EXPERIMENTAL AND THERAPEUTIC MEDICINE 24: 753, 2022

Molecular mechanisms of multidrug resistance in

clinically relevant enteropathogenic bacteria (Review)
JULIA ALEXSANDRA GONZÁLEZ‑VILLARREAL1, KATIA JAMILETH GONZÁLEZ‑LOZANO2,
ELVA TERESA ARÉCHIGA‑CARVAJAL2, JESÚS ANTONIO MORLETT‑CHÁVEZ3,
MIRIAM PAULINA LUÉVANOS‑ESCAREÑO4,
NAGAMANI BALAGURUSAMY5 and MAURICIO ANDRÉS SALINAS‑SANTANDER3

1
Faculty of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila 27275; 2Microbiology Department,
Phytopathology and Mycology Laboratory, Faculty of Biological Sciences, Genetic Manipulation Unit,
Autonomous University of Nuevo Leon, Monterrey, Nuevo León 66459; 3Research Department, Faculty of Medicine
Saltillo Unit, Autonomous University of Coahuila, Saltillo, Coahuila 25000; 4Bioprocesses Laboratory,
Faculty of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila 27275; 5Bioremediation
Laboratory, Faculty of Biological Sciences, Autonomous University of Coahuila, Torreón, Coahuila 27275, Mexico

Received June 27 2022; Accepted September 21, 2022

DOI: 10.3892/etm.2022.11689

Abstract. Multidrug resistant (MDR) enteropathogenic 4. Molecular mechanisms of multidrug resistance generation
bacteria are a growing problem within the clinical environ‑ 5. Drugs and bacterial response
ment due to their acquired tolerance to a wide range of 6. Mechanisms of antibiotics
antibiotics, thus causing severe illnesses and a tremendous 7. Mechanisms of drug resistance
economic impact in the healthcare sector. Due to its difficult 8. Treatment against multidrug‑resistant bacteria
treatment, knowledge and understanding of the molecular 9. Conclusions
mechanisms that confer this resistance are needed. The aim of
the present review is to describe the mechanisms of antibiotic
resistance from a genomic perspective observed in bacteria, 1. Introduction
including naturally acquired resistance. The present review
also discusses common pharmacological and alternative treat‑ Gastrointestinal diseases are the most frequent cause for
ments used in cases of infection caused by MDR bacteria, thus medical consultation and one of the leading causes of death
covering necessary information for the development of novel worldwide (1,2). In America, 77 million individuals get sick
antimicrobials and adjuvant molecules inhibiting bacterial annually due to food poisoning and, according to the World
proliferation. Health Organization (WHO), one in ten individuals get sick
each year for the same reason worldwide. As a result, ~420,000
individuals die on a yearly basis, of which ~30% are children
Contents under five years of age. It must be noted that diarrheal diseases
correspond to more than half of the cases of gastrointestinal
1. Introduction illnesses, for which ~95% of cases can be associated with
2. Multidrug resistant enteropathogenic bacteria Campylobacter spp., Escherichia coli and non‑typhoidal
3. Molecular mechanisms of multidrug resistance Salmonella spp. (3).
In 2017, the WHO published a list of drug resistant
bacteria for which there is a growing need to develop new
antibiotics as even the current most effective of them, such as
carbapenems and cephalosporins, are now ineffective. This
Correspondence to: Dr Mauricio Andrés Salinas‑Santander, list is divided into three categories (critical, high, and medium
Research Department, Faculty of Medicine Saltillo Unit,
priority) based on how urgently these antibiotics are needed.
Autonomous University of Coahuila, Calle Francisco Murguía
Sur 205, Zona Centro, Saltillo, Coahuila 25000, Mexico The critical priority group includes multidrug resistant
E‑mail: msalinsa@yahoo.com (MDR) bacteria that are especially dangerous for vulnerable
individuals, or individuals under specialized care, due to the
Key words: enteropathogenic bacteria, multidrug resistance, high risk of infection, complications, disease severity and
gastrointestinal disease, horizontal gene transfer, therapeutic mortality (4). Some of the bacteria included in this group
research are: Acinetobacter spp., Pseudomonas spp., Klebsiella spp.,
Escherichia coli, Serratia spp. and Proteus spp. (4), all of
which have different infection pathways in the host (5).
2 GONZÁLEZ-VILLARREAL et al: MOLECULAR MECHANISMS OF MULTIDRUG RESISTANCE IN BACTERIA

