Carbapenemase-producing Enterobacteriaceae in animals and methodologies for their detection

Abstract

Carbapenemase-producing bacteria are difficult to treat and pose an important threat for public health. Detecting and identifying them can be a challenging and time-consuming task. Due to the recent rise in prevalence of infections with these organisms, there is an increased demand for rapid and accurate detection methods. This review describes and contrasts current methods used for the identification and detection of carbapenemase-producing bacteria to help control their spread in animal populations and along the food chain. The methods discussed include cultures used for screening clinical samples and primary isolation, susceptibility testing, culture-based and molecular confirmation tests. Advantages and disadvantages as well as limitations of the methods are discussed.

Introduction

Carbapenems are a class of antibiotics that have a wide spectrum of activity against both Gram-positive and Gram-negative bacteria. They belong to the family β-lactams, which is also comprised of penicillins, cephalosporins, and monobactams. The first carbapenem, thienamycin, was discovered in 1976 as a natural product of Streptomyces cattleya (1). Its structure served as a basis for the subsequent development of more stable and effective semisynthetic carbapenems (Figure 1). The first of these new carbapenems is imipenem (Figure 2b), which presents a high affinity for penicillin-binding proteins (PBPs), although it must be coadministered with an inhibitor of dehydropeptidase-I to avoid the nephrotoxic effects of its degradation product (2).

Figure 1.

Structure of thienamycin, the first described, naturally produced carbapenem [adapted from (2)].

Figure 2.

Base structure of carbapenemase with a — β-lactam ring shown in blue, variable R₁ region present in all carbapenemases shown in red, and variable R₂ region present in meropenem, doripenem, and ertapenem shown in green; b — R₁ group for imipenem; and c — R₁ and applicable R₂ groups for meropenem, ertapenem, doripenem, and biapenem. Molecular structures were adapted from (2).

Following the development of imipenem, further advancements were made in the production of more stable synthetic carbapenems, such as meropenem, ertapenem, doripenem, and biapenem (Figure 2c) (2). While these new synthetic antibiotics have an additional methyl group, making them more stable, they have the same basic structure and mechanism of action (Figure 2a) (3). Carbapenems tend to be unstable in aqueous solution and have a short shelf life when not stored as dry powder (4), which may not only have clinical and pharmacological implications, but could also affect their use for research and diagnostic purposes.

Four main mechanisms have been described that confer resistance to carbapenems in Gram-negative bacteria. These include the production of carbapenemases, synergy between other β-lactamases and porin modifications, efflux pumps, and modifications to PBPs. These form the basis for the distinction between the broad category of carbapenem-resistant Enterobacteriaceae (CRE), which can be resistant to carbapenems by any of these mechanisms, and the more specific group of carbapenemase-producing Enterobacteriaceae (CPE or CP-CRE).

Carbapenemase-producing Enterobacteriaceae are of particular concern and epidemiological relevance because carbapenemase genes are frequently located on mobile genetic elements, such as transposons, integrons, and plasmids, and can be transferred horizontally between cells (5), while the other carbapenem resistance mechanisms are less prone to such transfer. The active transfer of carbapenemase-encoding plasmids among bacterial strains, species, and genera has been documented repeatedly, both locally as part of hospital outbreaks (6,7) and more broadly at the regional or national level (8). As a result, the 2015 guidelines of the Centers for Disease Control recommended that bacterial isolates be screened specifically for CPE and not only for CRE (9).

As with other β-lactamases, carbapenemases hydrolyze the β-lactam ring of penicillins, but they also hydrolyze cephalosporins, monobactams, and carbapenems (10). Carbapenemases belong to 3 Ambler classes: Class A — serine carbapenemases; Class B — metallo-β-lactamases (MBL); and Class D — OXA β-lactamases (oxacillinases) (11,12). Class A consists mainly of non- metallo-carbapenemase (NMC), non-metallo-carbapenemase-A/imipenem resistant (IMI), Serratia marcescens enzyme (SME), Klebsiella pneumoniae carbapenemase (KPC), and Guiana extended-spectrum (GES) (12). Class B consists primarily of β-lactamase active on imipenem (IMP), Verona integron-encoded metallo-β-lactamase (VIM), and New Delhi metallo-β-lactamase (NDM) (12). Finally, the main carbapenemases of the oxacillinase group are part of the OXA-48-like β-lactamases (13).

Carbapenemases can be encoded on either a plasmid or the chromosome (10,11). Although not formally carbapenemases, AmpC β-lactamases, such as CMY (class C) and some extended-spectrum β-lactamase (ESBL), can also cause carbapenem resistance, especially when combined with other resistance mechanisms, such as porin loss or efflux mechanisms (10,12). Porin modifications can cause a decrease in the diffusion rate of the antibiotics across the Gramnegative outer membrane at a rate that allows ESBLs and AmpC enzymes to sufficiently break down the remaining antibiotic to result in a fully resistant phenotype (14). Modifications in efflux pumps can cause an increase in antibiotic expulsion from the cell before the drug is able to bind to PBPs, thus rendering it incapable of harming the cell (14). Finally, modifications within the PBPs themselves prevent proper binding of the β-lactams to the targeted proteins, rendering them useless (14).

