Carbapenems
Carbapenems
Carbapenems
SCIENTIFIC OPINION
ABSTRACT
Carbapenems are broad-spectrum β-lactam antimicrobials used for the treatment of serious infections in humans.
To date only sporadic studies have reported the occurrence of carbapenemase-producing (CP) bacteria in food-
producing animals and their environment. The bacteria and enzymes isolated include VIM-1 producing
Escherichia coli and Salmonella Infantis from pigs and poultry in Germany, OXA-23-producing Acinetobacter
spp. from cattle and horses in France and Belgium, and NDM-producing Acinetobacter spp. from pigs and
poultry in China. In the German S. Infantis and E. coli isolates, the VIM-1-encoding genes were located on
IncHI2 plasmids. A methodology including selective culture is proposed for the detection of CP strains of
Enterobacteriaceae and Acinetobacter spp. The choice of selective media for the surveillance of carbapenem
resistance for testing animal and food samples needs to be experimentally evaluated and validated. Biochemical
and phenotypic tests for the confirmatory identification of CP bacteria are available. For CP bacteria in animals
and food, active/passive monitoring and/or targeted surveys should cover key zoonotic agents, animal pathogens
and indicator organisms. Priority should be given to broilers, fattening turkeys, fattening pigs, veal calves and
meat thereof. Because there are no data on the comparative efficacy of individual control options, prioritisation
is complex. Continued prohibition of the use of carbapenems in food-producing animals would be a simple and
effective option. As genes encoding carbapenemase production are mostly plasmid-mediated, and co-resistance
may be an important issue in the spread of such resistance mechanisms, decreasing the frequency of use of
antimicrobials in animal production in the EU in accordance with prudent use guidelines is also of high priority.
The effectiveness of any control measures should be monitored by targeted surveys, using selective isolation
methods and pre-enrichment of samples. Control measures should be proactively implemented at national and
international levels to prevent CP strains become widespread in livestock.
KEY WORDS
carbapenemases occurrence, detection , transmission, animals, control options
1
On request from EFSA, Question No EFSA-Q-2013-00010, adopted on 5 December 2013.
2
Panel members: Olivier Andreoletti, Dorte Lau Baggesen, Declan Bolton, Patrick Butaye, Paul Cook, Robert Davies,
Pablo S Fernández Escámez, John Griffin, Tine Hald, Arie Havelaar, Kostas Koutsoumanis, Roland Lindqvist, James
McLauchlin, Truls Nesbakken, Miguel Prieto, Antonia Ricci, Giuseppe Ru, Moez Sanaa, Marion Simmons, John Sofos
and John Threlfall. Correspondence: [email protected]
3
Acknowledgement: The Panel wishes to thank the members of the Working Group on Carbapenem resistance in food
animal ecosystems: Patrick Butaye, Teresa Coque, Beatriz Guerra-Román, Henrik Hasman, Annemarie Kasbohrer, Anna
Pelagia Magiorakos, Vivi Miriagou, Laurent Poirel and John Threlfall for the preparatory work on this scientific opinion
and EFSA staff: Ernesto Liebana and Pietro Stella for the support provided to this scientific opinion.
Suggested citation: EFSA BIOHAZ Panel (EFSA Panel on Biological Hazards), 2013. Scientific Opinion on Carbapenem
resistance in food animal ecosystems. EFSA Journal 2013;11(12):3501, 70 pp. doi:10.2903/j.efsa.2013.3501
Available online: www.efsa.europa.eu/efsajournal
SUMMARY
The European Food Safety Authority (EFSA) asked the Panel on Biological Hazards (BIOHAZ) to
provide a scientific opinion on the carbapenem resistance in food animal ecosystems. Carbapenems
are broad-spectrum beta (β)-lactam antimicrobials mostly used for the treatment of serious infections
in humans, frequently in hospitalised patients, and are considered last-line therapy for infections
caused by multidrug-resistant Gram-negative bacteria. In particular, the panel was asked: (i) to define
the carbapenemase-producing (CP) bacterial strains and genes relevant for public health and linked to
food-producing animals or food-borne transmission; (ii) to review the information on the
epidemiology of acquired resistance to carbapenems, including the genes coding for such resistance, in
food-producing animals and food; (iii) to perform a critical analysis of the methods (phenotypic and
genotypic) and the interpretive criteria currently used for detection (isolation and identification) and
characterisation of CP bacterial strains; (iv) to make recommendations for the harmonised monitoring
and reporting of resistance (phenotypic and genotypic) caused by carbapenemases in food and food-
producing animals in the EU; and finally, (v) to identify possible means of preventing or minimising
the further emergence and spread of CP bacterial strains transmitted via the food chain, including
consideration of the advantages and disadvantages of different options.
