase (Becton- Dickinson, NJ, USA). The assays were performed in sterile 96-well flat-bottom microtiter plates, each well containing 100 pl of bacteriocin and 100 pl of inoculum (turbidity corresponding to McFarland Standard No 1.0 for M. tuberculosis and McFarland Standard No 0.5 for M. abscessus) diluted 1/10 in 7H9. Bacteriocin controls without bacteria were also used to control the non-reduced resazurin produced by the bacteriocins alone (bacteriocin negative controls); additional controls were growth controls (wells inoculated with 200 pl inoculum of bacteria) and bacteria negative controls (200 pl 7H9 medium, no bacteria). After 7 days of incubation at 37°C, 30 pl of resazurin 0.01 % (wt/vol) was added to each well and re-incubated for 24-48h (M. tuberculosis) or for 3-5 days (M. abscessus) at 37°C for colour development.

Results were read visually by naked eye by observing a colour change of the resazurin from blue to pink. A change of colour to pink demonstrated bacterial growth. Thus, activity of the bacteriocin was recorded as blue colour.

Assessment of bacteriocin and antibiotics antimicrobial activity

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were determined following standard clinical laboratory methods (CLSI Clinical and Laboratory Standards Institute, 2018; NCCLS, 1999). First, the MIC values of RIF, OFLO, MFX and the bacteriocins (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) against M. tuberculosis H37Rv and the MDR-TB strain was determined using REMA as described above. For MIC determination, six serial two-fold dilutions of antibiotics or of the bacteriocin were prepared in a 96- well microtiter plate containing 100 pl of 7H9 medium (supplemented with 0.5% glycerol and 10% OADC). The range of concentrations tested was 2-0.0625 pg/ml for RIF, 8-0.25 pg/ml for OFLO, 2-0.062 pg/ml, and 100-3.12 pg/ml for the bacteriocins. The MIC values of CLA, TGC, IMI, and AMK against M. abscessus CCUG 41449 were similarly obtained. A growth control containing only bacteria and a sterile control were included in each plate, as described above. The plates were inoculated with 100 pl of inoculum (turbidity corresponding to McFarland Standard No 1 .0) diluted 1/10 in 7H9. Plates were incubated at 37°C for 7 days. After incubation, 30 pl of 0.01 % resazurin was added to each well and the plates were re-incubated for 24-48h at 37 °C. The MIC was defined as the lowest bacteriocin or antibiotic concentration that prevented a colour change of the resazurin from blue to pink. MBC determination was performed directly from the MIC plate. At day 9, blue wells above the MIC were chosen to determine the MBC. Briefly, 100 pl from each well comprising a concentration value corresponding to the MIC, and all the concentrations higher, were transferred to a tube and ten-fold dilutions were prepared in sterile distilled water and plated in duplicate on 7H10 agar (Sigma-Aldrich, MO, USA) for CFU counting. The plates were incubated at 37 °C for at least 21 days. The percentage of killed bacteria was calculated against the control and the MBC was defined as the lowest bacteriocin or antibiotic concentration that killed 99.9 % of bacteria. Each experiment was performed in duplicate. The ratio of MBC/MIC was determined by dividing the two values.

Determination of fractional inhibitory concentration (FIC index)

The FIC index determined by the checkerboard assay is the most widely used method to study drug interactions (Hsieh et al., 1993). Antibiotics-bacteriocin combination testing on M. tuberculosis H37Rv and the MDR-TB strain was performed by a modified checkerboard broth microdilution method that employs resazurin (Santos et al., 2018). Different concentrations of RIF, OFLO, MFX, and bacteriocins (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), at sub-inhibitory concentrations, were used individually or in combination to check the synergistic effect of bacteriocins and antibiotics. Antibiotics and bacteriocins were serially 2-fold diluted in sterile 96-well plates; 50 pl of bacteriocin and 50 pl of antibiotic solutions, prepared at a concentration eight times higher than the final concentration, were mixed into each well. In this way, antibiotic dilutions were mixed with the appropriate concentration of bacteriocin, thus obtaining a series for combination testing, so that each column contained 1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC, 1/32 MIC, 1/64 MIC and 1/128 MIC of one bacteriocin and one antibiotic. For M. tuberculosis H37Rv the final concentrations for RIF ranged from 0.25 to 0.0037 pg/ml, for OFLO from 0.5 to 0.007 pg/ml, and for MFX from 0.062 to 0.0019 pg/ml. For the MDR-TB strain the final concentrations for RIF and OFLO ranged from 4 to 0.062 pg/ml and for MFX from 0.25 to 0.007 pg/ml. For both strains the bacteriocin concentrations ranged from 25 to 0.39 pg/ml (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 4) or 50 to 0.78 pg/ml (SEQ ID NO: 3). A control corresponding to the MIC was included in each test.