This diversity of carbapenem resistance mechanisms and carbapenemases, as well as their variable effects on minimal inhibitory concentrations (MICs) in different bacterial host species (10,15,16), have made it difficult to detect CREs. Difficulty in reaching agreement on criteria for their detection and identification has resulted in variation across expert groups worldwide (15), particularly with regard to the specific carbapenem molecules and concentrations to use for screening for CRE and CPE and setting breakpoints for interpreting susceptibility tests. For example, a recent study assessing the accuracy of CPE detection using the methodologies of the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) showed that 13.8% and 1.6% of CPE isolates would have been missed when using their respective breakpoints (17). A study by Bulik et al (18) demonstrated the need for consistent breakpoints used across laboratories, e.g., CLSI versus US Food and Drug Administration, and the variability of accuracy of different tests, e.g., E-test, broth microdilution, and a variety of automated systems. These studies illustrate the challenges in detecting CPE and the need for accurate standardized detection methods that can be used worldwide.

Because of their broad spectrum of activity and the increasing frequency of resistance to other antimicrobial agents, carbapenems are considered to be critical for human medicine (19) and every attempt should be made to control the spread of CRE and of CPE in particular. The problem caused by the emergence of these bacteria is compounded by the frequent association of carbapenem resistance with multidrug resistance and the co-location of resistance genes for other antimicrobials on plasmids encoding CREs, thus leaving very few alternatives for treating an infection caused by these bacteria (20).

Although the prevalence of CRE and CPE is low in animals, it is increasing. They have now been found in companion animals, livestock, and even in wildlife and the environment. A more comprehensive list of references on their occurrence in animals is included in Table I. Carbapenemase genes are found worldwide in bacteria from humans and animals (10), although there may be some geographic variation in type and variant distribution (21). These data suggest that their prevalence in animals is increasing globally as is the potential for cross-species transfer.

Table I.

Geographical location of carbapenemase-producing Enterobacteriaceae (CPE) isolated from animal clinical samples.