The BIOHAZ Panel concluded that the production of carbapenemases may confer diverse β-lactam
resistance phenotypes depending on a variety of factors such as the bacterial species, variant of the
enzyme, expression level due to different promoters, copies of carbapenemase-encoding genes and the
presence of additional non-enzymatic resistance mechanisms. Although a wide variety of CP strains
with several different mechanisms of resistance to carbapenems and with a diversity of acquired genes
encoding carbapenem-hydrolyzing ß-lactamases have been identified in bacterial isolations from cases
of human infection worldwide, to date only sporadic studies have reported the occurrence of CP
bacteria in food-producing animals and their environment. The bacteria and enzymes therein include
VIM-1-producing Enterobacteriaceae (Escherichia coli and a putative strain of Salmonella Infantis
from pigs, poultry and their environment in Germany), OXA-23-producing Acinetobacter
genomospecies 15TU from cattle in France and Acinetobacter spp. from horses in Belgium, and
NDM-producing Acinetobacter spp. from pigs and poultry in China.
Genes encoding carbapenemase production in Acinetobacter spp. from animals have been located on
both the chromosome (Acinetobacter genomospecies 15TU from cattle in France) and on plasmids
(A. lwoffii and A. baumannii from pigs in China). In the putative S. Infantis and E. coli isolates
associated with food animals in Germany, the VIM-1-encoding genes were located on IncHI2
plasmids.
Factors that favour the emergence of carbapenem resistance include the increased consumption of
carbapenems in humans driven in part by the worldwide spread of ESBLs in Enterobacteriacae, the
location of carbapenem-encoding resistance genes on mobile genetic elements, and positive selection
due to co-resistance with other commonly-used antibiotics.
For the detection of carbapenemase-producing Enterobacteriacae (CPE) the screening cut-off values of
0.125 mg/l for meropenem, 1 mg/l for imipenem and 0.125 mg/l for ertapenem are recommended. If
disk diffusion is used, applying zone diameters of 25 mm for meropenem, 23 mm for imipenem, and
25 mm for ertapenem would be indicative of non-susceptibility to carbapenems. The Panel further
noted that pre-enrichment by incubation of samples in selective broth containing a carbapenem at a
low concentration (e.g. meropenem, 0.125 mg/L) may increase sensitivity. This methodology has not
yet been validated, and any method proposed would have to be subjected to thorough experimental
verification.
Biochemical and phenotypic tests for the confirmatory identification of CP bacteria among isolates
exhibiting non-susceptibility to carbapenems are available. The sensitivity and specificity of these
assays may vary considerably in different settings. The identity of the genes responsible for the
carbapenemase production should be determined by molecular methods. The Panel further considers
that plasmid and strain typing should be undertaken to acquire better knowledge on the epidemiology
of genes encoding carbapenemase production among bacteria from food-producing animal
populations, food thereof and evironmental samples.
The Panel noted that there are comprehensive requirements for the collection and reporting of
antimicrobial resistance (AMR) data, including resistance to carbapenems, laid down in European
legislation. Technical specifications and reporting manuals prepared by EFSA recommend reporting of
isolate-based data, and recommend mandatory phenotypic monitoring of Salmonella spp. and indicator
E. coli with a broadened test panel also covering meropenem. Active monitoring and/or additional
targeted surveys for CP bacteria in animals and food should cover key zoonotic agents and indicator
organisms of the commensal flora. Priority should be given to broilers, fattening turkeys, fattening
pigs, veal calves, and the derived fresh meat of domestic origin. Dairy cattle, raw milk and aquaculture
products may be also included in targeted surveys. For active monitoring all isolates of Salmonella
spp. and E. coli collected within the compulsory monitoring programme, as required by European
legislation, should be screened for meropenem resistance using standardized microdilution methods.