Different molecular mechanisms of bacterial resistance population dies, with only a small subpopulation persisting
to antibiotics have been described so far. Due to the impor‑ for a longer time (13‑15). Resistance is the ability to grow in
tance of this phenomenon in public health, the present review the presence of environmental stress or high concentrations of
gathers engaging and relevant information concerning the antibiotics, regardless of the treatment's duration, due to the
most common enteropathogenic bacteria in clinical practice increased MIC required to effectively destroy the microor‑
and describes the molecular mechanisms for the acquisition ganism (13‑16).
or de novo development of antibiotic resistance, thus seeking The acquisition of antibiotic or antimicrobial resistance is a
to enlighten the reader in this regard and provide a greater natural selection process of bacteria and thus considered as part
understanding of this process. of their evolutionary path. In this regard, the indiscriminate use of
Thorough research was conducted in the writing of the antibiotics exerts a high selective pressure on them, which results
present manuscript, primarily employing informatic tools such in genomic changes that translate into multidrug resistance, as
as PubMed (https: //pubmed.ncbi.nlm.nih.gov/), Scopus (https: seen with greater frequency in developing countries (15,17).
//www.scopus.com/home.uri), Scielo (https: //scielo.org/), Depending on the number or type of antimicrobials,
Medigraphic (https: //www.medigraphic.com/newMedi/) resistant bacteria can be classified as MDR, which occurs
and Science Direct (https: //www.sciencedirect.com/). The when clinically relevant microorganisms have developed
terms used in this search included: Enterobacteria, multidrug resistance to three or more classes of commonly used antibi‑
resistance, enteropathogenic bacteria, multidrug‑resistant otics and/or antimicrobials (18,19); extensively drug‑resistant
bacteria, bacterial drug resistance, horizontal gene transfer (XDR), microorganisms resistant to at least one agent of all
and gastrointestinal diseases. Inclusion criteria included antimicrobial classes; and pandrug‑resistant, which includes
English language and full‑length articles. Exclusion criteria: microorganisms resistant to all agents in all antimicrobial
Publications from 2012 to 2022 were prioritized. Older publi‑ classes (20). Most of these multidrug‑resistant bacteria are
cations were also reviewed and introduced in the present study typically gram‑negative enterobacteria representing an impor‑
if deemed relevant. A total of 99 research and review articles tant therapeutic challenge in the treatment of life‑threatening
were used in the present study (Fig. 1). infections (12,15).

2. Multidrug resistant enteropathogenic bacteria 3. Molecular mechanisms of multidrug resistance

The family Enterobacteriaceae includes several genus and Antibiotic resistance can be permanently maintained once it
species of both gram‑negative and ‑positive bacilli (for has been fixed in the genome or it can be just temporary if
example Enterococcus spp.), a number of which are present the selective pressure is absent causing non‑resistant bacteria
in water, soil, plants and the intestinal microbiota of humans to proliferate instead. Drug resistance often appears due to
and animals; however, their diversity is often dictated by the acquisition of exogenous DNA or through genomic DNA
geographical area (6) and often develop as opportunistic mutations (21).
pathogens causing severe infections in humans (Table I) (7). From an evolutionary perspective, bacteria have several
These bacteria are associated with 10‑20% cases of advantages over other organisms because they have short
infectious diarrhea in children worldwide (8,9). The majority replication time, large populations and capacity for horizontal
of patients affected by these bacteria only require an hydro‑ gene transfer, which enables bacteria to adopt, use, propagate
electrolytic imbalance intervention, caused by dehydration and fix advantageous genetic information between strains and
or antibiotics treatment, the latter of which diminishes the species, such as antibiotic resistance (22).
duration of the disease, reduces its transmission and prevents The limitation of both resources and nutrients within the
complications (10). In some cases, it is possible that severe environment is a decisive factor exerting great selective pres‑
infections can be caused by multidrug‑resistant enterobacteria sure on bacteria, forcing the stressed populations to adapt or
or by enterotoxin producing bacteria, and for this reason die. As a result, the genetic variations providing a survival
special epidemiological surveillance is necessary (10,11). A advantage become fixed in the bacterial population, thus
report made in 2017 revealed that antibiotic resistance in Latin taking another step in their evolution as a species (23,24).
America was as high as 45%, followed by Europe with 39%, The genetic evolution of bacteria mostly occurs due to
the US with 8%, and Canada with 5% (12). recombination events allowing gene acquisition, segment
In 2019 Levin‑Reisman et al (13) described the different duplication, fusion of homologous regions, functional domain
phenotypic traits enabling bacteria to acquire resistance to anti‑ exchange and gene deletion (24,25). Acquisition of exogenous
bacterial agents, such as tolerance, persistence and resistance. genomic material occurs via horizontal gene transfer (HGT),
Tolerance is the ability of a bacterial population to survive and which enables bacteria to absorb and incorporate genetic mate‑
grow under toxic conditions, such as high concentrations of rial of diverse origin, thus giving rise to different genotypes
antibiotics, thus prolonging treatment duration; notably, this between populations of the same species. Further, HGT events
acquired resistance may or may not be inherited to daughter can also confer pathogenicity factors related to virulence,
cells (13‑15). Persistence is the ability of bacteria to survive symbiosis, resistance and metabolism, among others (24,25).
a specific drug concentration, prolonging the duration of Three major mechanisms of HGT have been described
treatment unless corrected (13). These persistent bacteria until 2019: i) Natural transformation (26); ii) conjugation (27);
can withstand antibiotic treatment without affecting the and iii) transduction (28). However, Soler and Forterre (25)
drugs' minimum inhibitory concentration (MIC), presenting proposed a fourth mechanism called vesiduction in
a biphasic death curve because the majority of the bacterial 2020 (Fig. 2).
 EXPERIMENTAL AND THERAPEUTIC MEDICINE 24: 753, 2022 3

