Article 1

microorganisms
Review
A Review of Carbapenem Resistance in Enterobacterales and Its
Detection Techniques
Oznur Caliskan-Aydogan 1,2 and Evangelyn C. Alocilja 1,2, *
1 Department of Biosystems and Agricultural Engineering, Michigan State University,

East Lansing, MI 48824, USA; oznurca@msu.edu
2 Global Alliance for Rapid Diagnostics, Michigan State University, East Lansing, MI 48824, USA
* Correspondence: alocilja@msu.edu
Abstract: Infectious disease outbreaks have caused thousands of deaths and hospitalizations, along
with severe negative global economic impacts. Among these, infections caused by antimicrobial-
resistant microorganisms are a major growing concern. The misuse and overuse of antimicrobials
have resulted in the emergence of antimicrobial resistance (AMR) worldwide. Carbapenem-resistant
Enterobacterales (CRE) are among the bacteria that need urgent attention globally. The emergence
and spread of carbapenem-resistant bacteria are mainly due to the rapid dissemination of genes that
encode carbapenemases through horizontal gene transfer (HGT). The rapid dissemination enables
the development of host colonization and infection cases in humans who do not use the antibiotic
(carbapenem) or those who are hospitalized but interacting with environments and hosts colonized
with carbapenemase-producing (CP) bacteria. There are continuing efforts to characterize and
differentiate carbapenem-resistant bacteria from susceptible bacteria to allow for the appropriate
diagnosis, treatment, prevention, and control of infections. This review presents an overview of the
factors that cause the emergence of AMR, particularly CRE, where they have been reported, and then,
it outlines carbapenemases and how they are disseminated through humans, the environment, and
food systems. Then, current and emerging techniques for the detection and surveillance of AMR,
primarily CRE, and gaps in detection technologies are presented. This review can assist in developing
Citation: Caliskan-Aydogan, O.; prevention and control measures to minimize the spread of carbapenem resistance in the human
Alocilja, E.C. A Review of ecosystem, including hospitals, food supply chains, and water treatment facilities. Furthermore,
Carbapenem Resistance in the development of rapid and affordable detection techniques is helpful in controlling the negative
Enterobacterales and Its Detection
impact of infections caused by AMR/CRE. Since delays in diagnostics and appropriate antibiotic
Techniques. Microorganisms 2023, 11,
treatment for such infections lead to increased mortality rates and hospital costs, it is, therefore,
1491. https://doi.org/10.3390/
imperative that rapid tests be a priority.
microorganisms11061491
Academic Editors: Nadezhda Keywords: antibiotic resistance; carbapenem resistance; carbapenem-resistant Enterobacterales (CRE);
Fursova, Olga Khokhlova and carbapenemases; detection technology; surveillance
Angelina Kislichkina
Received: 3 April 2023

Revised: 23 May 2023
Accepted: 25 May 2023 1. Introduction
Published: 3 June 2023 Antimicrobial resistance (AMR) is acquired when microorganisms grow or survive
in the presence of antimicrobials or drugs designed to kill them [1]. AMR threatens the
effective prevention and treatment of a wide range of infections caused by pathogenic
bacteria, viruses, parasites, and fungi. AMR has been a serious threat to public health since
Copyright: © 2023 by the authors.
the beginning of the last decades [1–5].
Licensee MDPI, Basel, Switzerland.
AMR has a high potential to increase costs and destabilize the health infrastructure.
This article is an open access article
A recent report by the Centers for Disease Control and Prevention (CDC) in 2019 stated
distributed under the terms and
conditions of the Creative Commons
that AMR kills at least 1.27 million people worldwide and is associated with approximately
Attribution (CC BY) license (https://
5 million deaths [1,2]. In the United States (US), the CDC reported that AMR causes more
creativecommons.org/licenses/by/ than 2.8 million infections and 35,000 deaths annually [1], with a predicted annual cost of
4.0/). approximately USD 55 billion [1,6]. In Europe, AMR results in an estimated 25,000 deaths
Microorganisms 2023, 11, 1491. https://doi.org/10.3390/microorganisms11061491 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1491 2 of 26
and a cost of EUR 1.5 billion in health expenditures each year [4]. In accordance with
recent estimates, infections by antimicrobial-resistant microorganisms will annually result
in 10 million deaths, along with USD 100 trillion in costs, by the year 2050 [7]. The
problem of AMR is particularly urgent due to the high presence of unregulated antibiotics
in the market [1,3,8]. The misuse and overuse of antibiotics enable the emergence and
spread of resistance in bacteria, leading to more difficulty in controlling and treating such
infections [5].
1.1. Development of Antibiotic Resistance and Their Mechanisms

The first antibiotic, penicillin, was found by Alexander Fleming in 1928. With its release
into the market in 1941, penicillin-resistant bacteria (Staphylococcus aureus) increased in the
following year [1,9]. There has been a continued discovery of new antibiotics coupled with
the emergence of resistance, since bacteria find ways to survive and resist new antibiotics,
resulting in less-effective drugs. Antibiotics have different strategies and mechanisms for
bacterial death [1,9,10]. In general, bacteria have different mechanisms to become resistant
to antibiotics, and they are listed as follows: (1) restriction on the access of the antibiotic
by changing the entry pathways or limiting their number, (2) activation of efflux pumps
in their cell walls to remove the antibiotics that enter the cell, (3) changing or destroying
the antibiotic with enzymes, (4) alteration of the targets for the antibiotic by modification
of intracellular enzymes so that they can no longer latch onto it, and (5) the development
of a new cell process to bypass the effects of the antibiotic [3,4,11–13]. Bacteria can further
form biofilms on surfaces by extracellular enzymes to prevent antibiotics from penetrating
through the outer cell membrane, allowing the natural growth of the bacteria [14]. Thus,
once microorganisms are exposed to antibiotics, they adapt and grow in the presence
of antibiotics, similar to their adaptation to a new environment. They use their genetic
mechanisms to increase their adaption [4,8].
To understand the problem of antibiotic resistance, it is helpful to discuss how an-
tibiotic resistance is developed in a bacterial population, which is the main focus of in-
fections caused by antibiotic-resistant bacteria in clinical settings. Acquired resistance
is developed in a bacterial population that is originally susceptible to antibiotics either
by genetic mutations and the acquisition of resistant genes or through horizontal gene
transfer (HGT) [10,13,15–17]. In the former route, a subset of bacterial cells from susceptible
populations, in the presence of antibiotics, can develop mutations, allowing survival. When
the mutation emerges, resistant subpopulations become dominant [13–15] as a result of
bacterial multiplication (vertical evolution), passing the gene on to their generations [14,15].
A few studies on the enrichment of resistance genes and mutation selection showed that
the time scale of acquiring antibiotic resistance was extended up to 24 transfers [18],
20 days [19], and 1000 generations, which take 10–15 days [20].
Genes that encode resistance are commonly transferable; acquiring genes by HGT is
a significant driver of bacterial evolution and is primarily responsible for the spread of
antibiotic resistance [13–15,21]. The transmission of genetic materials between microor-
ganisms occurs through three main routes: (1) transformation, incorporation of free DNA
from the environment into the chromosome; (2) transduction, phage-mediated transfer of
DNA from infected cells through virus particles; and (3) conjugation, transfer of plasmids
from one bacterium to another. Conjugation is the most efficient and common way to share
genetic information, always leaving behind a copy of the resistant gene, resulting in the
emergence and spread of AMR [11,13–15]. Resistant genes can also be acquired by trans-
posons or integrons linked with mobile genetic elements (MGE). Transposons, specialized
fragments of DNA, carry several resistant genes but cannot replicate by themselves. They
can move within the genome, facilitating resistant gene migration from the chromosome to
the plasmid [14,15]. Integrons can also encode several resistant genes but are not movable.
Thus, encoding mechanisms are based on the capture of resistant genes and the excision
of the genes within and from the integrons. This is one of the efficient mechanisms of the
accumulation of resistant genes. Integrons also provide a mechanism for adding new genes
for adding new genes into the bacterial chromosome and are mostly carried in plasmids,
increasing the horizontal mobility of the antibiotic-resistant genes [13,15,17].
into the bacterial chromosome and are mostly carried in plasmids, increasing the horizontal
mobility of the antibiotic-resistant genes [13,15,17].
1.2. Factors Converging Emergence and Transmission of Antibiotic Resistance
The development
1.2. Factors of antibiotic
Converging Emergence andresistance
Transmission worldwide
of Antibioticis increasing
Resistance due to the misuse
and The
excessive use of antibiotics and antifungals, global
development of antibiotic resistance worldwide is increasing trade networks,due medical
to thetourism,
misuse
poorexcessive
and sanitation useconditions,
of antibioticsimproper waste management
and antifungals, global trade systems, and urbanization
networks, medical tourism, [21–
26]. Remarkably, the overuse and misuse of antibiotics in healthcare,
poor sanitation conditions, improper waste management systems, and urbanization [21–26]. veterinary medicine,
agriculture, the
Remarkably, andoveruse
aquaculture and their
and misuse release to the
of antibiotics environment
in healthcare, contribute
veterinary to the emer-
medicine, agri-
gence and spread of AMR [21,23–26]. Significant sources of antimicrobial-resistant
culture, and aquaculture and their release to the environment contribute to the emergence bacte-
ria include
and spread of healthcare settings Significant
AMR [21,23–26]. and the environment. AMR can be transmitted
sources of antimicrobial-resistant through
bacteria in-
contact
clude with people,
healthcare animals,
settings andenvironment.
and the contaminatedAMR watercan or foods [27]. In addition,
be transmitted throughintestinal
contact
commensal
with people, bacteria
animals, have been reportedwater
and contaminated as a or
significant
foods [27]. reservoir of antimicrobial-re-
In addition, intestinal com-
sistant bacteria
mensal bacteria and
havegenes
been(ARGs).
reportedDue as atosignificant
HGT and the prior use
reservoir of antibiotics, the com-
of antimicrobial-resistant
mensal flora
bacteria of humans
and genes (ARGs).andDueanimals
to HGTcan and
acquire
the ARGs;
prior use theoffecal carriage the
antibiotics, of resistant
commensal bac-
teria and ARGs leads to their emergence and spreads in the community,
flora of humans and animals can acquire ARGs; the fecal carriage of resistant bacteria and environment,
animal,
ARGs andtofoods
leads [27,28]. Forand
their emergence example,
spreadsthe surveillance
in the community, of environment,
human fecal animal,
carriageandhas
shown
foods a significant
[27,28]. increase
For example, theinsurveillance
intestinal ARG carriage
of human fecalworldwide
carriage has [27]. In another
shown exam-
a significant
ple, animal
increase guts can ARG
in intestinal contaminate
carriageits products [27].
worldwide during In animal
anotherslaughtering
example, animal and food
guts pro-
can
cessing. The its
contaminate handling
products and consumption
during of contaminated
animal slaughtering food processing.
and food or contact with Theanimals
handling or
theirconsumption
and surroundingsof(fertilizer)
contaminated causefood
the spread of AMR
or contact with in the community
animals and environ-
or their surroundings
ment (soilcause
(fertilizer) and water), alongofwith
the spread AMR fruits, vegetables,
in the community etc.and
[1]. Thus, wastewater
environment (soil from human
and water),
activities,
along with healthcare services,etc.
fruits, vegetables, and[1].general
Thus, population-collected
wastewater from human wastewater
activities, treatment
health-
plants
care (WWTP)
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general of antimicrobials, commensal
population-collected wastewater and pathogen
treatment bacteria,
plants (WWTP) antibi-
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otic-resistant
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bacteria, or missing
antibiotic-resistant regu-
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latory
and ARGs status
[26].and
Due practices on WWTP
to inefficient, systems, or
inappropriate, contaminated
missing regulatoryurban wastewater, sewage
status and practices
sludge, manure, sediment, and reclaimed water result in their accumulation and spread
on WWTP systems, contaminated urban wastewater, sewage sludge, manure, sediment,
in the
and environment
reclaimed water and community
result [11,26]. As seen
in their accumulation in Figure
and spread 1, everything
in the environment is connected
and com-
in a complex
munity [11,26].web; the health
As seen of people
in Figure is connected
1, everything to the health
is connected of animals
in a complex web;and the
the envi-
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of animals facilities,
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mals are allfacilities,
healthcare impacted, affecting progress
environments, food, in healthcare
farms, and lifeare
and animals expectancy [1,26].affecting
all impacted,
progress in healthcare and life expectancy [1,26].
Figure1.1.The
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andspread
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AMR causes threats to anyone regardless of age, to immunocompromised people,