Specific targeted surveys for the detection of CP organisms in the food animal ecosystem should be
implemented at the EU level. For passive monitoring, diagnostic isolates of veterinary origin (at least
those classified as microbiologically resistant to 3rd- or 4th-generation cephalosporins on the basis of
epidemiological cut-off values) should be subjected to phenotypic testing for carbapenem resistance
and carbapenemase production, and subsequent molecular identification and characterization of the
carbapenemase production genes present. For correct interpretation of results relating to carbapenem
resistance, the sampling strategy (active vs. passive monitoring) and the selection procedure applied
for each isolate (randomly selected isolate vs. isolate from selective media) should be reported. Results
of phenotypic methods used for testing for carbapenemase production as well as the results from
further characterisation of resistance genes should be reported for each isolate. Methods involving pre-
enrichment and selective plating should be used in specific surveys to increase sensitivity for
populations with a low prevalence of CP microorganisms.
Because there are no data on the comparative efficicacy of individual control options in reducing the
potential public health risks caused by CP bacteria related to food-producing animals, prioritisation is
complex. At present, carbapenems are not licensed for use in food-producing animals in the EU and
other parts of the world, and therefore one simple and effective control option to minimise the further
emergence and possible spread of such strains transmitted via the food chain would be to continue to
prohibit the use of carbapenems in food-producing animals. Furthermore, as genes encoding
carbapenemase production are mostly plasmid-mediated, and co-resistance may be an important issue
in the spread of such plasmid-mediated resistance mechanisms, decreasing the frequency of use of
antimicrobials in animal production in the EU in accordance with prudent use guidelines is also of
high priority. The effectiveness of any control measures should be monitored on a regular basis by
targeted surveys of food-producing animals and foods for CP bacteria, using selective isolation
methods and pre-enrichment of samples as necessary.
Finally the Panel strongly recommends that control measures to contain the spread of CP bacteria in
food-producing animals should be proactively implemented at national and international levels. Such
plans should be agreed to prevent CP strains become widespread in livestock.
TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 2
Table of contents ...................................................................................................................................... 4
Background as provided by EFSA ........................................................................................................... 6
Terms of reference as provided by EFSA ................................................................................................ 7
Assessment ............................................................................................................................................... 8
1. Introduction ..................................................................................................................................... 8
1.1. Structure of and activity of penems and carbapenems ............................................................ 8
1.2. Carbapenem resistance.......................................................................................................... 10
1.2.1. Resistance due to production of carbapenemases ............................................................. 10
1.2.2. Resistance due to other mechanisms ................................................................................ 13
1.3. Scope of opinion ................................................................................................................... 13
1.4. Significance and public health threat of human infections with carbapenemase-producing
bacteria .............................................................................................................................................. 13
1.5. Risk factors ........................................................................................................................... 14
1.6. Incidence of human infection................................................................................................ 15
1.6.1. Carbapenem-resistant Klebsiella pneumoniae from EARS-Net data ............................... 16
1.6.2. Carbapenem resistance in the United States ..................................................................... 16
1.6.3. Carbapenem resistance worldwide ................................................................................... 16
1.7. Reservoirs of carbapenemase-producing bacteria and encoding genes ................................ 16
1.7.1. Reservoirs of bacteria ....................................................................................................... 16
1.7.2. Reservoirs for carbapenemase production-encoding genes .............................................. 17
1.8. Summary and conclusions .................................................................................................... 20
2. The epidemiology of carbapenemase-mediated resistance in food-producing animals and foods 20
2.1. Carbapenemase-producing microorganisms relevant for public health that have been linked
to food-producing animals, foodborne transmission and wildlife...................................................... 21
2.1.1. Carbapenemase-producing microorganisms from food-producing animals and their