Continent	Year	Gene	Country	Bacterial species	Animal species	Clinical sample	Reference
North America	2008–2009	NDM-1	USA	Escherichia coli	Dog	Wound	(98)
					Dog	Nose
					Dog	Urine
					Cat	Urine
	2009–2013	OXA-48	USA	Escherichia coli	Dog	Unknown clinical source	(99)
					Cat	Unknown clinical source
South America	2017	KPC-2	Brazil	Escherichia coli	Dog	Urine	(100)
Asia	2013	OXA-48	Lebanon	Escherichia coli	Fowl	Rectum	(101)
	2013–2015	OXA-48	China	Escherichia coli	Dog	Diagnostic sample	(102)
	2013	NDM-1	China	Escherichia coli	Dog	Anus	(27)
	2014–2016	NDM-1	India	Escherichia coli	Swine	Rectum	(103)
	Unknown	NDM-1	India	Escherichia coli	Dog	Scrotum	(104)
	2014–2016	NDM-5	India	Escherichia coli	Swine	Rectum	(103)
	2015	NDM-5	China	Escherichia coli	Dairy cattle	Feces	(105)
				Klebsiella pneumonia	Dairy cattle	Feces	(106)
				Escherichia coli	Duck	Rectum	(107)
					Cat	Rectum	(108)
	2017	NDM-5	South Korea	Escherichia coli	Dog	Rectum	(109)
	2015	NDM-17	China	Escherichia coli	Poultry	Cloaca	(110)
	2015	VIM-2	China	Escherichia coli	Dairy cattle	Feces	(105)
Africa	2014	OXA-181	Egypt	Escherichia coli	Dairy cattle	Rectum	(111)
	2014	OXA-48	Egypt	Klebsiella pneumonia	Poultry	Organs	(112)
				Escherichia coli	Dairy cattle	Rectum	(111)
	2014–2015	OXA-48	Algeria	Escherichia coli	Dog	Rectum	(113)
				Escherichia coli	Cat	Rectum
	2014–2016	OXA-48	Algeria	Escherichia. Coli	Wild boar	Rectum	(114)
				Klebsiella pneumonia	Wild boar	Feces
	2015	OXA-48	Algeria	Escherichia coli	Bird	Feces	(115)
	2015–2016	OXA-48	Algeria	Escherichia coli	Dog	Rectum	(116)
				Enterobacter cloacae	Bird	Rectum
				Enterobacter cloacae	Horse	Rectum
				Enterobacter cloacae	Dog	Rectum
				Klebsiella pneumonia	Cat	Rectum
	2014–2015	NDM-5	Algeria	Escherichia coli	Dog	Rectum	(113)
	2015	NDM-5	Algeria	Escherichia coli	Dairy cattle	Milk	(117)
	2014	NDM	Egypt	Klebsiella pneumonia	Poultry	Organs	(112)
	2014	KPC	Egypt	Klebsiella pneumoniae	Poultry	Organs	(112)
Oceania	2012	IMP-38	Australia	Citrobacter freundii	Bird	Cloaca	(118)
	2012	IMP-4	Australia	Escherichia coli	Bird	Cloaca
				Escherichia fergusonii	Bird	Cloaca
				Klebsiella pneumoniae	Bird	Cloaca
				Kluyvera georgiana	Bird	Cloaca
				Enterobacter aerogenes	Bird	Cloaca
				Enterobacter cloacae	Bird	Cloaca
				Citrobacter braakii	Bird	Cloaca
				Proteus mirabilis	Bird	Cloaca
				Proteus penneri	Bird	Cloaca
	2016	IMP-4	Australia	Salmonella enterica Typhimurium	Cat	Rectum	(85)
Europe	2009–2011	OXA-48	Germany	Klebsiella pneumoniae	Dog	Diagnostic sample	(119)
				Enterobacter cloacae	Dog	Diagnostic sample
				Klebsiella pneumoniae	Cat	Diagnostic sample
				Klebsiella pneumoniae	Horse	Diagnostic sample
	2009–2012	OXA-48	Germany	Klebsiella pneumoniae	Dog	Diagnostic sample	(23)
				Enterobacter cloacae	Dog	Diagnostic sample
				Escherichia coli	Dog	Diagnostic sample
				Klebsiella oxytoca	Dog	Diagnostic sample
				Klebsiella pneumoniae	Cat	Diagnostic sample
				Enterobacter cloacae	Cat	Diagnostic sample
				Escherichia coli	Cat	Diagnostic sample
	2010	OXA-48	Germany	Klebsiella pneumoniae	Guinea pig	Diagnostic sample	(23)
				Klebsiella pneumoniae	Rat	Diagnostic sample
				Klebsiella pneumoniae	Mouse	Diagnostic sample
				Klebsiella pneumoniae	Rabbit	Diagnostic sample
	2012	OXA-48	Germany	Escherichia coli	Dog	Diagnostic sample	(120)
				Klebsiella pneumoniae	Dog	Diagnostic sample
	2015	OXA-48	France	Escherichia coli	Dog	Rectum	(121)
	2015–2016	OXA-48	Spain	Klebsiella pneumonia	Bird	Cloaca	(122)
				Klebsiella pneumoniae	Bird	Cloaca
				Klebsiella pneumoniae	Bird	Cloaca
				Escherichia coli	Bird	Cloaca
				Enterobacter cloacae	Bird	Cloaca
	2012	OXA-23	Belgium	Acinetobacter sp.	Horse	Feces	(123)
	2016	OXA-181	Italy	Escherichia coli	Swine	Feces	(83)
	2014	KPC-2	Spain	Escherichia coli	Bird	Cloaca	(124)
	2011	VIM-1	Germany	Salmonella Infantis	Swine	Feces	(125)
	2014	VIM-1	Spain	Escherichia coli	Bird	Cloaca	(124)
	2014–2015	VIM-1	Spain	Klebsiella pneumoniae	Dog	Rectum	(24)
	2015	VIM-1	Germany	Escherichia coli	Swine	Colon	(126)
	2016	VIM-1	Germany	Salmonella Infantis	Swine	Diagnostic sample	(127)
	2015–2016	NDM-5	UK	Escherichia coli	Dog	Wound	(128)
	2018	NDM-5	Switzerland	Escherichia coli	Dog	Wound	(129)
	2015	NDM	Finland	Escherichia coli	Dog	Ear	(26)
	N/A	NDM-1	Germany	Salmonella Corvallis	Bird	Unknown	(130)

NDM — New Delhi metallo-β-lactamase; OXA — oxacillinases; KPC — Klebsiella pneumoniae carbapenemase; VIM — Verona integron-encoded metallo-β-lactamase.

To the best of our knowledge, no study has been published to date at the local level in Canada describing the presence of CPE in food animals. In the context of companion animals, we conducted a short study in Canada in 2018 with 64 fecal samples from dogs in Guelph and Toronto, Ontario. Approximately 1 g of feces was first enriched in tryptic soy broth containing 0.25 μg/mL of ertapenem and then plated onto CARBA SMART agar and MacConkey agar plates for detection of carbapenemase-producers. Isolates growing on these plates were tested with the modified Hodge test (MHT), using ertapenem disks to increase sensitivity. No CPE was detected with this approach, which suggests that they were still absent or infrequent (< 6% using an exact binomial 95% confidence interval) among dogs in southern Ontario at that time.