and to people with chronic illnesses [1,29]. AMR also puts at risk those who receive
modern healthcare advances, such as joint replacements, organ transplants, cancer ther-
apy, etc. These procedures have a risk of infection, and effective antibiotics may not be
available [1,30]. AMR is a global crisis, and new forms of resistance emerge and rapidly
spread across countries and continents through people, goods, and animals. One billion
people travel through international borders every year, and a global effort is necessary to
slow the emergence and spread of AMR [1,5].
2. Urgent Threat of Infections by Antimicrobial Resistant Bacteria:

Carbapenem-Resistant Bacteria
The World Health Organization (WHO) and CDC reported the current and future
threat of infections by antimicrobial-resistant microorganisms with a high level of con-
cern [1,31]. Carbapenem-resistant Acinetobacter baumannii (CRA), carbapenem-resistant
Pseudomonas aeruginosa (CRP), and carbapenem-resistant Enterobacterales (CRE) have been
listed as critical priority pathogens by the WHO [31]. In addition, CRE and CRA have been
reported as the most urgent threats by the CDC since 2019 [1]. Particularly, CRE results in
1100 deaths and 13,100 infections in the USA [1], with a high fraction of these infections
potentially resulting in death due to limited antibiotic therapies [1,22,30].
Carbapenems, a broad-spectrum β-lactam antibiotic, are structurally related to peni-
cillin [32]. Carbapenems have a carbon instead of a sulfone at the fourth position of the
β-lactam ring, differing from other β-lactams. The unique structure plays a major role in
their stability against β-lactamases [33]. Carbapenems are not easily diffusible through
the cell wall, but they enter the bacteria through outer membrane proteins (porins). Then,
carbapenems degrade the cell wall at the penicillin-binding proteins (PBPs) via the β-lactam
ring. The mode of action weakens the glycan backbone in the cell wall due to autolysis,
and the cell is destroyed because of osmotic pressure [32–34].
Carbapenems have been used as last-line agents against Gram-negative, Gram-positive,
and anaerobic bacteria [33]. The last-resort antibiotics were approved for clinical use in hu-
mans and released into the market in 1985 [1,35,36]. Carbapenems may occasionally be used
for pets under certain conditions, according to the Animal Medicinal Drug Use Clarification
Act (AMDUCA) [37,38]. Among carbapenems, ertapenem and panipenem have limited
use against non-fermentative Gram-negative bacteria but are appropriate for community-
acquired infections. Carbapenems, including imipenem, meropenem, doripenem, and
biapenem, have been widely used in hospital-acquired infections. These carbapenems
are typically reserved for use in patients infected with multi-drug resistant (MDR) bac-
teria, including extended-spectrum β-lactamase (ESBL)-producing and ampicillinase C
(AmpC)-producing bacterial infections [22,23,34], such as complicated intraabdominal and
urinary infections, bloodstream and skin infections, community-acquired and nosocomial
pneumonia, meningitis, and febrile pneumonia [35,39,40].
Carbapenem-resistant bacteria were first described in 1996 with the identification of
carbapenemase-producing Klebsiella pneumoniae [11,23]. In the last decade, the emergence
and spread of carbapenem-resistant bacteria have globally increased. For example, many
infections caused by CRE are mostly seen in patients in hospitals, long-term care facilities,
and long-term acute care hospitals [41–43]. Such infections are high risk for patients using
ventilators, urinary catheters, intravenous catheters, and long-term antibiotic treatment
and for immunocompromised patients [41]. A significant fraction of these infections result
in death due to limited treatment options [1,22,30,44]. Specifically, bloodstream infection
by CRE causes a high mortality rate in pediatric populations [40,45]. The characteristics,
mechanisms, and outcomes of carbapenem-resistant bacteria are thus crucial to prevent
and manage such infections [46].
CRE are Enterobacterales resistant to at least one of the carbapenem antibiotics based
on their antibiotic susceptibility profile (phenotypic definition) [41]. There are different
mechanisms (e.g., genotypic); carbapenem resistance mainly develops when bacteria (1)
acquire structural changes in penicillin-binding proteins (PBPs), (2) show a decrease or loss
of specific outer membrane porins that filter carbapenems from reaching their site of action,
(3) activate the efflux pumps to remove the antibiotics and regulate the intramembrane
environment, and (4) acquire β-lactamases and carbapenemases to degrade or hydrolyze
carbapenems and other β-lactam antibiotics (e.g., penicillins and cephalosporins) [32–34,41].
In addition, carbapenem resistance can be acquired by a combination of CTX-M (activity
against cefotaxime) and AmpC enzymes, allowing low-level carbapenem resistance. Fur-
ther, the combination of the β-lactamase expression and porin gene mutations is associated
with high-level carbapenem resistance, attenuating therapy responses [47].
Overall, CRE can become resistant through chromosomal mutations in the porin gene
(non-carbapenemase-producing CRE) and/or the production of carbapenem hydrolyzing-
enzymes (carbapenemase-producing (CP) CRE) [41]. The presence or expression of the
gene coding carbapenemase is usually sufficient for carbapenem resistance, covering
30% of CRE. Thus, CP-CRE is a subset of all CRE [22,41]. These genes are often on
mobile genetic elements, leading to their rapid spread and resulting in infections and
colonization [1,14,41,48]. Many CRE-colonized individuals do not develop infections;
however, they can still spread the bacteria [41]. Similarly, the transfer of genetic elements
can occur in the food chain and the environment [1,14,41]. Therefore, routine tests for
these carbapenemases through the Antibiotic Resistance Laboratory Network and CDC
laboratories are conducted to prevent and control their emergence and spread [41].
2.1. Carbapenemases
A large variety of carbapenemases have been classified into three groups: Ambler
Classes A, B, and D β-lactamases, based on hydrolytic and inhibitor profiles using active
catalytic substrates of serine or zinc [13,23,32,34,49]. The characteristics of the three most
common classes of carbapenemases are detailed and listed in Table 1 [23,49].
Class A enzymes, serine β-lactamases, hydrolyze a broad variety of β-lactam an-
tibiotics, including carbapenems, cephalosporins, penicillin, and aztreonam [49]. These
enzymes were identified as chromosomally encoded and plasmid-encoded types [49]. Some
of the chromosomally encoded genes are NmcA (not metalloenzyme carbapenemase A),
SME (Serratia marcescencens enzyme), IMI-1 (imipenem hydrolyzing β-lactamase), and
SFC-1 (Serratia fonticola carbapenemase-1). The plasmid-encoded genes are KPC (Kleb-
siella pneumoniae carbapenemase), IMI (Imipenem-hydrolyzing beta-lactamase), and GES
(Guiana extended spectrum) [23,32]. Among these, the KPC type is the most prevalent
enzyme and causes outbreaks in many Asian, African, North American, and European
countries [23,32]. KPC gene is mainly located within a 10-kb length, mobile transposon
Tn4401, frequently established on conjugative plasmids. The link of blaKPC with plasmids
and transposons assists in intraspecies gene transfer and the dissemination of the gene [50].
Several KPC variants have rapidly increased, and 84 KPC alleles have been recorded in
the GenBank database [51]. Of these, KPC-2 and -3 are the most common enzymes world-
wide, and 22 KPC variants have also conferred ESBL-, CTX-M-, or ceftazidime-avibactam
(CZA)-resistance in their gene position. For example, the KPC-2 gene was carried on
the NTEKPC -Ib transposon on plasmids with a 15-bp insertion, which also harbored the
resistance gene, CZA resistance [47,51]. Overall, KPC types are mostly found in Klebsiella
pneumoniae, Klebsiella oxytoca, E. coli, and Serratia marcescens, as well as in Enterobacter,
Salmonella, and Proteus species [13,23,32]. Their rapid spread and diverse variants severely
threaten human health and impact therapeutic efficacy [13,32,51].
Class B enzymes are known as Metallo-β-lactamases (MBL) since they utilize metal
ions (usually Zinc) as a cofactor to attack the enzyme’s active site (β-lactam ring). There
are 10 types of MBLs; the most important ones include New Delhi Metallo-beta-lactamase
(NDM), Verona Integron-Encoded Metallo-beta-lactamase (VIM), and Imipenemase
(IMP) [23,32,41,52]. They hydrolyze all current β-lactam antibiotics, except for monobac-
tams (e.g., aztreonam) [53]. IMP was first reported in Japan in S. marcescens in the early
1990s [13], and over 85 sequence variants have been described [53]. IMP variants are found
in Acinetobacter and Pseudomonas species, as well as in the Enterobacteriaceae family [13,32].