Transduction. Transduction is mediated by bacterial viruses

called bacteriophages. When a phage infection culminates in
bacterial lysis, some viral particles can encapsulate bacterial
DNA fragments, thus producing transducer particles. Upon
subsequent infection, the transducer particles inject bacte‑
rial DNA into the next bacterium host, which may acquire
new genetic traits after adopting the exogenous DNA (35).
Generalized transduction occurs when any of the bacterial
genes maintains the same probability of being encapsulated in
a transducing particle and transferred into a recipient. On the
other hand, specialized transduction defines the transference
of specific genes, such as those located next to the bacterio‑
phage's DNA (30,36,37).

Vesiduction. Vesiduction was proposed in 2020 by

Soler and Forterre (25). It involves the donation and/or
Figure 1. Flow chart of the bibliographic search.
acquisition of exogenous material from extracellular vesicles,
a phenomenon that has been observed in all three domains
of life. Vesicles are secreted through the cell membrane
of Gram‑positive bacteria and the outer cell membrane of
Natural transformation. This phenomenon represents the Gram‑negative bacteria. The precise mechanism is not yet fully
active transfer of genes from extracellular free DNA from understood, and it may be possible that it differs according to
lysed bacteria to a living, competent bacterium that captures the composition of the cell wall and the proteins used in the
and incorporates it into its genome through DNA recombina‑ construction of the vesicles (38,39). These vesicles can fulfill
tion. This process contributes to genetic variability, shapes different physiological roles that are not mutually exclusive
evolution and, in the case of pathogenic bacteria, it is respon‑ with genetic material transference, such as the transport of
sible for their adaptation to host cells, fosters the spread of peptidoglycan hydrolases or toxins, and other effector proteins
antibiotics resistance, promotes antigenic variation and leads that may be involved in the elimination of concurrent microor‑
to the acquisition of new virulence factors (29‑31). In addition, ganisms through competition or pathogenicity. Some vesicles
natural transformation also promotes DNA exchange between can also transport intercellular communication molecules (39).
taxonomically distant bacteria (29‑31). Regardless of their inherent differences, all of the previ‑
In 2018, Ellison et al (31) demonstrated the ability of ously mentioned mechanisms for genetic acquisition can be
bacteria to capture and introduce free DNA molecules driven by RecA‑dependent recombination, illegitimate recom‑
through surface appendages known as competing pili. Using bination, transposition or integration (25).
Vibrio cholerae as a model, type IV competing pili were
demonstrated to be able to capture and bind double‑stranded 4. Molecular mechanisms of multidrug resistance generation
DNA from the extracellular space. Once bound to DNA, the
pili retracts and mobilizes the captured DNA molecule towards There are different mechanisms of natural resistance that can
the cell surface, where it is finally internalized. appear through other pathways; however, these are usually
induced by the presence or prolonged exposure of hazardous
Conjugation. This process explains the exchange of genetic molecules (such as antibiotics) resulting in the prolif‑
material from donor bacteria with an adjacent recipient through eration of those populations with advantageous biological
a sexual pilus or physical contact, requiring the formation of a changes (40,41). The majority of these mechanisms are specifi‑
pore between both bacteria while connected as a mating pair. cally developed by bacteria to generate resistance to antibiotics
The exchanged genetic elements can usually provide resistance or antimicrobials and may involve the modification of existing
to drugs, antiseptics and/or disinfectants (30,32). genomic material through spontaneous mutations that might
The occurrence of a conjugation event, and thus of an effec‑ be punctual or massive. These resistant populations thrive
tive DNA transfer, requires cell‑cell contact between a donor thanks to the action of bactericidal molecules eliminating
and a recipient cell. There are two types of genetic elements than the cells lacking tolerance or resistance; in other words, the
can be exchanged during this conjugation: i) Conjugative plas‑ microorganisms are forced to evolve in order to survive (21).
mids, found in free form within the intracellular space and with This selective pressure has become the standard in areas such
autonomous replication capacity; and ii) integrated‑conjugative as hospitals, biohazard waste disposal areas, pharmaceutical
elements, or conjugative transposons, that can integrate into the industry effluents, wastewater, manure treated soils, animal
genome of the recipient cell. Since these plasmids were part of breeding and aquaculture areas (21).
the donor's genome prior to the exchange, the latter are rarely,
if ever, found free in the cytoplasm and do not replicate autono‑ Inherent (natural) resistance. Natural resistance to drugs,
mously (33,34). In the majority of bacteria, conjugation occurs by antibiotics or antimicrobials is a trait often shared between
transferring single‑stranded DNA molecules through the type IV different species of microorganisms, which may be due to the
secretion system contained in the conjugative element, which can same physiology or spontaneous genetic mutations regardless
also transfer DNA from bacteria to eukaryotes (33,34). of previous exposure to these molecules (42,43). An example
4 GONZÁLEZ-VILLARREAL et al: MOLECULAR MECHANISMS OF MULTIDRUG RESISTANCE IN BACTERIA