Carbapenemase-producing bacteria have been transferred between companion animals and humans in many cases and are a growing public health concern (22–26). In this context, the case of a carbapenem-resistant Escherichia coli isolate resistant to all antimicrobials except tigecycline and polymyxins reported recently in a young dog is of great concern (27). As part of the control measures to limit the spread and selection of CREs and CPEs, the use of carbapenems should be strictly restricted, if not avoided completely, in animals in general (28,29). Despite such measures, CRE and CPE may still occur in animals, in part because of the potential for co-selection by other antimicrobial agents. The detection of CPE carriers and CPE infections in animals is therefore warranted and adequate methodologies for this purpose need to be further developed and agreed upon.

The relatively low carbapenem MICs of some CPEs (15,30) and the need for further tests, in addition to basic susceptibility testing to differentiate them among CREs, may make CPEs difficult to identify. For the same reasons, it is also difficult to detect CPE carriers for epidemiological and preventive purposes. If present in low numbers, CPEs can easily be overshadowed by other bacteria and missed with standard detection procedures, thus allowing further transmission while remaining undetected (10). The second part of this review will therefore discuss the methodologies used to identify and detect CPEs and their challenges.

Methodologies and applications

Susceptibility testing methods

Antimicrobial susceptibility tests (ASTs) provide quantitative evidence as to whether or not an isolate has reduced susceptibility to a particular carbapenem. The results of such tests are used primarily to guide treatment modalities and predict therapeutic success (10). Although these results can also be used to identify CREs for epidemiological and preventive purposes, additional tests are needed to reliably identify CPEs and to identify the type of carbapenemase responsible for the observed decreased susceptibility.

The main ASTs include disk diffusion, broth microdilution (BMD), agar dilution, gradient methods (E-test), and a variety of commercial automated systems. An important challenge with all these methods is choosing the adequate carbapenem molecule and clinical breakpoints or epidemiological cut-off values for the purpose under consideration. This choice would appear relatively straightforward for clinical diagnostic purposes, for which expert groups, such as the Clinical and Laboratory Standards Institute (CLSI) (31,32) and the European Committee on Microbial Susceptibility Testing [EUCAST (http://eucast.org)], among others, provide well-established and validated guidelines and standards.

This is more challenging for epidemiological and surveillance purposes in which it is important to detect any carbapenemase, even if associated with minor changes in MICs. For these purposes, it is critical to choose adequate molecule(s). The AST methodology chosen is critical and cut-off values to sensitively and specifically identify isolates with reduced susceptibility are more difficult to set. This is particularly challenging for OXA-48-like enzymes, which can be easily missed with traditional susceptibility testing techniques because of the relatively low MICs they afford (33).

Many studies have been conducted to compare the effectiveness of a variety of ASTs for identifying CPEs. Unfortunately, the diversity of objectives, populations under study, i.e., the distribution of different carbapenemases and bacterial species in the samples, and interpretation criteria used for these studies make them extremely difficult to compare and almost impossible to draw clear general conclusions on the respective performances of ASTs. Nevertheless, researchers seem to agree that tests based on ertapenem (and perhaps imipenem) are the most sensitive for detecting CPEs, although they present a relatively low specificity (34). Meropenem seems to present the best overall compromise in terms of sensitivity and specificity and is now recommended by both EUCAST (http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms_170711.pdf) and CLSI (31,32) for screening isolates for reduced susceptibility to carbapenems and possible presence of carbapenemases. In order to reach maximum sensitivity, it is recommended that adequate species-specific cut-off values be used to differentiate CREs from wild-type organisms in the context of epidemiological investigations (17). These organism-specific epidemiological cut-off values can be found on the EUCAST website (https://mic.eucast.org/Eucast2/), although some caution may be needed in their use (http://www.eucast.org/mic_distributions_and_ecoffs/). Alternatively, they can be generated using software such as ECOFFinder (https://clsi.org/meetings/microbiology/ecoffinder/) (35).

For practical and financial reasons, only disk diffusion and BMD are usually used in veterinary medicine and in surveillance programs involving animals. Broth microdilution is often used as a reference method. Several studies using ertapenem (36) and meropenem (15) or variable combinations of carbapenems (37,38) for comparing ASTs suggest a good correlation between disk diffusion and BMD, thus supporting the use of disk diffusion for identifying CREs and CPEs in animals. The instability of carbapenems in aqueous solution (3,4,39) further supports the use of disk diffusion, in which these drugs are more stable.

Confirmation tests for carbapenemase-producers

As previously stated, the inability of classical antimicrobial susceptibility tests (ASTs) to clearly differentiate between carbapenemase-producing Enterobacteriaceae (CPEs) and other types of carbapenem-resistant Enterobacteriaceae (CREs) has emphasized the need for alternative methods for identifying CREs. Multiple methods have been developed in response to this need with variable success. The main approaches to date include: i) tests based on carbapenemase inhibitors; ii) growth tests based on inactivation of carbapenems; iii) tests based on detection of carbapenem hydrolysis products; iv) immunochromatographic tests to detect carbapenemases; and v) Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF). These methods are discussed here and their advantages and disadvantages are summarized in Table II.

Table II.

Advantages and disadvantages of testing methods that confirm carbapenemase production in bacteria.