VIM was then identified in P. auregionasa in Verona, Italy, in late 1997, and over 69 variants
have been described [53]. VIM variants are mostly found in Pseudomonas, Acinetobacter, and
Enterobacteriaceae species, which are globally distributed [13,32,53]. Recently, NDM was the
most prevalent MBL, first identified in Klebsiellea pneumoniae and E. coli isolated from a pa-
tient who traveled from India to Sweden in 2008 [13,53]. There have been 29 NDM variants
described, and NDM-1 is the most prevalent type. NDM variants are generally dominant in
Klebsiella pneumoniae, E. coli, Acinetobacter baumannii, and Pseudomonas aeruginosa [32,52,53].
Class B enzymes are usually found in plasmid vectors or other mobile genetic ele-
ments [49]. For instance, IMP and VIM are mostly integron-associated; they are encoded by
gene cassettes within class 1 or 3 integrons that may be embedded in transposons, allowing
insertion into the bacterial plasmids [53]. NDM is not integron-associated; it has been ob-
served in plasmids rapidly disseminated worldwide [52,53]. Additionally, NDM-producing
bacteria can have both NDM-1 and a type IV secretion system (T4SS) gene cluster in plas-
mids, showing high virulence [52]. Further, NDM-producing bacteria may harbor other
carbapenemases in plasmids (e.g., KPC, VIM, and OXA types) and ESBLs [13,41,47,52].
Thus, the emergence of NDM-producing bacteria with increasing variants is a significant
threat to public health.
Table 1. The most common carbapenemases in bacteria with their gene location [23,49,53].
Ambler Class Representative Gene No of Variants Gene Location Bacterial Origins

KPC (Klebsiella pneumoniae carbapenemase) >84 Plasmid K. pneumoniae
GES (Guiana extended spectrum) >27 Plasmid P. aeruginosa
IMI (Imipenem-hydrolysing beta-lactamase) >9 Chromosome E. cloacae
A
SME (Serratia marcescencens enzyme) >5 Chromosome S. marcescencens
SFC (Serratia fonticola carbapenemase-1) >1 Chromosome S. fonticola
NMC-A (not metalloenzyme carbapenemase A) >1 Chromosome E. cloacae
NDM (New Delhi metallo-lactamase) >29 Plasmid K. pneumoniae
VIM (Verona integron-encoded
>69 Plasmid P. aeruginosa
metallo-lactamase)
B
IMP (Imipenemase), >85 Plasmid S. marcescencens
GIM (German imipenemase) >2 Plasmid P. aeruginosa
SIM (Seoul imipenemase) >1 Plasmid P. aeruginosa
D OXA (Oxacillin-hydrolyzing carbapenemase) >40 Plasmid K. pneumoniae
Class D enzymes, serine β-lactamases, are oxacillinase or oxacillin-hydrolyzing en-

zymes (OXA), comprising over 200 enzymes. OXA rapidly mutates and expands its
spectrum activity; the most prevalent carbapenem-hydrolyzing enzymes are OXA-48 and
OXA-181 in over 40 carbapenemase variants [36]. OXA-48 was first identified in Kleb-
siella pneumoniae in Turkey in 2001 [54,55]. Plasmids are the primary genetic elements for
the transmission and propagation of the genes; the most frequent hosts for OXA-48 are
self-conjugative 60- to 70-kb plasmids [55]. Currently, OXA-48 and OXA-101 variants are
mostly dominant in Klebsiella pneumoniae in Turkey, the Middle East, North Africa, and
Europe [13,32,36,55]. However, it should be noted that OXA-producing bacteria often have
low-level resistance due to weak expression, which is risky for false positive detection and
suitable treatment options [55].
The genes coding carbapenemase in β-lactamase (bla) are defined as blaKPC , blaNDM ,
blaOXA-48 , blaVIM , and blaIMP [13,34,56]. These genes are found in many bacteria, such as E.
coli, K. pneumoniae, Salmonella, Acinetobacter, and Pseudomonas. These bacteria are isolated not
only from humans but also animals, food supplies, and water sources worldwide [23,48,57],
detailed in the next section.
2.2. Dissemination of the Carbapenemases in Humans, Animals, Foods, and Environment