Table I. Common enteropathogenic bacteria in clinical practice and their symptoms.

Bacteria Antibiotic resistance Pathology Mechanism of action (Refs.)

Klebsiella pneumoniae Carbapenems, β‑lactams, Acute Diarrheic Syndrome, Adherence and biofilm (8,85,86)
aminoglycosides, urinary tract infections, formation by type 1 and
quinolones, tigecycline, cystitis, pneumonia, type 3 pili.
polymyxin. endocarditis, septicemia.
Escherichia coli Cephalosporins, Acute watery diarrhea, A/E and changes of the (2,5,8,87)
fluoroquinolones. bloody diarrhea. host apical enterocyte
membrane. Activation of
T3SS and formation of A/E.
Shigella spp. Cephalosporins, ampicillin, Bloody diarrhea. T3SS encoded on a large (5,10,88,89)
co‑trimoxazole, nalidixic plasmid and transport of
acid. effector proteins.
Salmonella spp. Quinolones, nalidixic acid. Acute watery diarrhea, Activation of T3SS and (5,10,90)
bloody diarrhea, enteric transport of effector
fever. proteins.
Campylobacter spp. Quinolones, tetracycline. Enteric fever, acute watery Presence of flagella, high (10,90,91)
diarrhea, bloody diarrhea. molecular weight plasmids,
surface adhesins and
chemotactic factors.
Vibrio cholerae Ampicillin, nalidixic acid, Acute liquid diarrhea. Biofilms formation and (90,92,93)
co‑trimoxazole. production of
extended‑spectrum
β‑lactamases.
Aeromonas spp. Beta‑lactams, tetracyclines, Acute watery diarrhea, Travel by the blood to the (2,94,95)
glycylcyclines, bloody diarrhea. first organ it finds where
fluoroquinolones, it produces the toxic
aminoglycosides, enterotoxin aerolysin.
sulfamethoxazole‑
trimethoprim.
Yersinia enterocolitica Nalidixic acid. Enteric fever, bloody Activation of T3SS and (5,10,90,96)
diarrhea. transport of effector
proteins and/or apoptosis.
Yersinia forms
microcolonies and starts
replication.
Staphylococcus aureus Penicillin, methicillin. Acute liquid diarrhea. Inoculation into an open (2,90,97)
wound. adhesion and
invasion of host epithelial
cells by microbial surface
components recognizing
adhesive matrix molecules.
Enterococcus spp. Vancomycin, Beta‑lactams, Sepsis, endocarditis, When pathologic alterations (18,98,99)
glycopeptides, urinary tract infections. are caused by either direct
aminoglycosides, toxin activity or indirectly
tetracyclines, quinolones, by bystander damage from
macrolides. the inflammatory response,
enterococci are able to
outpace host defenses,
multiply at rates that are
faster than clearance, and
overwhelm the host.

A/E, attaching/effacing lesion; T3SS, Type III secretion system.