Thus, the following combinations were tested against M. tuberculosis H37Rv and MDR-TB strains: bacteriocin (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4) + rifampicin (RIF), bacteriocin (SEQ ID NO:1 , SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4) + ofloxacin (OFLO), and bacteriocin (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4) + moxifloxacin (MFX).

Combinations were tested in duplicate. Each well was inoculated with 100 pl of inoculum (turbidity corresponding to McFarland Standard No 1 .0) of M. tuberculosis H37Rv or the MDR-TB strain, diluted 1/10, and incubated for 7 days at 37°C. Then, 0.01 % resazurin was added to each well and plates were re-incubated 24-48h for the final reading. The checkerboard assay was repeated two times. The FIC index was determined using Formula I as described earlier herein: ... with:

“A” corresponding to RIF, OFLO or MFX, and “B” to the respective bacteriocin; and "combination” corresponding to the individual combinations (bacteriocin + RIF, OFLO, or MFX). The FIC index was interpreted as follows: -Presence of a synergistic effect at a FIC index of < 0.5;

-Presence of an additive effect at a FIC index of > 0.5 and < 1 . The same protocol and determination procedure were used to test combinations of the bacteriocins (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) with CLA, TGC, IMI, and AMK against M. abscessus CCUG 41449.

Example 1 . Determination of MIC, MBC and MBC/MIC ratio of bacteriocins against M. tuberculosis

In Example 1 , the MIC value, MBC value, and MBC/MIC value of a bacteriocin peptide represented by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:4 against M. tuberculosis H37Rv and the MDR-TB strain was determined as described in the General Procedures. The results are shown in Tables 3 and 4: Table 3. CFU counting after exposure to the four tested bacteriocins

Table 4. MIC value, MBC value, and MBC/MIC ratio of tested compounds

The MIC value of rifampicin (RIF), ofloxacin (OFLO), and moxifloxacin (MFX) against both strains was also determined in the same way. The MIC of RIF was 0.25 pg/ml against M. tuberculosis H37Rv and was >0.5 pg/ml (8 pg/ml) against the MDR-TB strain. The MIC of OFLO was 0.5 pg/ml against M. tuberculosis H37Rv and was 4 pg/ml against the MDR-TB strain. The MIC of MFX was 0.062 pg/ml against M.tuberculosis H37Rv and 0.25 pg/ml against the MDR-TB strain. These values confirm the antibiotic resistance of the tested strains.

The results demonstrate that bacteriocins described herein are able to effectively inhibit bacteria of the genus Mycobacterium, in particular antibiotic-resistant strains.

Example 2. Determination of synergistic effect of bacteriocin and antibiotics combinations against M. tuberculosis

Determination of synergistic effect of the combination of a bacteriocin represented by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 with either RIF, OFLO, or MFX against M. tuberculosis H37Rv and the MDR- TB strain was performed by determination of the FIC index as described in the General Procedures. The MIC decrease over the compound alone was also determined. The results are shown in Tables 5 and 6:

Table 5. FIC index and MIC-fold change decrease demonstrated by combinations of bacteriocins with RIF or OFLO

Table 6. FIC index and MIC-fold change decrease demonstrated by combinations of bacteriocins with MFX

The results demonstrate strong synergistic effects when the bacteriocins described herein are combined with rifampicin (RIF), ofloxacin (OFLO), or moxifloxacin (MFX) against bacteria of the genus Mycobacterium, in particular against antibiotic-resistant strains.

Example 3. Determination of MIC of bacteriocins against M. abscessus

In Example 3, the MIC value of a bacteriocin peptide represented by SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:4 against M. abscessus CCUG 41449 was determined as described in the General Procedures. The results are shown in Table 7:

Table 7. MIC value of tested compounds

The MIC value of clarithromycin (CLA), tigecycline (TGC), imipenem (IMI), and amikacin (AMK) against M. abscessus CCUG 41449 was also determined in the same way. The MIC of CLA was 2 pg/ml, the MIC of TGC was 1 pg/ml, the MIC of IMI was 4 pg/ml, and the MIC of AMK was 8 pg/ml.

Example 4. Determination of synergistic effect of bacteriocin and antibiotics combinations against M. abscessus

Determination of synergistic effect of the combination of a bacteriocin represented by SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 4 with either CLA, TGC, IMI, or AMK against M. abscessus CCUG 41449 was performed by determination of the FIC index as described in the General Procedures. The results are shown in Table 8:

Table 8. FIC index demonstrated by combinations of bacteriocins with CLA, TGC, IMI, or AMK

These results demonstrate the synergistic effect of the bacteriocins with antibiotics against non-tuberculous Mycobacteria. References

Skrahina, A. et al. Multidrug-resistant tuberculosis in Belarus: the size of the problem and associated risk factors.

Bull World Health Organ 1 ;91 (1): 36-45, 2013.

Palomino, J.C. et al. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 46:2720-2, 2002.

Martin, A. et al. Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second- line drugs: rapid, simple, and inexpensive method. Antimicrob Agents Chemother 47:3616-9, 2003.