Several studies have shown that healthcare settings can lead to the spread of CP
pathogens in humans [23,29,58]. Frequent hospital visits and long-term stays in healthcare
facilities represent a high risk of colonization and infection development with CP bacteria,
particularly with CP-CRE [23,29]. For instance, KPC-producing K. pneumoniae caused
hospital outbreaks in many European countries such as Greece, Italy, Spain, France, and
Germany [59–61]; NDM and KPC-producing K. pneumoniae were identified in transplanted
patients in Brazil [62]; CP-CRE were found to spread in hospital and community settings
in Africa [63–65] and Asia [58,66,67]. Another factor of CP-CRE spread is international
travel and medical tourism [23]. For instance, KPC-producing K. pneumoniae and E. cloacae
were isolated from patients in New York who had recently traveled from France and
Greece [68,69]. In another example, NDM-producing K. pneumoniae and E. coli were isolated
from Sweden and UK patients who recently traveled to India [70,71].
Among CP-CRE, E. coli and K. pneumoniae have been disseminated globally at an
alarming rate in the medical community as critical human pathogens [72,73]. For instance,
KPC-producing K. pneumoniae has been found in more than 100 different sequence types
(STs). Particularly, K. pneumoniae ST258 is predominant and primarily associated with
KPC-2 and KPC-3 production. ST258 comprises two distinct lineages, clades I and II, and
ST258 is a hybrid clonal complex created by a large recombination event between ST11 and
ST442 [73]. Further, ST11, ST340, and ST512 are single-locus variants of ST258 and harbor
carbapenemases. ST11 is closely related to ST258, which is associated with KPC, NDM,
VIM, IMP, and OXA-48 production [73].
Further, carbapenem resistance in pathogenic E. coli is a major concern because of
limited therapy. For instance, E. coli ST131, causing severe urinary infections, has been
linked to the rapid global increase in AMR among E. coli strains [72]. Further, FimH30
lineage and virotype C are the common lineage among ST131, contributing to the spread
of ST131 associated with carbapenemases. ST131 is most likely responsible for the global
distribution of E.coli with KPC, NDM, and OXA-48 production [72]. These sequence types
of E. coli and K. pneumoniae pose a major threat to public health because of their worldwide
distribution [72,73].
Additionally, hospitals or health-care settings are a reservoir for CP bacteria. Car-
bapenem residues in human excreta can get into hospital sewage. Due to the selection of a
low concentration of antibiotics, bacteria in hospital effluent can become resistant to car-
bapenems [23]. Hospital sewage may act as a reservoir for resistance genes, where bacteria
likely acquire resistance through HGT [23,29]. Likewise, antibiotic residues and resistant
genes released into municipal wastewater could contribute to the selection of CRE and
their dissemination to ground and surface water, spreading them to the environment [23].
For example, CP E. coli, E. cloacae, K. pneumoniae, and Citrobacter freundii were found in the
river and hospital sewage in Portugal [74], China [75], Vietnam [76], and Australia [77,78].
VIM- and KPC-producing E. coli were found in seven waste water treatment plants in
the USA [79]; OXA-48 carrying CRE in tap water was found in six states in the USA [78].
In addition, KPC-producing Salmonella was found in human feces, hospital sewage, and
effluent in the USA and Brazil [80,81].
Another possible way of CP bacteria transmission to animals and farms is through
direct contact with colonized hosts (human and animal) and a contaminated environment
(surface water, ground water, soil) [23]. CP bacteria (E. coli, K. pneumoniae, Salmonella,
Acinetobacter, Pseudomonas) have been detected in farm animals, poultry, fish, mollusks,
and wild birds and animals [23,48,57,82–88]. The transmission of CP bacteria also alerts
food safety, particularly CRE in the food-chain. For instance, CP bacteria were isolated in
meat (beef, chicken, pork), seafood (clam, fish, prawn), and vegetables (lettuce, spinach,
Chinese cabbage, roselle) [42,89–94]. These studies showed major carbapenemases (NDM,
VIM, and KPC) present in foods. The presence of CP bacteria in the food chain mainly
contributes to their spread worldwide due to the global food trade, posing a risk to human
health [93].
Various environmental, microbiological, and clinical investigations have shown that

CP-CRE can widely spread in the community, animal and agricultural products, and
the environment [23,38,83,95–97]. For the early detection and optimal management of the
spread and emergence of CRE, some recommendations include (1) the necessity of screening
and rapid diagnostic tools for patients who may have visited countries or hospitals with
frequent infection by CRE, (2) specific policies and prioritizing funding for the control and
management of infections by CRE, (3) clear strategies indicating the use of carbapenems,
and (4) international co-operation to reduce the global spread of CRE [98]. As infections
caused by particularly CRE are a global concern, the rapid detection of the causative
bacteria is of utmost importance [38,46,98].
3. Current and Emerging Detection Techniques of CRE

Diagnostic tests assist in screening or monitoring specific infections or conditions to
control and prevent CRE spread in the community. However, diagnostic AST protocols usu-
ally start with identifying the bacteria species in selective media, followed by growth in the
presence of antibiotics (carbapenem) for determining their antibiotic-resistant profile [1,99].
However, each hour of delay in obtaining a correct diagnosis and appropriate antibiotic
treatment of infections by CRE increases the mortality rate by approximately 8% [100]. For
instance, delayed diagnosis and treatment in CP-CRE raise the mortality risk from 0.9% to
3.7%, hospital cost from ~USD 10,000 to ~USD 25,000, and hospital stay from 5.1 days to
8.5 days [43,101]. Thus, rapid and accurate detection is a significant step in controlling and
preventing such microbial infections. Several culture-based, rapid phenotypic, genotypic
methods, and biosensors have been developed to detect carbapenem resistance, including
carbapenem-hydrolyzing enzymes, detailed in this section with advantages and limitations.
3.1. Culture-Based Methods

The antimicrobial susceptibility testing (AST) is widely used in clinical and public
health laboratories to assess the antimicrobial resistance profiles of target microorganisms.
The standard culture-based AST methods include broth and agar dilution tests, disk
diffusion, and E-tests. These tests, approved by the Food and Drug Administration (FDA),
involve the isolation of pure cultures of the potential pathogens, followed by testing
these bacteria on media with minimum inhibitory concentration (MIC) levels [99,102–104].
Specifically, disk diffusion is a gold standard for AST; bacteria are inoculated on agar
plates with a single antibiotic disk and then incubated to determine the resistant profile.
Among carbapenems, imipenem, meropenem, and ertapenem have been commonly used
for the early detection of carbapenem resistance; ertapenem has been described as the most
sensitive indicator [105]. To determine the susceptible, intermediate, and resistant profile
of tested bacteria, the most widely used standard interpretation of AST and breakpoints
are recommended by the Clinical and Laboratory Standards Institute (CLSI) and European
Committee for Antimicrobial Susceptibility Testing (EUCAST) [106].
AST disk diffusion and E-test combination with specific inhibitors have been used
to differentiate the carbapenem-hydrolyzing enzymes from two main types, KPC and
MBLs [107]. Examples of this are the addition of chelating agents, such as EDTA, in the
broth microdilution and E-test aids in confirming the presence of MBLs with binding zinc
ions and inhibiting MBL activities [49,108]. The sensitivity and specificity of this test are
reported to be >82% and >97%, respectively [107]. Similarly, phenyl-boronic acid (PBA) is
incorporated into the E-test for KPC identification by the inhibition of KPC activity. This
test’s sensitivity and specificity are reported as 92% and 100%, respectively [107]. Multidisc
diffusion tests with inhibitors of specific enzyme types, including clavulanate for ESBL and
cloxacillin for AmpC, are also used to differentiate enzymes [32,107,108].
The modified Hodge test (MHT) was developed to identify the presence of carbapen-
emases [109,110]. The MHT was first introduced in 2010 for detecting carbapenemase
genes and was widely used because of its ability to detect KPC producers. In this method,
the suspected bacteria are inoculated by swabbing a straight line from the edge of the
meropenem disk on Mueller Hilton Agar (MHA) that is pre-inoculated with susceptible E.
coli. The plates are incubated overnight, and the cloverleaf-like zone is observed for CP
isolates. The use of this method was recommended by CLSI in 2009. It has good sensitivity
for other carbapenemases (VIM, IMP, and OXA-48), although its performance in detecting
NDM enzymes was found to be lower [108,110]. Overall, its sensitivity and specificity were
found to be 69% [110] and 93–98% [107], respectively.
The carbapenemase inactivation method (CIM) has been recently introduced by
CLSI (2016) with higher accuracy and accessibility [107,108,111]. This method is initiated
by a suspension of bacteria in a broth and incubation with a meropenem disk (2–4 h); if the
isolate produces the enzymes, the meropenem in the disk is degraded. The disk in the broth
can then be placed on MHA streaked with susceptible E. coli and incubated, which detects
carbapenemase activity with no zone or a narrow zone diameter of <19 nm [107,108,111].
This method showed high concordance with results obtained by a PCR test, which is used
in many clinical and public health laboratories [107,108]. The sensitivity and specificity of
the CIM method were over 95% [107,111].
Specific media have also been designed for CP strain screening [32,112,113]. For
example, Chromogenic Media and Brilliance CRE Agar are used for the initial detection
of CRE strains in colonized and infected patients, with 76.5% sensitivity. CHROM agar
KPC is used to screen for KPC and VIM-producing Enterobacteriaceae, but it can detect
high-level resistance with 43% sensitivity. SUPERCARBA medium is mainly used for
KPC and OXA-48 producers and is applicable to detect low-level resistance with higher
sensitivity (96.5%) [112–114]. ID Carba and Colorex KPC media were designed for CP
Enterobacteriaceae [112]. All these selective media are directly applicable to patient samples;
however, they have lower specificity (>50%) depending on the enzyme type [112,114,115].
The mentioned culture-based methods are cost-effective and widely applicable. Among
these methods, the CIM has a higher sensitivity and specificity in identifying and typing
carbapenemases. However, they are labor-intensive and require time-consuming steps
to isolate pure cultures, taking days to weeks to determine the resistance profile of the
suspected bacteria [99,102,103].
3.2. Rapid Phenotypic Methods