 EXPERIMENTAL AND THERAPEUTIC MEDICINE 24: 753, 2022 5

Figure 2. Molecular mechanisms of multidrug resistance acquired by horizontal gene transfer.

of intrinsic antibiotics resistance conferred by physiology can this change can become fixed in the population through
be seen in bacteria of the Mycoplasma genus, whose members vertical gene transfer and become a dominant trait (21,48).
are highly resistant to drugs targeting the cell wall, such as The frequency of spontaneous mutations related to antibi‑
β ‑lactams and glycopeptides (44,45); although some antibi‑ otic resistance occur at a rate of 1x10 ‑5 to 2x10 ‑8 in members
otics normally have difficulty crossing the outer membrane of the Chlamydiaceae and Helicobacter pylori (49,50).
of Gram‑negative bacteria. For example, vancomycin inhibits Though this would appear to be a rare event, in reality
cell wall synthesis by targeting d‑Ala‑d‑Ala peptide precursor antibiotic resistance appears in bacterial populations within
units of Gram‑positive bacteria, thus preventing the assembly a relatively short period of time, accelerating further when
of peptidoglycan layers and transpeptidation (46). By contrast, exposed to a selective agent due to exponential growth rate
this antibiotic cannot affect Gram‑negative bacteria since it and the number of cells generated per replication cycle (51).
is unable to cross the outer membrane, and thus kept from For example, the gastric pathogen Helicobacter pylori can
accessing the cell wall (46). Even though these events occur have different mutations in the 23S rRNA, gyrA and rpoB
naturally in the environment, (47) it must be mentioned that genes, which are responsible for resistance to clarithro‑
the intrinsic resistance to antibiotics is not considered as a mycin, ciprofloxacin and rifampicin, respectively (50). The
clinical problem because previously developed antibiotics do capacity of Chlamydia trachomatis to resist antibiotics such
not target these bacteria. as azithromycin, tetracycline and fluoroquinolone has also
been attributed to spontaneous mutations (52). Although this
Spontaneous mutations. Spontaneous mutations occur by mutation rate is not even across the board, there are bacterial
random nucleotide changes that induce different effects; subpopulations with a significant tendency to acquire and
for example, amino acid sequence variations that may lead accumulate spontaneous mutations, which is why they often
to altered phenotypes. These mutations can be caused by present a greater number of mutation events compared with
DNA replication errors or through the action of mutagenic what is commonly observed (21). These subpopulations are
agents. It must be noted that acquired mutations are often known as hypermutable and, although not all spontaneous
detrimental, so these are usually not inherited, are rarely mutations confer antibiotics resistance, this hypermuta‑
widespread and often are just isolated events (21,48). bility is directly proportional with the increased resistance
However, when a mutation provides a biological advantage, capacity (21).
6 GONZÁLEZ-VILLARREAL et al: MOLECULAR MECHANISMS OF MULTIDRUG RESISTANCE IN BACTERIA