Testing method	Example	Advantages	Disadvantages	Reference
Carbapenemase inhibitor-based tests	Boronic acid derivatives	Inhibits class-A carbapenemases, such as KPC, commercially available disks	Does not inhibit other classes of carbapenemases	(40)
	Dipicolinic acid and EDTA	Inhibits MBLs, commercially available disks	Does not inhibit other classes of carbapenemases	(41,42)
	Temocillin	Indicates the presence of OXA-48 like enzymes	Not conclusive, optimal inhibitor combinations have yet to be determined	(43)
	Avibactam	Inhibits OXA-48 like enzymes, can be used in addition to temocillin	Optimal inhibitor combinations have yet to be determined	(44)
	Cloxacillin	Inhibits AmpC enzymes, used to identify CREs with AmpC production and porin modifications	Optimal inhibitor combinations have yet to be determined	(45)
Growth tests based on carbapenem inactivation	Modified Hodge test (MHT)	Results read after overnight incubation, visual identification of carbapenemase production via clover leaf pattern, inexpensive	Variable sensitivity and specificity depending on agent used, inoculum and β-lactamase being identified, low sensitivity for MBLs and low specificity for CREs, best used in parallel with other methods	(30,48,49)
	Carbapenem inactivation method (CIM)	Results read after overnight incubation, visual identification of carbapenemase production via lack of zone of inhibition, inexpensive, high sensitivity and specificity, can be combined with inhibitors for β-lactam type identification (e.g., cloxacillin, boronic acid, sodium mercaptoacetate)	Sensitivity and specificity of combination methods have yet to be elucidated	(48,51–53)
Detection of carbapenem hydrolysis products	Carba NP test	Results read after 2 h of incubation, modifications have improved sensitivity and maintained high specificity, variations of tests are commercially available and affordable	Low sensitivity for detection of OXA-48-like producers	(48,51,55–58)
Immunochromatographic tests (ICT)		Tests identifying multiple carbapenemases have recently been developed and validated, results are read within minutes, high sensitivity and specificity, can be used with clinical samples, e.g., blood culture	High cost, unaffordable for routine screening of CPE and surveillance programs in animals	(60,61)
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)		Equipment present in diagnostic laboratories, detection of OXA-48 like producers has been improved by adding ammonium bicarbonate, the use of inhibitors is strongly suggested to identify specific carbapenemase producers	Struggles to identify OXA-48-like producers, routine use for surveillance laboratories is not practical due to lack of automation and standardization	(65,66)

Inhibitor-based methods

While inhibitor-based tests cannot identify all CRE classes, they provide useful information for at least some of them. These tests are used in conjunction with ASTs, mostly as part of disk diffusion tests, for which disks containing combinations of carbapenems and inhibitors are now commercially available. They can also be conducted in broth dilution methods, for which inhibitor tablets are also commercially available. There are 2 main types of inhibitors for carbapenemases. The first is composed of boronic acid derivatives that inhibit the activity of class-A carbapenemases and are used particularly to identify Klebseilla pneumonia carbapenenase (KPC) producers (40). The second includes dipicolinic acid and ethylenediaminetetraacetic acid (EDTA), which are chelating agents inhibiting metallo-β-lactamases (MBLs) (41,42).

The use of temocillin has been suggested to obtain more precise information about other types of carbapenemases and carbapenem resistance mechanisms since decreased susceptibility to this agent indicates, the presence of OXA-48-like enzymes, although not entirely conclusively (43). Avibactam specifically inhibits the activity of OXA-48-like enzymes on temocillin and has been suggested as an additional inhibition test to identify them (44). Cloxacillin, which inhibits AmpC enzymes, has also been recommended to identify CREs with combinations of AmpC production and porin modification (45). Although not fully validated and not always reliably identifying certain types of CPEs and CREs, combinations of these tests form the basis of some useful CPE confirmation schemes (46,47). See also the EUCAST guidelines available at: http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms_v1.0_20131211.pdf.

Growth tests based on inactivation of carbapenems

The 2 main tests for carbapenemases in this category are the modified Hodge test (MHT) (30) and the carbapenem inactivation method (CIM). For the MHT, a plate is first inoculated with a lawn of a carbapenem-susceptible indicator strain and a carbapenem-impregnated disk is placed in its center. Before overnight incubation, streaks of the strains to test for the presence of carbapenemase are drawn from the periphery of the plate to the disk. Results are read after overnight incubation. If a test strain is producing a carbapenemase, the enzyme will diffuse around the streak, allowing the indicator strain to grow around the streak closer to the carbapenem disk, which leads to the appearance of the “clover leaf” pattern shown in Figure 3a. Nothing will diffuse from CREs with resistance mechanisms other than carbapenemases and the border of the inhibition zone of the susceptible strain will remain unchanged around the streak.

Figure 3.