Automated AST systems, which are rapid culture-based methods, have been de-
veloped to shorten the required time to detect antimicrobial resistance [103,105]. For
example, FDA-approved commercial automated instruments are MicroScanWalkAway
and Vitek-1/Vitek-2, which measure bacterial growth in the presence of antibiotics by
recording bacterial turbidity using a photometer [32,103]. Further, BD Phoenix measures
bacterial growth in the presence of antibiotics by recording bacterial turbidity and col-
orimetric changes. Sensititrere records bacterial growth with antibiotics by measuring
fluorescence [103]. Besides imaging-based technologies, automated microscopes, such as
multiplexed automated digital microscopy (MADM) that is FDA approved, single cell-
morphological analysis (SCMA), oCelloscope, Fluorescence microscopy, and cell lysis-based
methods are also used. These automated microscopes measure the phenotypic response,
changes in bacterial growth rate, and the cellular morphology and structure profile of
bacteria in the presence of antibiotics [32,103,104].
Optical techniques have also been developed, which measure the physical and bio-
chemical profile of bacterial cells [116]. For example, forward laser light scatters (FLLS) and
rapid electro-optical technology have been used to measure bacterial numbers by optical
density and to estimate cell density and size using light scattering of the cell particles in a
liquid [99,103,117]. Another optical technique, flow cytometry (FC), is used for cell count-
ing and the detection of a biomarker using changes in morpho-functional and physiological
characteristics of cells [103,118,119]. Additionally, Raman spectroscopic analysis has been
recently used to measure and compare the spectra of bacteria in the presence of antibiotics
to distinguish resistant strains [99,104,120]. Further, several miniaturized lab-on-a-chip
systems have also been fabricated using microfluidic techniques, substituting agar to mea-
sure the growth of pure bacteria in the presence of antibiotics for rapid testing [99,103]. An
ultraviolet (UV) spectrophotometric method was developed to measure the carbapenem
imipenem hydrolysis activity of CP bacteria [121]. Lastly, bioluminescence-based detection
assays (BCDA) have also been developed for CP bacteria based on adenosine triphosphate
(ATP) level differences in culture media. Such assays are rapid (<2.5 h) and accurate, with
higher specificity and sensitivity [122]. However, the applicability of this technique in
matrices is low due to reduced sensitivity [108,121–123].
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-
TOF MS) in optical techniques has recently become popular for identifying pathogens
and resistant bacteria due to its distinct fingerprint spectra [104,118]. To determine the
antimicrobial resistant profile in pathogens, MALDI-TOF MS identifies (1) the antimicrobial-
resistant clonal group (e.g., carbapenem-resistant E. coli), the modified antimicrobial drug
(e.g., carbapenemase activity), the modified antimicrobial target (e.g., lipid A modification),
the direct detection of the AMR determinant (e.g., KPC-2 β-lactamases), and biomarkers co-
expressed with the AMR determinants (e.g., blaKPC carrying plasmid) [124]. This technique
identifies specific resistant profiles (e.g., KPC and MBLs) of bacteria at the species and
genus level from single isolated colonies within 1–4 h, with 72.5–100% sensitivity and
98–100% specificity; however, it has issues regarding OXA-48 identification [40,123,124].
Further, the combination of automation and the implementation of a user-friendly interface
recently made MALDI-TOF MS popular in clinical laboratories [123,125–127].
Colorimetric assays have also been developed as rapid, simple, and cost-effective
phenotypic methods for detecting CP bacteria based on their carbapenemase hydrolytic
activity [60,107,108]. The Carba NP test (2 h) measures the hydrolysis of imipenem, leading
to changes in pH and resulting in a color change from red to yellow/orange. The sensitivity
of this test was found to be 73–100% for most carbapenemases, but it performed poorly in
the detection of the OXA-48 enzyme [60,107,108]. The Carba NP test has been recommended
for use as a first-line test for screening carbapenemase activity by the CLSI in the US.
The RAPIDEC Carba NP test (first commercial test), β-CARBA test, Rapid CARB screen,
Rapid Carb Blue kit, and Neo-CARB kit have been used to detect CP bacteria within 2 h,
with varying sensitivity (>70%) and specificity (>89%) from the pure culture [108,123,128].
However, these assays require pure cultures and are dependent on the growth rate of the
bacteria [123].
Many of these rapid phenotypic techniques and automated systems still require
pure cultures; thus, sample preparation and pre-treatment steps require several hours to
days [116,123]. Last but not least, these techniques require costly equipment, complex data
analysis, and skilled personnel, which limits their applicability in low-resource setting
laboratories [99,103,104,118].
3.3. Genotypic Methods

Molecular AST methods are effective techniques to detect specific resistant genes in
a short time from matrices without the need for a tedious bacterial culture and a long
incubation time [116,123]. Among these, PCR-based methods, DNA microarray and chips,
whole genome sequencing (WGS), loop-mediated isothermal amplification (LAMP), and
fluorescence in situ hybridization (FISH) methods are the main techniques used for the
detection of the antimicrobial-resistant profile [99,104,118].
PCR-based methods are among the most efficient and widely used rapid molecular
tools to quantify and profile genes encoding resistance in species and genus levels. This
method amplifies the target nucleic acid sequence using specific primers that anneal to
single-stranded DNA after denaturing the target DNA at a high temperature [129,130].
Advancements in PCR offer a more rapid and robust variation of this technique, such as
real-time or quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), digital PCR,
multiplex PCR (mPCR), and automated PCR. For instance, mPCR offers the advantage
of the simultaneous detection of multiple resistant genes through the use of multiple sets
of primers [99,118,123,131]. Real-time PCR, or qPCR, allows for the rapid simultaneous
detection and quantification of amplified PCR products using fluorescent dyes, eliminating
gel electrophoresis [99,132,133]. Automated systems of PCR or qPCR are commercially
available and automatically purify the sample, concentrate DNA, and amplify and detect
major bacterial genes, confirming antibiotic resistance in less than two hours [99,134].
For two decades, PCR-based techniques have been used as the gold standard for the
detection of β-lactam resistant genes in Enterobacteriaceae. For example, multiplex PCR was
developed to detect 11 acquired genes encoding carbapenemase (blaIMP , blaVIM, blaNDM ,
blaSPM , blaAIM , blaDIM , blaGIM , blaSIM blaKPC, blaBIC , and blaOXA-48 ) using three different
multiplex reaction mixtures [108,123,135]. Several automated systems were also developed
to identify the target genes [108,123,135]. Real-time multiplex PCR or qPCR systems allow
a combination of amplification and detection in a single step, limiting contamination risks.
GeneXpert is an automated real-time PCR platform that uses the Carba-R assay and can
detect and quantify numerous bacterial species and several carbapenemase genes from
rectal samples [123,136]. The Check-Direct assay has a panel of different multiplex real-
time PCR kits using several probes, including narrow and broad-spectrum B-lactamase
genes [108,123,137]. A broad range of multiplex PCR panels was developed to increase their
analytical performance without requiring skilled personnel. However, these PCR-based
tests are expensive, limiting their use in low-resource laboratories [123].
Other molecular methods such as FISH, microarray, WGS, and LAMP assays have
also been used for detecting carbapenem resistance [99,118,138]. FISH is a technique
for detecting specific RNA or DNA sequences using dye-labeled oligonucleotide probes
visualized by fluorescence microscopy [139]. Microarray-based methods utilize multiple
spots on a solid support chip for different oligonucleotides corresponding to resistant genes
to detect labeled DNA fragments in a single assay [140]. In the whole genome sequencing
(WGS) technique, a whole bacterial sequence is screened for antibiotic-resistant genes and
compared with known genes in publicly available databases, allowing the prediction of
existing and emerging phenotypic and genotypic resistance [141]. Lastly, the LAMP assay
is a simple amplification technique that resolves PCR temperature cycling using a single
temperature for target gene amplification. This method produces a large number of DNA
copies in a short period [142,143]. LAMP has been used as an alternative to PCR due to its
simplicity and cost-effectiveness, especially in low-resource setting laboratories. However,
the technique still requires a complex primer design [99,104,138].
Emerging molecular techniques and automated systems have been improved to
reduce costs and the detection system for β-lactam resistant genes [108,123]. Luminex tech,
for example, is a well-established approach based on a colored microsphere-based flow
cytometry assay. The method can detect specific alleles, antibodies, or peptides from a single
colony [144]. The multiplex oligonucleotide ligation-PCR procedure assists in detecting
β-lactam resistant genes and their variations with higher sensitivity and specificity (100%
and 99.4%) within 5 h [123]. Further, the LAMP method, using hydroxy naphtol blue
dye (LAMP-HNB) and microarray techniques, detects genes encoding carbapenemase
with higher specificity and sensitivity at 100% and >90%, respectively [99,108,142,143].
Multiplexed paper-based Bac-PAC is another assay used to categorize the AMR profile of
individual strains of CRE by providing a colorimetric readout [145]. The RNA-targeted
molecular approach, NucliSENS EasyQKPC test, has also been used for detecting blaKPC
variants within 2 h, at a 93.3% sensitivity and 99% specificity [146]. Another technique,
PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS),
has been used to accurately measure exact molecular masses. With advanced software,
the sequence of DNA fragments is reconstructed for accurate identification as well as
subtyping of the resistant genes. This technique has been used to identify blaKPC genes
directly from clinical samples in 4–6 h [147]. Lastly, whole genome sequencing methods
have been used as the most reliable technique to detect carbapenemase, but the high cost,
longer turn-around time, and complex data management limit their use [99,108,148].
Genotypic methods generally offer key advantages, including higher sensitivity and
specificity in a short time, increasing their real-world applicability. However, these methods
require costly reagents and equipment and need skilled operators [99,103,116,149]. In
addition, their sensitivity and selectivity can be affected by specimen debris, resulting
in the inhibition of the reaction or false positives [99,123]. Another limitation of many
molecular assays is that only known genes can be targeted; phenotypic resistance may
be missed by molecular assays, but WGS can help discover novel genes [99,104,116,129].
Further, the limited number of targeted genes is a challenge in molecular tests due to the
diversity of carbapenemase-encoding genes. Thus, the target gene is mainly based on the
most relevant variant in each geographical area. For example, several commercial kits
stated have been developed to detect blaKPC , the most prevalent carbapenemase in the USA,
and may not be used for other genes [123].
3.4. Rapid Serological (Immunological) Methods