Duplications. Gene duplications are often overlooked as membrane are specific for each microbial group because their
the primary source of functional genomic diversity, origi‑ function depends on the lipid content of such membrane;
nating new functions from a pre‑existing gene. In addition, however, these drugs can sometimes be toxic, thus limiting
the generation of genetic copies derives into elements that their use (56,57). For example, Daptomycin can rupture the
can evolve independently due to inexistent selective pres‑ cell membrane due to depolarization, whereas that polymyxins
sure, further diversifying their functions (53). For example, bind to the lipid fraction of the membrane's lipopolysaccharide
the Plasmodium falciparum multidrug resistance protein layer, thus causing its disintegration (57).
transporter 1 gene plays an important role in the parasite's
resistance to drugs due to the strong correlation between the Nucleic acid synthesis inhibition. Nucleic acid synthesis is
number of copies and the resistance to artemisinin‑based important for the survival of living beings, including bacteria.
therapies, an anti‑malaria drug used to reduce its mortality The cellular processes responsible for cell replication and
rate since the year 2000. By 2020, Calçada et al (54) reported bacterial conservation can be negatively affected due to the
the threat of resistance against this drug due to the appearance interruption of this process by drugs that block DNA replica‑
of new duplication events and the presence of single nucleotide tion or transcription (56,57). In this regard, antibiotics such
polymorphisms in current strains. as quinolones interfere with the functionality of the helicase
enzyme preventing the function of unwinding DNA, effecting
5. Drugs and bacterial response the process of DNA replication and repair. On the other hand,
they can exert their action by inhibiting topoisomerase II and
As aforementioned, the genetic elements leading to drug IV of bacteria, preventing the synthesis of RNA (61).
resistance can be spread between different microorganisms in
different manner, from the horizontal (transformation, trans‑ Metabolic inhibitors. Some drugs act against important meta‑
duction, conjugation or vesiduction) to vertical gene transfer bolic processes for survival, such as the folic acid pathway,
(from mother to daughter cells) of either intrinsic or extrinsi‑ which is necessary to produce important precursors in DNA
cally acquired genomic modifications, such as spontaneous synthesis (57). In this case, sulphonamides and trimethoprim
mutations, duplications, insertions, deletions or transpositions. release similar substrates to those produced and used by
The mechanism of antibiotics resistance is highly dependent the bacteria in its normal metabolism (56,57). Each of these
on the way the drug itself works against the bacterium, drugs is responsible for inhibiting different stages of folic acid
regardless of how this resistance was acquired, thus deriving metabolism. For example, the sulphonamides competitively
in different survival pathways that may limit the absorption inhibit dihydropteroate synthase, binding to it with greater
of drugs, modify the target molecules, directly inactivate the affinity compared with the substrate produced by the bacteria;
drug and/or secrete it into the microenvironment (Fig. 3) (55). while trimethoprim is responsible for inhibiting dihydrofolate
reductase at a later stage of folic acid synthesis (56,57,59).
6. Mechanisms of antibiotics
Protein synthesis inhibition. Proteins play a role in various
Antibiotics with the capacity to inhibit or kill a wide range cellular structures and physiological processes; therefore,
of bacteria are known as broad‑spectrum antibiotics, whereas their synthesis is fundamental for survival (56,57). For this
that those that only affect certain types of bacteria are known reason, drugs that inhibit protein biosynthesis by targeting the
as narrow‑spectrum antibiotics. Antibiotics typically target the 70S prokaryotic ribosome (30S and 50S ribosomal subunits)
structure or metabolic processes of bacteria, preventing their constitute the largest class of antibiotics (56,57,59).
replication (56‑58). In this regard, the most common mecha‑
nisms consist in the inhibition of cell wall synthesis, DNA 30S subunit inhibitors. Antibiotics such as tetracycline,
replication or transcription, protein synthesis, metabolic path‑ aminoglycosides and streptomycin target and inhibit the 30S
ways or directly through cell membrane degradation (41,57). ribosome, blocking the passage of aminoacyl‑tRNA towards
the ribosome (57,59).
Cell wall synthesis inhibition. The majority of bacterial cells
are surrounded by a rigid peptidoglycan layer consisting 50S subunit inhibitors. Antibiotics targeting the 50S ribosomal
of long sugar polymers linked through peptide bonds. This subunit can act in two different ways, by blocking protein
structure is needed for survival as it protects the bacteria translation (oxazolidinones) or by blocking the elongation
from osmotic pressure and other hostile conditions from the phase of protein synthesis (for example, macrolides). However,
environment (56,57,59). Drugs such as penicillin and cephalo‑ the latter may be ineffective when the elongation phase has
sporins inhibit the formation of peptide bonds in the bacterial advanced significantly (57,59). Natural antibiotics such as
cell wall, thus effectively killing the microorganism (56). By aminoglycosides are considered as bactericidal, whereas
contrast, glycopeptides inhibit bacterial growth by inhibiting macrolides, tetracyclines, chloramphenicol, streptogramins
peptidoglycan synthesis (56,57). and spectinomycin are considered as bacteriostatic (57).

Cell membrane function inhibition. In comparison with 7. Mechanisms of drug resistance

gram‑positive bacteria, gram‑negatives have a greater resis‑
tance to antimicrobials due to the existence of an external Antibiotics outlet, secretion or efflux pumps. Some bacteria
cell membrane regulating both intracellular and extracellular have exporter proteins on their cell membrane that can rapidly
substance flow (56,60). The drugs targeting this external cell transport the antibiotics from the cytoplasmic membrane in
 EXPERIMENTAL AND THERAPEUTIC MEDICINE 24: 753, 2022 7

Figure 3. Antibiotics and bacterial resistance.