Carbapenem-resistant Enterobacteriaceae (CRE) confirmation tests based on inactivation of carbapenemases: a — modified Hodge test using a meropenem disk (test strains counter-clockwise from top: NDM-producer, KPC-producer, OXA-48-producer, and CMY-producer); and b — carbapenemase inactivation method (counter-clockwise from top right: NDM-producer, KPC-producer, OXA-48-producer, and CMY-producer).

The effectiveness of the modified Hodge test (MHT) for detecting CPE has been examined in multiple studies and it has been shown to provide both false positive and false negative results under some conditions. Sensitivity and specificity vary among others as a function of the agent used for the test (meropenem vs. ertapenem), inoculum used, and β-lactamases being investigated (48). Although the MHT is inexpensive and practical, both the sensitivity and specificity may be unsatisfactory. For example, problems may be encountered in its sensitivity in detecting MBLs such as NDM (30,48) and in its specificity in the presence of CREs resistant due to the combination of extended-spectrum β-lactamases and AmpC enzymes (48,49). Care should therefore be taken when using this test alone. It may be necessary to conduct other tests in parallel to compensate for these weaknesses (49). In evaluating the significance of MHT results, it may also be helpful to know the geographical epidemiology of CPE and the local distribution of carbapenemase variants in order to predict whether or not it is identifying the most prevalent CPEs locally (49).

For the carbapenem inactivation method (CIM), a carbapenem disk, usually meropenem, is incubated with a suspension of the isolate under consideration and the disk is subsequently used for a disk diffusion test with a carbapenem-susceptible strain. If the isolate produces a carbapenemase, the antibiotic in the disk will be inactivated and no zone of inhibition will be observed around the disk in the subsequent disk diffusion test (Figure 3b). If the isolate is resistant to carbapenems because of a mechanism other than a carbapenemase, the disk will still contain enough carbapenem to induce the presence of an inhibition zone with the susceptible strain.

The CIM is a simple and inexpensive test with a relatively high sensitivity and specificity (48,50). Yamada et al (48) compared the effectiveness of 3 CPE detection methods (MHT, Carba NP, and CIM) and found that the CIM had the highest concordance rate to the results of the reference polymerase chain reaction (PCR). These results are corroborated by another study by Tijet et al (51), who also found a high sensitivity and specificity for CIM. The performance of CIM has recently been improved with different suspension media and incubation times (52,53). The CIM procedure can be further altered by adding inhibitors, such as cloxacillin, boronic acid, and sodium mercaptoacetate, to identify KPC and MBLs. While the sensitivity and specificity of these combination methods have yet to be assessed, they are worth further investigation (48).

Tests based on detection of carbapenem hydrolysis products

These tests consist of variations of the Carba NP test, which is a biochemical assay that detects hydrolysis of a carbapenem by bacterial extracts via a pH indicator (54). Modifications of the test have been proposed that either use different lysis procedures or entirely circumvent the need for bacterial extracts (55,56), while apparently improving its sensitivity and maintaining its high specificity (56,57).

Multiple studies have demonstrated satisfactory performances for this test, although one of its main weaknesses may be its relatively low sensitivity for detecting OXA-48-like producers (48,51,58). The main advantage of the Carba NP test and its derivatives over other phenotypic tests is that it can be read after 2 h of incubation. Multiple variants of the test are now commercially available and seem to represent a fast and affordable method for identifying carbapenemase producers in clinical laboratories.

Immunochromatographic tests (ICTs)

Immunochromatographic tests are routinely used for point-of-care diagnostics in the form of lateral flow immunoassay. The principals of ICTs, including in veterinary medicine, are reviewed in Koczula and Gallotta (59). Although a number of ICTs for detecting a single carbapenemase or pairs have been described for characterizing bacterial cultures, they have only recently been developed and validated to detect a sufficiently broad panel of enzymes, i.e., NDM-, KPC-, IMP-, VIM-type, and OXA-48-like carbapenemases (60).

Immunochromatographic tests provide results within minutes and seem to present high levels of sensitivity, including for OXA-48-like enzymes, and specificity (60). They seem to even be applicable directly to blood culture for rapid human diagnostics (61). These types of tests are still too expensive, however, for routine screening of CPEs and surveillance programs in animals.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)

This type of spectrometry is now used routinely in diagnostic laboratories for rapidly identifying bacterial isolates. Recent studies have attempted to use it for detecting β-lactamases and carbapenemases in particular. Two approaches have been developed: one assesses the presence of specific peaks for the hydrolysis products of carbapenems when incubated together with the isolate under investigation (62,63) and the other is based on signature peaks for products of plasmids associated with carbapenemases (64). Although it may have interesting applications for investigating outbreaks, this method is much narrower in scope and only the first approach will be discussed here.

As with other methods, the hydrolysis product detection method by MALDI-TOF has also struggled with OXA-48-like producers. This sensitivity issue was improved, however, by adding ammonium bicarbonate (NH₄HCO₃) to the reaction buffer (65). As with other tests, the use of inhibitors to further identify the group of carbapenemases responsible for the observed hydrolysis has also been suggested for MALDI-TOF-based approaches (66). Despite these promising results, the routine use of MALDI-TOF for identifying carbapenemase-producers in diagnostic and surveillance laboratories may have to wait until automated and standardized protocols and instrument settings are established.