Immunological assays rely on antibody–antigen reactions to detect bacteria, providing
rapid results at a moderate cost [150]. A few methods, including latex agglutination and
immunochromatographic assays, have generally been used for the detection of methicillin-
resistant Staphylococcus aureus (MRSA) [99]. Enzyme-linked immunosorbent assay (ELISA)
has also been used to detect either genes or proteins by the combination of the PCR am-
plification of samples [99,138], which aids in detecting MRSA and CRE [99]. Additionally,
a lateral-flow immunochromatographic assay, OXA-48K-SeT, was designed based on the
immunological capture of two epitopes that are specific to OXA-48 variants, using col-
loidal GNPs in 15 min. It has 100% sensitivity and specificity with a detection limit of
106 CFU/mL [150]. In another study, a lateral-flow immunochromatographic assay was
used to detect the carbapenem-resistant gene, blaOXA-23-like , using multiple cross displace-
ment amplification from pure culture as well as clinical sputum samples [151].
Further, a multiplex immunochromatographic test, ICT RESIST-4 O.K.N.V. K-SeT,
uses monoclonal antibodies to rapidly detect OXA-48 variants, KPC, NDM, and VIM
carbapenemases. This assay has 99.2% sensitivity and 100% specificity from pure culture
on Mueller Hilton Agar (MHA) [152]. The immunological assays depend on the level of
protein production; accurate results require an enrichment of 18 h [150,152]. However,
the diversity of carbapenemase can prevent further developments since initially designed
antibodies may not be applicable for targeted antigenic site modification [123]. Although it
is a rapid test, it is still costly [123,152], with lower sensitivity and specificity in complex
matrices [99,123,138].
3.5. Biosensing Techniques

Biosensors, as analytical devices, have emerged as alternative techniques for sim-
ple, rapid, cost-effective, and reliable pathogen detection. Biosensors utilize biological
or chemical reactions and convert the recognition event into measurable signals for the
detection of the target analyte [153,154]. Biosensor types are classified based on their
data output system, target analyte, and label dependence [155]. Mainly, biosensors are
classified as thermal, mechanical, electrochemical, and optical based on their operating
mechanism [118]. Several biosensor applications, particularly electrochemical and opti-
cal biosensors, are well documented, but few studies have been developed for antibiotic
resistance, especially for carbapenem-resistant bacteria. Biosensor platforms often share
the same mechanism, advantages, and disadvantages, and for the purpose of this brief
overview, popular electrochemical and optical biosensors are elaborated on. Examples of
antibiotic-resistant detection studies are discussed.
Electrochemical biosensors utilize the electrical response of bacterial cells; the im-
mobilized bio-recognition element on an electrode interacts with the target, resulting in
an electrical signal. Various recognition elements (antibody, phage, aptamer, DNA, etc.),
nanoparticles, and signal processing techniques have been used [118,155]. For example,
DNA-based biosensors typically use single-stranded DNA immobilized on an electrode
with a sequence complementary to the target DNA. The difference between the electrical
properties of single-stranded DNA and hybridized double-stranded DNA assists in the
detection of the specific target using cyclic voltammetry [156]. Numerous electrochemical
biosensors have been used to detect antibiotic resistance. Examples include a study that
combined nitrogen-doped graphene with GNPs to detect the human multidrug-resistant
gene MDR1 [157]. In another study, an electrochemical DNA-sensing system identified
MRSA based on a MNP/DNA/AuNP hybridization complex using a chronoamperometric
signal [158]. Another electrochemical sensor utilized an antibody conjugated with MNPs
to detect MRSA from nasal swabs [159]. A label-free electrochemical biosensor detected a
PCR amplified blaNDM gene in carbapenem-resistant Citrobacter freundii using impedance
spectroscopy [160]. In another study, blaKPC detection was achieved in K. pneumoniae and E.
coli using voltammetry techniques and sandwich hybridization assays in 45 min at a level
of 104 CFU/mL [43].
Optical biosensors are widely used platforms for bacterial detection and rely on
measuring absorbance, fluorescence, Raman scattering, surface plasmon resonance (SPR),
and colorimetry [118]. These biosensors are highly sensitive but can be costly. Surface
plasmon resonance (SPR) is the most commonly used assay, which utilizes refractive index
measurements due to the excitation of the surface plasmon waves by the interaction of an
analyte with its ligand [161]. The technique mainly uses antibodies or DNA as a recognition
element. For example, the immobilized single-stranded DNA sequences on the surface bind
to their complementary sequence upon hybridization, resulting in a change in plasmon
resonance [161]. Raman scattering techniques are also common and measure molecular vi-
brational, rotational, and low-frequency modes, providing characteristic information about
carbohydrates, lipids, proteins, and nucleic acids [118,120,138]. However, they require
a higher bacterial concentration and are limited in differentiating closer spectral signals.
Recently, the Surface-Enhanced Raman Scattering technique (SERS) has been developed
using nanoparticles that enhance Raman signals [120]. This assay can differentiate strains
of carbapenem-resistant and susceptible E.coli using silver nanoparticles [120], gold nanos-
tars [162], and gold and silver nanorods [163] by comparing their SERS spectral signature
with higher specificity and sensitivity. However, these optical biosensors require complex
and multivariate data analysis.
The performance of these optical and electrochemical biosensors often depends on the
detection limit, sensitivity, specificity, reproducibility, interference response, response time,
storage, and operational stability [153,154]. These platforms are highly rapid and often
sensitive to their target bacteria. They also reduce or eliminate isolation and culture times,
allowing direct measurements from clinical and biological samples [118,138]. However,
sensitivity at low-level bacterial loads is still challenging for many biosensor platforms,
along with their costly and complex techniques for signal measurements and analysis [118].
Plasmonic biosensors that allow colorimetric detection are noteworthy; they offer
rapid and simple visual detection within one hour without the necessity of complex
and costly equipment [164–166]. For example, a study used a plasmonic nanosensor for
the colorimetric detection of CP pathogens using gold nanoparticles (GNP) based on
carbapenemase activity and pH changes [167]. Here, the GNPs changed color in response
to pH and turned to purple, blue, or gray, from red, within 15 min. CRE was detected at a
concentration of more than 105 CFU/mL directly from urine and sputum samples within
2.5–3 h. The results were easily distinguished visually and confirmed quantitatively using
vis-NIR spectroscopy [167]. Further, DNA-based plasmonic biosensors using GNPs have
extensively been used to detect target bacterial DNA. For instance, thiol-capped GNPs were
used to detect Klebsiella pneumoniae within one hour using an amplified K2A gene [165]
and the unamplified DNA of uropathogenic E. coli [168]. Dextrin-coated GNPs were used
earlier to detect the unamplified DNA of E. coli O157:H7 [164], E.coli [169], Salmonella
Enteritidis [170], and Pseudoperonospora cubensis [171] within 30 min. Further, dextrin-
coated GNPs have recently been used to detect KPC-producing bacteria (~103 CFU/mL)
from clinical isolates with 79% sensitivity and 97% specificity [172]. Plasmonic biosensors
allow the detection of pathogens and resistant bacteria in a short time without complex
and costly equipment requirements. However, further attention is needed to detect the
resistant genes from clinical and biological samples to improve their accessibility and
applicability [164,172].
4. Surveillance Systems for Control of Antimicrobial Resistance