gram‑positive bacteria and from the intermembrane space function as a physical barrier that reduces the permeability
in gram‑negative bacteria to the exterior of the cell without of a number of drugs (53,65). Notably, this envelope can
the help of energy‑dependent efflux pumps, thus preventing also be targeted by antibiotics (53,65). The outer membrane
the antibiotics from reaching their target (30,62,63). There of gram‑negative bacteria is populated by proteins called
are two groups of efflux pumps, some of them are specific porins, which determine its permeability and allow the entry
whereas others can secrete diverse substances. These pumps of hydrophilic compounds into the cell. The absence or low
are classified according to energy source and function; in number of porins can also prevent the entry of antimicrobial
this regard, the first group uses ATP as an energy source and molecules, thus hindering their action in the cytoplasm and/or
functions through hydrolysis (63,64). By contrast, the second cell envelope (62,63).
group uses the mobile force of protons as an energy source,
enhancing secretion through the electrochemical potential Active site alterations. Bacteria have the ability to form
of the membrane (63,64). A total of five families of efflux metabolic substances that compete with antimicrobial drugs
pumps have been described within the second group: i) The for the active site, preventing it from binding due to loss of
multidrug and toxic extrusion family; ii) the major facilitator affinity (30,63). There are two types of modifications in this
super family; iii) the resistance nodulations cell division regard as follows.
(RND) family; iv) the small MDR family; and v) the multidrug Penicillin‑Binding‑Protein (PBP) modification. Observed
endosomal transporter family (62‑64). These efflux pumps are in Gram‑positive bacteria, this effect is caused by variations
widely distributed among gram‑positive and ‑negative bacteria, in the peptidoglycan gene, which modify the antimicrobial
except for the RND poly‑selective superfamily, which is found binding site in the cell wall (30,62).
gram‑negative bacteria with very high frequency (62,63). Ribosomal modification. The ermA and ermB genes can
These efflux pumps play a notable role in multidrug resistance modify the ribosome's active site through methylation. These
due to their capacity to secrete a wide range of structurally modifications occur in the 30S and 50S subunits of the 70S
unrelated drugs and molecules (62‑64). ribosome, affecting the target site of drugs such as aminogly‑
cosides, macrolides, tetracyclines and lincosamides (30,66).
Permeability alterations in the outer cell membrane. The
majority of antibiotics penetrate the bacterial membrane and Enzymatic modification or inactivation of antibiotics. This
target diverse intracellular processes; therefore, the concentra‑ is the most common mechanism of resistance observed in
tion of antibiotics within the cell can be affected by alterations bacteria. It is achieved through the expression of enzymes with
in the lipid bilayer of the membrane, modifying either the the capacity to modify the active component of the antibiotics,
cell's diameter or number of porins (30,65). The bacterial cell thus reducing their effectiveness. Three mechanisms have
envelope provides a selective barrier allowing the exchange been reported so far: i) Redox reactions; ii) group transfer;
of nutrients and signaling molecules with the microenviron‑ and iii) enzymatic hydrolysis. The latter is the primary
ment. This envelope is formed by the cell wall and the plasma mechanism of resistance, with the clearest example being the
membrane, and, in Gram‑negatives, it provides an additional hydrolysis of the beta‑lactam ring of antibiotics. The enzymes
8 GONZÁLEZ-VILLARREAL et al: MOLECULAR MECHANISMS OF MULTIDRUG RESISTANCE IN BACTERIA