DNA-based methods

Deoxyribonucleic acid (DNA)-based methods are the gold standard for detecting CPEs (5,10,33). They are more commonly used for research purposes than for clinical diagnostics and surveillance, particularly in animals, due to their high cost, frequent lack of standardization, and the need for specialized equipment and personnel (5). Although not necessarily able to detect new carbapenemase genes and variants (10), they are invaluable and essential procedures for characterizing resistant bacteria and can improve on current detection methods by providing accurate and reliable comparative results.

Polymerase chain reaction (PCR)

This is the most commonly used tool to detect the presence of a gene of interest. Besides being fast, PCR is also able to detect carbapenemase genes that may be difficult to detect otherwise, such as those for OXA-48-like enzymes (33). There are too many PCR protocols for carbapenemase genes in the literature to be described here. As for other antimicrobial resistance (AMR) genes, multiple variations and combinations of conventional (end-point), real-time, single, and multiplex protocols have been described, some of which are available commercially (67). These protocols have targeted all the major groups of carbapenemases, including KPC, NDM, OXA-48 and OXA-48-like, VIM, IMP, SME, and GES.

Bialvaei et al (68) review PCR methods and protocols for carbapenemase detection. Criteria and strain sets used to validate these numerous PCR tests are extremely variable and their performance in the real world should be carefully assessed before their use. Some official recommendations for primers for detecting the major carbapenemase genes are available, such as those from the European Union Reference Laboratory for Antimicrobial Resistance, available at: https://www.eurl-ar.eu/CustomerData/Files/Folders/25-resourcer/459_primerliste-til-web-07-11-2018.pdf.

Polymerase chain reaction can be a first line test leading to further investigation, when, for example, testing bacterial isolates for surveillance (69,70). This method can also be used to directly screen clinical samples (71,72) with relatively low detection limits (73) and can also confirm results of a previous test that suggests the presence of carbapenemase production (28). Finally, while PCR cannot necessarily identify the targeted carbapenemase gene to the variant level, sequencing of the PCR products can generally be used to identify them, thus providing much more detailed information for epidemiological investigations.

Microarrays

While providing acceptable, but slightly longer turnover times, microarrays have a major advantage over PCR-based approaches. Microarrays are able to test for the presence of a large number of genes at the same time and can also differentiate between variants on the basis of single nucleotide polymorphism (67). Multiple research groups have developed microarrays for the detection of AMR genes. Cross-validation against a collection of isolates with well-characterized carbapenemase genes has demonstrated excellent sensitivity and specificity (74,75). Microarray-based approaches have also been developed and assessed for direct testing of clinical samples (76,77). Due to the need to constantly adapt to new genes and gene variants, as well as for specialized equipment, however, only a few of these “homemade” and commercial platforms have withstood time.

New technologies combining DNA amplification and microarray technologies have also been commercialized that promise to increase sensitivity of detection, while maintaining specificity (78). Unfortunately, these platforms remain relatively expensive and have not been widely adopted for surveillance of AMR in animal populations.

Genome sequencing

Whole genome sequencing (WGS) has become mainstream for epidemiological research and surveillance recently as a result of major technical progress. This has allowed bacterial strains to be characterized to a level unthinkable a decade or 2 ago. It is now possible to investigate outbreaks and identify antimicrobial resistance genes, as well as assess virulence potential of a strain in almost real time through WGS (79). Despite many efforts in the development of dedicated bioinformatics tools, the short reads provided by earlier and current mainstream WGS platforms, e.g., https://www.illumina.com/, do not allow the consistent assembly of plasmids. Newer methods providing longer reads, such as the Oxford Nanopore platform (https://nanoporetech.com/) and the Pacific Biosciences platform (https://www.pacb.com/), are now providing the information needed to overcome the problems with long repeats in sequences that were hampering plasmid assembly.

This provides researchers with the tools to decipher the epidemiology of AMR and carbapenem resistance in particular at all levels, from the local (80) to global (21) spread of CRE and CPE clones, as well as the spread of CPE-encoding plasmids and associated CPE determinants (8). Future progress in the use of these same sequencing platforms for metagenomics studies is bound to drastically deepen our understanding of AMR epidemiology, including of CREs and CPEs (81). Unfortunately, because of cost and the need for specifically trained, highly qualified personnel, WGS is not yet a viable methodology for routine diagnostic and broad surveillance purposes. It is an essential tool, however, for understanding the epidemiology of antimicrobial resistance and the mobile genetic elements on which the carbapenemase genes are carried.