The WHO initiated the Global Antimicrobial Resistance and Surveillance System
(GLASS) in 2015 to strengthen knowledge and develop strategies against AMR [173]. The
GLASS has supported a standardized approach for collecting, analyzing, and sharing data
regarding antimicrobial resistance at global, national, regional, and local levels. The system
provides surveillance approaches with epidemiological, clinical, and population-level data.
It incorporates data on AMR in humans, antimicrobial medicines, and AMR in the food
chain and the environment [173].
The GLASS has partnerships with the WHO AMR Surveillance and Quality Assess-
ment Collaborating Centers Network (WHO AMR Surveillance CC Network) [173]. This
network has a strong collaboration with AMR regional networks, such as the Central Asian
and European Surveillance of Antimicrobial Resistance (CAESAR), the European Antimi-
crobial Resistance Surveillance Network (EARS-Net), the Latin American Network for
Antimicrobial Resistance Surveillance (Rede Latinoamericana de Vigilancia de la Resisten-
cia a los Antimicrobianos (ReLAVRA)), and the Western Pacific Regional Antimicrobial
Consumption Surveillance System (WPRACSS) [173].
The CDC also tracks AMR threats and collects data on human infections, pathogens,
and risk factors with domestic and international partners. This allows for strengthening
and sharing among networks of the collected data submitted to WHO [56]. AMR tracking
systems of the CDC are the National Antimicrobial Resistance and Monitoring Systems
for Enteric Bacteria (NARMS) and the Antibiotic Resistance Laboratory Network (AR Lab
Network). The NARM was established in 1996 by the CDC, FDA, and the United States
Department of Agriculture (USDA) in partnership with the government to track antibiotic
resistance in pathogens from humans, retail meats, and food-producing animals [56,174].
The AR Lab Network was established in 2016, which supports lab testing in healthcare,
community, food, and the environment (e.g., water and soil) [56]. The Global Antimicrobial
Resistance Laboratory and Response Network (Global AR Lab and Response Network) of
the CDC was established in 2021 to improve the detection of existing and emerging AMR
threats in humans, foods, animals, and the environment globally [56].
The CDC tracks resistant bacteria (Salmonella, Shigella, Campylobacter, Vibrio, and E.
coli O157) in infected patients [56]. The FDA checks retail meats from grocery stores
(chicken, ground beef, ground turkey, pork, shrimp, tilapia, and salmon) for Salmonella,
E. coli, Campylobacter, Vibrio, Enterococcus, and Aeromonas. The USDA, along with FSIS
and Agricultural Research Services (ARS), tracks Salmonella, E.coli, Campylobacter, Vibrio,
and Enterococcus in food animals at slaughter (chickens, turkeys, cattle, swine) [56,174].
The CDC tests these specified bacterial isolates to determine their resistance profile and
routinely tests 12 classes of antibiotics depending on the bacterial type. Among the antibi-
otics, aminoglycosides, penicillin, carbapenems, macrolides, β-lactam combination agents,
cephems, and tetracyclines are commonly used for AST tests. Recently, carbapenems have
been added to the list to test the AMR profile in Salmonella, Shigella, E.coli, and Vibrio [56].
Due to the major threat of CP-CRE infections and the colonization to public health with
global economic and security implications, their rapid diagnostic surveillance is of utmost
importance [22,56,175].
The clinical laboratory improvement amendments (CLIA) monitor the regulation of
laboratory testing and the certification of clinical laboratories by the Center for Medicare
and Medicaid Services (CMS) before any diagnostic testing [176]. Here, CDC plays a role in
providing analysis and research protocols, developing technical standards and laboratory
practice guidelines, monitoring proficiency testing practices, etc. [176]. Clinical laboratories
are to rapidly provide reliable laboratory data to healthcare providers and determine
the cause of infections to implement appropriate treatments. In clinical laboratories, the
identification of these infections has been conducted by culture or culture-independent
diagnostic tests (CIDT) using commercial antigen-based or DNA-based methods [177,178].

Culturing methods have been used to obtain isolates forwarded from clinical laboratories
to public health laboratories for additional testing, such as resistant profiles, serotyping,
and DNA fingerprints [178].
Healthcare and clinical laboratories establish protocols that rapidly notify the health
department, healthcare provider, and infection control staff and work with public health
departments on the submission of specimens for testing [179]. For example, clinical labora-
tories screen their resistance profile with ASTs, phenotypic carbapenemase tests (CIM, or
CarbaNP test), MALDI-TOF tests for fingerprints, and PCR-tests to detect carbapenemase
genes. The isolates can then be sent to public health laboratories to confirm the identity of
bacterial species and to perform additional susceptibility and genomic testing to character-
ize the isolates. In case of unusual resistance, the isolates can further be sent to regional
laboratories for additional testing. This allows for detecting existing and emerging types
of antibiotic resistance, tracking resistance changes, identifying outbreaks, and generating
stronger data to protect against future resistance threats [179].
The surveillance programs also aid in preventing and controlling possible future
outbreaks from food and water sources. Thus, foodborne pathogens and their resistant
profile, including CP bacteria, are tracked in many countries. However, there is now a
need to monitor non-animal products, such as fresh vegetables and fruits. There is also
an emerging need to monitor nonpathogen bacteria that can be a reservoir for antibiotic-
resistant genes [35,96]. Thus, the rapid identification of CP organisms, regardless of their
pathogenicity in biological samples and its implementation of the surveillance program, is
essential to prevent and control possible future endemics or pandemics.
5. Gaps in Detection Technology

Several phenotypic and genotypic methods and biosensors have been developed to
detect pathogens and antimicrobial-resistant bacteria with advancements in automation and
nanotechnology. All these techniques have advantages and disadvantages in cost, rapidity,
simplicity, reliability, and applicability (Table 2). There have been new emerging phenotypic,
genotypic, and biosensor techniques to detect carbapenemases in a simple and cost-effective
manner. However, these detection techniques need to be developed further by improving
their sensitivity, specificity, and testing on clinical and biological samples to increase their
real-world applicability and accessibility. The plasmonic biosensors typically do not require
highly trained personnel, as is usually the case with conventional and rapid phenotypic
and molecular techniques. This enhances their applicability in low-resource settings for
on-site detection [164,172]. Since the estimated material cost of the plasmonic biosensor
is as low as ~USD 2 per test [172], while rapid molecular and phenotypic tests range
USD 23–150 and USD 2–10, respectively [123]. Such rapid and cost-effective techniques
as screening/diagnostic tests should be implemented in clinical and public health and
agricultural and food testing laboratories, especially in low-resource laboratories.
In addition to concerns about accessible detection assays, pre-analytical sample pro-
cessing, including the separation, enrichment, and purification of bacteria from biological
and clinical matrices, is often a significant challenge before detection. Therefore, the
concentration of bacteria is essential to ensure sufficient numbers of cells for rapid de-
tection [100,180,181]. Current bacterial separation techniques in biological and clinical
matrices include physical methods, such as centrifugation and filtration, and chemical and
biological methods, such as dielectrophoresis, metal hydroxides, and magnetic nanopar-
ticles, detailed in the literature [180–182]. However, separation techniques have not been
extensively documented for resistant bacteria. While some examples exist for separating
resistant bacteria from pure cultures [159,183–185], data are scarce for biological samples.
This could be related to the fact that the antibiotic-resistant bacteria profile has more com-
monly been tested on pure cultures after retrieving and identifying them. Therefore, the
rapid extraction of AMR bacteria directly from clinical and biological samples is needed for
their rapid detection.
Table 2. The advantages and limitations of the current and emerging detection techniques for the most common carbapenemases (carbapenem-hydrolyzing
enzymes).
Techniques Advantages Limitations