of gram‑negative bacteria typically originate from a plasmid thus causing a serious dilemma between the selection of a
or have a transposon origin with constitutive and periplasmic broad‑spectrum drug, which could induce greater drug resis‑
expression. By contrast, this resistance is solely provided by tance, or a narrow‑spectrum drug, which could be completely
a plasmid in gram‑positive bacteria, which can be inducible ineffective (71,73). Regardless of its potential shortcomings,
and/or extracellular (40,65). the latter could supply important information on the pathogen's
susceptibility to certain antimicrobials (71,73). Several factors
Biofilm formation. Biofilms are structured aggregations must be evaluated during the selection of antibiotics treatment,
of bacterial cells enclosed in a self‑synthesized extracel‑ including susceptibility, risk of developing resistance, poten‑
lular matrix composed of different macromolecules such as tial side effects, comorbidities, local epidemiology and clinical
proteins, nucleic acids and polysaccharides (63,67). Biofilms severity (10,71,74).
bacterial production protects them from ultraviolet light,
dehydration, immune system or certain antibiotics. There are Combination antibiotic therapy, The combined therapy of
three important steps in biofilm formation: i) Adhesion, in this antibiotics enables the synergistic effect of one or more drugs,
phase bacteria can attach to any give surface; ii) growth and potentially increasing the probability of an effective treatment
maturation, occurs when bacteria secrete an exopolysaccha‑ and lowering the risk of bacterial resistance. However, the
rides matrix and mature from microcolonies to multi‑layered results of drug synergy tests observed in vitro do not always
cell clusters; and iii) shedding, which can be either active translate well into a clinical setting (71,75).
(initiated by the bacteria) or passive (caused by external
factors) (30,62). Amongst the most common pathogens that Alternative treatments. Alternative treatments can also
develop biofilms are S. aureus, P. aeruginosa, A. baumannii be implemented in addition to antibiotics therapy if their
and K. pneumonia (30,62). contribution proves safe for the patient and does not enable
the development of bacterial resistance, for example, phage
Target site overexpression. This mechanism has been described therapy or competing microorganisms (76). There are some
in clinical isolates of mycobacteria with promoter duplica‑ reports demonstrating the benefits of these treatments against
tions. This often results in the overexpression of genes that multidrug‑resistant pathogens, even suggesting they could be
may include mutations affecting the target site of antibiotics or used as replacements for common drugs (76).
antimicrobials (30). In this regard, Martinez et al (68) describe
the presence of plasmids in E. coli that provide resistance to Phage therapy. Bacteriophages are bacteria‑specific viruses
amoxicillin‑clavulanate as a result of the hyperproduction of that can infect bacteria through the binding of specific recep‑
plasmid‑determined TEM‑1 P‑lactamase. TEM‑1 β‑lactamase tors on the cell's surface and injecting their genetic material.
is a known determinant of resistance to antibiotics, such as Once infected, the phage can go through a lysogenic cycle,
penicillin, cephalosporins and their derivatives, including where the phage's genome is integrated in the bacterial chro‑
second, third and fourth generation cephalosporins, mono‑ mosome as an endogenous prophage, spreading horizontally
bactams and β‑lactamase inhibitors. This enzyme inactivates during cell division. The virus can remain latent for prolonged
the aforementioned compounds by hydrolyzing their lactam periods of time during this cycle; however, environmental or
rings (69,70). cellular stress factors can re‑activate the phage and induce
its lytic cycle, in which the viral genome is no longer inte‑
8. Treatment against multidrug‑resistant bacteria grated in the bacterial chromosome and goes into a massive
replication event, finally causing cell death after the phage's
Some of the first‑line drugs used in the treatment of serious lytic proteins hydrolyze the cell wall. These liberated phage
infections caused by Enterobacteriaceae include penicillin, particles can then infect other bacteria and start the lytic cycle
cephalosporins, carbapenems, fluoroquinolones, monobactams again (76‑78). It must be mentioned that the infective capacity
and, occasionally, aminoglycosides. However, bacterial resis‑ of bacteriophages is constrained to particular bacterial species,
tance against these drugs is rapidly becoming widespread, thus resulting in a reduced spectrum. Although this could be
thus making difficult these treatment (20,71). In some cases, considered a shortcoming, it could also be considered as a
second‑line drugs are more effective against enterobacteria, positive aspect since they are unable to affect the intestinal
as would be the case with polymyxins, tigecycline, aminogly‑ microbiota or the host (76,78).
cosides and fosfomycin (72). Pathogenic bacteria have evolved
different strategies to overcome the host's response by avoiding Probiotics, prebiotics and synbiotics. Probiotics are live micro‑
highly competitive environments. For example, the mucosal organisms that play a beneficial role if administered in adequate
barrier can be breached by mucinases, such as the Pic enzyme amounts, regardless of those present in the essential diet or
from Shigella and enteroaggregative from Escherichia coli naturally in the intestinal microbiota of the host (79‑81). On the
(EAEC). Notably, the Pic gene can be found in a ‘pathoge‑ other hand, prebiotics are non‑digestible compounds (non‑starch
nicity island’ flanked by insert‑like EAEC elements that have polysaccharides and non‑digestible oligosaccharides) present in
been acquired through horizontal gene transfer (24). the daily diet and which help stimulate the growth or activity
of the intestinal microbiota, favoring the development of
Empirical treatment with antibiotics. As a first line deci‑ beneficial microorganisms (79,80,82). Finally, synbiotics are
sion, empirical therapy becomes essential in the treatment a composition of the previous two and which are often found
of serious infections caused by bacteria. However, the emer‑ in the form of pharmaceutical or food preparations containing
gence of bacterial resistance complicates its implementation, one or more probiotic organisms and prebiotic compounds in
 EXPERIMENTAL AND THERAPEUTIC MEDICINE 24: 753, 2022 9

order to provide a synergistic effect on the prebiotics, enhancing Ethics approval and consent to participate
the development, activity and nutritional properties of the
probiotics. The inclusion of synbiotics increases the density of Not applicable.
probiotics and their health benefits (80,82). The probiotics used
in clinical treatments are mainly composed of Gram‑positive Patient consent for publication
strains such as Lactobacillus that are resistant to the human
digestive process. The administration of these microorganisms Not applicable.
improves the epithelial barrier function, promotes the growth
of beneficial bacteria, the proliferation of epithelial cells in the Competing interests
host (by upregulation of cell growth and downregulation of
apoptosis), prevents the adhesion and colonization of patho‑ The authors declare that they have no competing interests.
genic microorganisms and toxins, improves lactose digestion,
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