environment ................................................................................................................................... 21
2.1.2. Carbapenemase-producing Salmonella spp. isolates from cases of human infections ..... 22
2.1.3. Carbapenemase-producing Salmonella spp. isolates from wildlife .................................. 23
2.2. Carbapenemase-encoding genes relevant for public health that have been linked to food-
producing animals, foodborne transmission and wildlife .................................................................. 23
2.3. Mobile genetic elements involved in transmission of carbapenem resistance relevant for
public health that have been linked to food-producing animals, foodborne transmission and
wildlife .............................................................................................................................................. 24
2.4. Data on occurrence of carbapenem resistance in food-producing animals and foods. ......... 25
2.4.1. Harmonised EU monitoring of resistance: EFSA Community Summary Report. ........... 25
2.4.2. Data from other published studies designed to detect carbapenem resistance ................. 25
2.5. Evidence of transmission of carbapenemase-producing strains and/or carbapenem
resistance genes to humans by consumption or handling of contaminated food or through the food
animal production environment. ........................................................................................................ 27
2.6. Summary and conclusions .................................................................................................... 27
3. Critical analysis of the methods for detection of carbapenemase-producing bacteria (isolation and
identification), and characterisation of carbapenemase-encoding genes and associated mobile
elements.................................................................................................................................................. 28
3.1. Selective isolation of carbapenemase-producers .................................................................. 28
3.2. Interpretative criteria for phenotypic testing for non-susceptibility to carbapenems. ........... 34
3.3. Phenotypic and biochemical confirmation of carbapenemases ............................................ 35
3.4. Characterisation of carbapenemases and typing of plasmids carrying carbapenemase-
encoding genes ................................................................................................................................... 37
3.4.1. Molecular detection and characterization of carbapenemase genes. ................................ 37
3.4.2. Detection and characterization of plasmids harbouring carbapenemase genes. ............... 39
3.5. Molecular typing of isolates. ................................................................................................. 39
Carbapenemases are now seen as a new and potentially emerging problem in food-producing animals.
The prevalence of such resistance in bacteria from animals is largely unknown. To date three
publications have reported the presence of carbapenem resistance in bacteria from animals, including
food-producing animals (pigs, bovines and horses) in three different EU Member States (Germany,
France and Belgium). Most significantly from a food safety perspective, a carbapenemase has been
recently identified in Salmonella in different pig and broiler farm origins in Germany. This is most
probably only the tip of the iceberg since there is limited surveillance on carbapenem resistance
amongst bacteria from food-producing animals. Carbapenems are usually not included in the antibiotic
panels of national surveillance programmes, nor in the panels of diagnostic laboratories dealing with
veterinary samples.
EFSA (BIOMO Unit) has recently produced Technical specifications on the harmonised monitoring
and reporting of antimicrobial resistance in Salmonella, Campylobacter and indicator Escherichia coli
and Enterococcus spp. bacteria transmitted through food4. In this report there is reference already to
the methods needed for screening for this type of resistance:
Actions to limit the spread of resistance are only possible when the prevalence of such resistance is
low. This is particularly relevant to carbapenems. It is therefore of utmost importance to initiate
studies to determine when and where this resistance is present, and to limit its dissemination
throughout the food chain.
Assess the public health risk posed by carbapenemase-producing bacterial strains in food-
producing animals and food.
4
http://www.efsa.europa.eu/en/efsajournal/doc/2742.pdf
It is the intention to request participation of ECDC and EU Reference laboratories in this working
group.
1. Define the carbapenemase-producing bacterial strains and genes relevant for public health and
linked to food-producing animals or food-borne transmission;
3. Perform a critical analysis of the methods (phenotypic and genotypic) and the interpretive
criteria currently used for detection (isolation and identification) and characterisation of
carbapenemase-producing bacterial strains.
5. Identify possible means of preventing or minimising the further emergence and spread of
carbapenemase-producing bacterial strains transmitted via the food chain, including
consideration of the advantages and disadvantages of different options.
ASSESSMENT
1. Introduction
Carbapenems are broad-spectrum beta(β)-lactam antimicrobials mostly used for the treatment of
serious infections, frequently in hospitalised patients, and are considered last-line therapy for
infections caused by multidrug-resistant (MDR) Gram-negative bacteria, e.g. the extended-spectrum
β-lactamase (ESBL)-producing Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp.