Culture-based methods Simple and cost-effective Time-consuming (>24 h)
Insufficient for OXA-48
1. Improved AST tests: E-test or disk diffusion test [32,107,108] Detect KPC and MBLs with good sensitivity (>82%) and specificity (>95%)
Require specific reagents and pure culture
Insufficient for MBLs
2. Modified Hodge Test (MHT) * [108,110] Detects KPC with good sensitivity (>69%) and specificity (>90%)
Requires pure culture
Detect all carbapenemases with
3. Carbapenem-inactivation methods (CIM) * [107,108] Require pure culture
higher sensitivity (>90%) and specificity (>95%)
4. Selective media: SUPERCARBA, Colorex KPC, ID Carba, CHROM agar Detect carbapenemases from direct patient samples
Variable sensitivity (40–96.5%) and specificity (>50%)
KPC, etc. [112–114] SUPERCARBA has higher sensitivity (>96.5%)
Rapid phenotypic methods Rapid (<24 h) Costly equipment
Detect carbapenemases with good sensitivity (>70%) and specificity (>80%)
Insufficient for OXA-48
1. Colorimetric assay: CarbaNP test and its automated kits * [60,107,108] Simple, rapid (<2 h), and cost-effective
Require pure culture
No equipment requirement
Rapidly (1–4 h) detects KPC and MBLs with good sensitivity (>72.5%) and Requires data analysis
2. MALDI-TOF MS * [123,125,126] specificity (>95%) Insufficient for OXA-48
Low-measurement cost and simple Requires single isolated colonies
3. Emerging techniques: BCDA, FC, microfluidic techniques, and Raman Simple and rapid (<4 h) Lower applicability on specimens
spectroscopic techniques [116,119,120,122,123] Good sensitivity (>80%) and specificity (>90%) from pure culture Insufficient work on carbapenemases
Genotypic methods Rapid and highly specific (>90%) and sensitive (>90%) Costly and complex equipment
1. PCR-based methods: qPCR, RT-PCR, mPCR, automated PCR (Xpert Gold standard and rapid (<4 h) High technical requirements and specific reagents
system, Check-Direct, and Carba-R-assay) [123,131,135] * Detect and type all carbapenemases directly from specimens High measurement cost
Simple and moderate cost
2. Loop-mediated isothermal amplification (LAMP) [123,142] Specific reagents and complex primer design
Applicable in low-resource settings
Longer turn-around time
3. Whole genome sequencing (WGS) [123,141] * Discovers a new resistance mechanism
Complex data management
4. Emerging techniques: FISH, microarray techniques, PCR-ESI-MS, and Rapid (<6 h) Require specific equipment and reagents
NucliSENS EasyQKPC [116,123,143] Detect carbapenemases Insufficient work on carbapenemases
Immunological Methods
Rapid and moderate cost
Enzyme-linked immunosorbent assay (ELISA), an Complex and difficult antibody design due to antigenic site modification
Poor sensitivity and specificity directly from specimens
Immunochromatographic assay [99,123,138,151]
Biosensors: Emerging Technology Rapid, Simple, and Cost-effective Specific Equipment
1. Electrochemical assays: Impedimetric, potentiometric, and Require equipment for signal processing and data analysis
Detect carbapenemases
voltammetric [43,156,160] Insufficient work on AMR and carbapenemase detection from pure culture
Moderate cost
2. Optical assays: Raman scattering, SPR, and SERS [118,120,138,161] and specimens
Rapid, simple, and cost-effective
Insufficient work on AMR and carbapenemase detection from pure culture
2.1. Plasmonic biosensors [167,172] Detect carbapenemases with good sensitivity (78%) and specificity (97%)
and specimens
No equipment requirement
* Techniques have been used in diagnostic laboratories (clinical and public health laboratories). AST: antibiotic susceptibility test, MALDI-TOF MS: matrix-assisted laser des-
orption/ionization time-of-flight mass spectrometry, BCDA: bioluminescence-based detection assays, FC: flow cytometry, FISH: fluorescence in situ hybridization, PCR-ESI-MS:
PCR amplification coupled with electrospray ionization mass spectrometry, NucliSENS EasyQKPC: RNA-targeted molecular approach, SPR: surface plasmon resonance; SERS:
Surface-Enhanced Raman Scattering technique.
Among the separation techniques, chemical and biological separation processes have
become popular because of their speed, simplicity, and cost-effectiveness. Within these,
magnetic nanoparticles (MNPs) have commonly been used to rapidly and effectively ex-
tract bacteria from food and clinical samples without centrifugation and filtration [186,187].
MNPs draw attention because of their low-cost, stability, benign nature, biocompatibility,
and functionalization with recognition moieties [149,180,186,187]. Such as many chemical
and biological separation techniques, MNP-bacterial cell adhesion relies on cell surface char-
acteristics, such as surface charge, hydrophobic or hydrophilic interactions, and antibody
or lectin binding sites [180,188,189]. Therefore, cell surface characteristics are important in
understanding cell adhesion mechanisms for bacterial extraction and detection.
Cell surface characteristics of the resistant bacteria were investigated in several stud-
ies. For instance, some studies showed that the biochemical components of antibiotic-
resistant cells are different from those of susceptible bacteria. Raman spectrum is one
example where bacterial differentiation or detection was achieved using unique fingerprint
patterns [190,191]. Further, various studies have shown that alteration in the biosynthesis of
cell wall material, membrane components, and cytoplasmic contents can result in changes in
cell surface characteristics, including cell morphology and surface charge [192–196]. These
cell morphological characteristics are utilized for detecting the bacterial-resistant profile
using AST techniques [102–104]. In addition, cell surface charge characteristics are mostly
utilized for studying cell attachment, bacterial capture, and detection [194,195,197,198].
Research is still ongoing on understanding the cell characteristics of antimicrobial-resistant
bacteria to discover new potential factors. The cell surface properties of carbapenem-
resistant bacteria, in particular, have not been documented well. The changes in cell surface
characteristics can impact their adhesion or attachment to substrate surfaces depending
on their interactions with the environment [194]. Therefore, cell surface characteristics
need further attention in relation to cell adhesion mechanisms for developing or improving
rapid extraction and detection assays.
6. Conclusions and Future Perspective

The emergence and spread of carbapenem-resistant bacteria are a global health issue.
Even though carbapenems are used in human medicine, several studies showed that
carbapenem-resistant bacteria are found in humans, food-producing animals, foods, water
sources, etc., due to the rapid dissemination in the complex web, affecting the health of
people. There have been continuing efforts to develop rapid and cost-effective detection
methods to prevent and control their spread in the community. Current rapid phenotypic
and genotypic methods often require pure culture, costly and complex equipment, and
skilled personnel. Immunological and conventional biosensor assays offer rapid and cost-
effective detection, but they still require complex techniques for signal measurements and
analysis. Recently, plasmonic biosensors have shown promise as a cost-effective, rapid, and
simple detection technique by eliminating complex and costly equipment. The biosensors
need further attention for their increased applicability and accessibility, especially in low-
resource settings.
For rapid and cost-effective detection, simple and rapid bacterial separation plays a
major role. The current separation techniques on bacteria need further investigation on
their effectiveness for resistant bacteria. In addition, cell surface characteristics affect their
separation and cell attachment properties. Overall, further studies are needed to enlighten
their cell surface characteristics, bacterial attachment, and separation techniques to develop
rapid and cost-effective detection assays. These assays can assist as screening or diagnostic
tests in low-resource settings.
Author Contributions: O.C.-A. and E.C.A. had the idea for the article and suggested the review
topic. O.C.-A. participated in the collection of data from the literature, critiqued the literature, and
drafting the manuscript. The final manuscript was prepared with contributions from all authors. All
authors have read and agreed to the published version of the manuscript.
Funding: Research related to this article was supported by the Targeted Support Grant for Technology
Development (TSGTD), Michigan State University Foundation, the USDA Hatch project 02782, and
the USDA-NIFA project 2022-67017-36982.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Saad Sharief for many helpful discussions and manuscript reviews.
Furthermore, the Turkish Ministry of Education sponsored O. Caliskan-Aydogan’s Ph.D. program at
Michigan State University.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

1 Department of Biosystems and Agricultural Engineering, Michigan State University,

Received: 3 April 2023

Microorganisms 2023, 11, 1491. https://doi.org/10.3390/microorganisms11061491 https://www.mdpi.com/journal/microorganisms

1.1. Development of Antibiotic Resistance and Their Mechanisms

Microorganisms 2023, 11, 1491 3 of 26

AMR causes threats to anyone regardless of age, to immunocompromised people,

2. Urgent Threat of Infections by Antimicrobial Resistant Bacteria:

in Acinetobacter and Pseudomonas species, as well as in the Enterobacteriaceae family [13,32].

Ambler Class Representative Gene No of Variants Gene Location Bacterial Origins

Class D enzymes, serine β-lactamases, are oxacillinase or oxacillin-hydrolyzing en-

2.2. Dissemination of the Carbapenemases in Humans, Animals, Foods, and Environment

Various environmental, microbiological, and clinical investigations have shown that

3. Current and Emerging Detection Techniques of CRE

3.1. Culture-Based Methods

3.2. Rapid Phenotypic Methods

3.3. Genotypic Methods

3.4. Rapid Serological (Immunological) Methods

3.5. Biosensing Techniques

4. Surveillance Systems for Control of Antimicrobial Resistance

diagnostic tests (CIDT) using commercial antigen-based or DNA-based methods [177,178].

5. Gaps in Detection Technology

Techniques Advantages Limitations

6. Conclusions and Future Perspective

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