CLSI Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, Nocardia spp., and other aerobic actinomycetes, 3rd ed, CLSI standard document M24. Clinical and Laboratory Standards Institute, Wayne, PA., 2018.

NCCLS. Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline. NCCLS Document M26-A. 1999. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087, USA, 1999.

Hsieh, M.H. et al. Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infect Dis 16:343-349, 1993. Santos, N.C.S. et al. Combinatory activity of linezolid and levofloxacin with antituberculosis drugs in Mycobacterium tuberculosis. Tuberculosis (Edinb). 111 :41-44, 201.

Claims

47 Claims

1. A bacteriocin peptide or peptidomimetic, wherein said peptide or peptidomimetic comprises, consists essentially of, or consists of a peptide or peptidomimetic represented by the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or by an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identity or similarity with SED ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, for use in the treatment, prevention and/or delaying of an infection and/or a disease in a subject, wherein said infection and/or disease is caused by a bacterium of the genus Mycobacterium.

2. A bacteriocin peptide or peptidomimetic for use according to claim 1 , wherein said bacterium is selected from a species belonging to the Mycobacterium tuberculosis complex or a nontuberculous Mycobacterium.

3. A bacteriocin peptide or peptidomimetic for use according to claim 1 or 2, wherein said bacterium is Mycobacterium tuberculosis.

4. A bacteriocin peptide or peptidomimetic for use according to claim 1 or 2, wherein said bacterium is Mycobacterium abscessus.

5. A bacteriocin peptide or peptidomimetic for use according to any one of claims 1-3, wherein said infection and/or disease is tuberculosis.

6. A bacteriocin peptide or peptidomimetic for use according to any one of claims 1-5, wherein said bacterium is antibiotic-resistant.

7. A bacteriocin peptide or peptidomimetic for use according to claim 6, wherein the antibiotic resistance is caused by a mutation in a gene selected from rpoB, gyrA, or gyrB.

8. A bacteriocin peptide or peptidomimetic for use according to claim 6 or 7, wherein said antibiotic is a polyketide, preferably is rifampicin.

9. A bacteriocin peptide or peptidomimetic for use according to claim 6 or 7, wherein said antibiotic is a fluoroquinolone, preferably is ofloxacin or moxifloxacin.

10. A bacteriocin peptide or peptidomimetic for use according to claim 6, wherein said antibiotic is a macrolide, preferably is clarithromycin.

11. A bacteriocin peptide or peptidomimetic for use according to claim 6, wherein said antibiotic is a tetracycline or glycylcycline, preferably is tigecycline.

12. A bacteriocin peptide or peptidomimetic for use according to claim 6, wherein said antibiotic is a -lactam or a carbapenem, preferably is imipenem.

13. A bacteriocin peptide or peptidomimetic for use according to claim 6, wherein said antibiotic is a aminoglycoside, preferably is amikacin. 48

14. A bacteriocin peptide or peptidomimetic for use according to any one of claims 1-13, wherein an antibiotic is further used, preferably selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a -lactam, a carbapenem, an aminoglycoside, or a combination thereof, more preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

15. A composition comprising a bacteriocin peptide or peptidomimetic as defined in any one of claims 1-13.

16. A composition according to claim 15, wherein said composition further comprises an antibiotic, preferably selected from a polyketide, a fluroquinolone, a macrolide, a tetracycline, a glycylcycline, a -lactam, a carbapenem, an aminoglycoside, or a combination thereof, more preferably selected from rifampicin, ofloxacin, moxifloxacin, clarithromycin, tigecycline, imipenem, amikacin, or a combination thereof.

17. A composition according to claim 15 or 16, wherein said composition is a pharmaceutical composition optionally further comprising one or more antimicrobial compounds and/or pharmaceutically acceptable ingredients, for use as defined in any one of claims 1-14.

18. A bacteriocin peptide or peptidomimetic for use according to any one of claims 1-14, or a pharmaceutical composition for use according to claim 17, wherein administration of said peptide, peptidomimetic, or composition is selected from intrapulmonary, oral, or parenteral administration.

19. A composition according to claim 15 or 16, wherein said composition is suitable for disinfecting a surface contaminated with a bacterium of the genus Mycobacterium, optionally further comprising one or more antimicrobial compounds and/or a solvent.

20. An ex-vivo method of disinfecting a surface comprising contacting said surface with a bacteriocin peptide or peptidomimetic as defined in any one of claims 1-14, or with a composition as defined in claim 19.

21. An ex-vivo method according to claim 20, wherein said surface is contaminated with a bacterium of the genus Mycobacterium.

22. An ex-vivo method according to claim 21 wherein said bacterium is selected from a species belonging to the Mycobacterium tuberculosis complex or a nontuberculous Mycobacterium.

23. An ex-vivo method according to claim 22, wherein said bacterium is Mycobacterium tuberculosis.

24. An ex-vivo method according to claim 22, wherein said bacterium is Mycobacterium abscessus.