The first studies that used WGS for CPEs from animals have started trickling in and can be found in the literature. They have helped characterize Salmonella and E. coli CPE isolates and their plasmids encoding, for instance, KPC-4 (82), OXA-181 (83), VIM-1 (84), and IMP-4 (85). In one study, WGS allowed the authors to demonstrate the identity of NDM-5-carrying E. coli in dogs and a member of their owner’s family, thus suggesting a possible transfer between them (26). In another fascinating study, WGS allowed researchers to follow the persistence and transfer of an NDM-1-encoding plasmid in chickens (86). The authors were able to assess not only the dynamics of this plasmid in its original Salmonella Corvallis host, but also to follow its transfer to E. coli and Klebsiella pneumonia strains in vivo and its microevolution through recombination events (86). These studies on CPE in animals demonstrate the potential of WGS for the study of carbapenem resistance in bacteria from animals and for understanding its epidemiology in a more global context, including the animal/human/environment interface.

Media for CRE screening

Selective culture media are used to enhance the growth of bacteria that have a certain characteristic, while inhibiting the growth of unwanted bacteria. They can be used either as enrichment broth to increase the concentration of the bacteria of interest and increase sensitivity of detection or as a selective agar to more easily isolate and identify the target organism. Selective media often include indicators that allow colonies of specific organisms to be identified among those growing on a plate. Enrichment broth and selective agar can also be used sequentially to optimize sensitivity and specificity.

In the particular case of CRE, the instability of carbapenems in liquid solution has reduced the suitability of ready-made enrichment broth for CRE detection and has led to the use of carbapenem disks dipped into nutrient broth immediately before inoculation (87). The use of such enrichment broth has significantly increased the sensitivity of CRE-carrier detection in humans (88,89) and similar advantages can also be expected when assessing for the presence of carrier animals.

A variety of selective-indicator agar media for CRE is available on the market. Most of those currently available, e.g., CHROMagar, mSuperCARBA, CHROMID CARBA, Brilliance CRE Agar, can identify E. coli and also provide putative differentiation of other CREs, such as fermenters from non-fermenter species, e.g., Pseudomonas spp. and Acinetobacter spp. While there are too many evaluations and comparative studies to cite here, a few examples are provided in Table III.

Table III.

Comparison of selective culture media for isolating and detecting CPE.

Selective culture media	Sensitivity (%)	Specificity (%)	Material	Reference
Brilliance CRE	75.9	98.1	Pure	(90)
Brilliance CRE	76.3	57.1	Pure	(93)
Brilliance CRE	78 to 82	60 to 66	Pure	(94)
Brilliance CRE	94	71	Pure	(95)
Brilliance CRE + Brilliance ESBL	98.1	67.6	Pure	(90)
ChomID ESBL	87.7	24.2	Pure	(96)
ChomID ESBL	96 to 97	6 to 19	Pure	(94)
SuperCARBA	96.5	76.3	Pure	(93)
SuperCARBA	95.6	82.2	Pure	(96)
CHROMagar KPC	100	98.4	Mixed	(97)
CHROMagar KPC	40.3	85.5	Pure	(96)
CHROMagar KPC	43	67.8	Pure	(92)
ChromArt CRE	100	55.8	Pure	(90)
BBL CHROMagar CPE	88.5	86.1	Pure	(90)
ChromID CARBA SMART	90.7	89.1	Pure	(90)
ChromID Carba	91 to 96	76 to 89	Pure	(94)

CPE — carbapenemase-producing Enterobacteriaceae; ESBL — extended-spectrum β-lactamase; KPC — Klebsiella pneumoniae carbapenemase.

Challenges arise when selecting the correct carbapenem molecule and its concentration in the media, in the face of the diversity of MICs associated with the multiple types of carbapenemase-organism combinations present in the field (90,91). It seems that none of the commercially available media has been able to consistently achieve a combination of both sensitivity and specificity clearly above 90% when screening for CPE in general. This has led to the use of selective agar to target specific types of CREs, such as CHROMagar KPC, CHROMID OXA-48, CHROMID CARBA (for KPC and MBLs), or combinations of media in a single plate, such as CHROMID CARBA SMART agar. It has recently been suggested that the new CHROMagar mSuperCARBA agar has significantly improved the detection of CRE from all the major carbapenemase-type producers at once (92). Further evaluation with clinical samples instead of pure cultures and on a larger scale is needed, however, to confirm these results for practical clinical applications (90).

In conclusion, there are many challenges in detecting CPEs and there is no single, cost-effective test that will detect every CPE. Promising strides have been made in identifying key features and combinations of tests to optimize the performance of detection methods, while reducing cost and time. These recent advances make CPE detection and surveillance in animals more practicable and affordable.

Carbapenemase-producing Enterobacteriaceae (CPEs) have only recently been discovered in animals and may still be infrequent. Animals need to be screened for CPEs more frequently. Using some of the most specific and sensitive methods now available and described in this article, it is possible to thoroughly and reliably assess the current situation and monitor it in the future. This may be particularly important for assessing the dynamic transmission of these organisms between animals and humans, and vice versa, as well as their persistence once animals are colonized.