Carbapenems have been isolated from fermentation products of various streptomycetes. Thienamycin,
isolated from Streptomyces cattleya, was the first among the naturally occurring carbapenems. The
compound exhibited excellent activity against both Gram-positive and Gram-negative bacteria but was
unstable in aqueous solutions. Thus, thienamycin derivatives with adequate stability have been
developed by the pharmaceutical industry. The latter group includes all clinically available
carbapenems, such as imipenem, meropenem, ertapenem and doripenem.
All carbapenems exhibit broad antimicrobial spectra against a variety of Gram-positive, Gram-
negative and anaerobic microorganisms. Compared with imipenem, meropenem and doripenem, the
spectrum of activity of ertapenem is slightly more limited, lacking activity against non-fermenters
(Pseudomonas spp. and Acinetobacter spp.).
Carbapenems are vital for the effective therapy of community-acquired and healthcare-associated
infections caused by MDR bacteria. Resistance to this class of antibiotic compromises the therapeutic
options for patients by leaving only a few or in some cases no other antimicrobials with which to treat.
These may often exhibit variable effectiveness and frequently provoke significant adverse reactions.
There are only a very limited number of new antimicrobial agents in the antibiotic pipeline; thus the
problem resulting from the lack of availability of effective antibiotics for therapy is considerably
accentuated (ECDC and EMEA, 2009).
Carbapenems Thienamycin
Imipenem
Meropenem
Ertapenem
Doripenem
Penems Faropenem
Acquired carbapenem resistance is due to the acquisition of exogenous genetic material containing
gene(s) coding for carbapenemase production (CP) (i.e. enzymes capable of hydrolysing carbapenems
and conferring detectable increase in levels of resistance to carbapenems). In this respect, carbapenem
Minimal Inhibitory Concentration (MIC) may vary widely, ranging from full susceptibility to high-
level resistance according to the Clinical Laboratory Standards Institute (CLSI) or the European Union
Committee for Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints and according to
bacterial species (see Section 3.2 below).
For the purpose of this document, carbapenem-non-susceptible (CNS) bacteria are defined as those
exhibiting MICs above the epidemiological cut-off values (ECOFF) established by EUCAST (see
Table 5 below), and the term „acquired carbapenemases‟ is used to indicate carbapenemases produced
by bacteria as a result of the acquisition of gene(s) coding for their production.
One of the milestones in the emergence of carbapenemases in Enterobacteriaceae was the detection of
a novel carbapenemase, designated as „Klebsiella pneumoniae carbapenemase‟ (KPC) according to the
bacterial species from which it was originally isolated (Yigit et al., 2001). Such carbapenem-
hydrolysing ß-lactamases confer resistance to almost all ß-lactams (Nordmann et al., 2011a).
Subsequently, most acquired carbapenemases have been detected and reported in carbapenemase-
producing Enterobacteriaceae (CPE) globally (Nordmann et al., 2009; Bush and Jacoby, 2010;
Nordmann et al., 2011a). The broad resistance profile in bacteria that is associated with the production
of carbapenemases is significant in human infections, and poses a major on-going public health threat.
It is important: (i) to assess whether animals, particularly food-producing animals, and more generally
the environment around animal production, might also provide the conditions required for the
emergence of carbapenemase-mediated resistance; (ii) to evaluate whether specific reservoirs for CP
bacteria or carbapenemase-encoding genes exist; and (iii) to identify factors that could favour the
emergence and/or spread of carbapenemase-mediated resistance in food-producing animals and
derived foods, and thus to humans through the food chain.
1.4. Significance and public health threat of human infections with carbapenemase-
producing bacteria
Acquired carbapenemases have been found in all screened species of Enterobacteriaceae and also in
non-fermenters, such as Acinetobacter spp. and Pseudomonas spp. (Patel and Bonomo, 2013).
Enterobacteriaceae are a family of Gram-negative bacilli that are part of the normal flora of the human
and animal intestine and are the cause of community-acquired and healthcare-associated infections.
Amongst the Enterobacteriaceae, certain species, within which resistance to antimicrobials is of
particular concern, require special mention.
immunologically compromised, treatment can be necessary. Salmonella spp. can also act as a reservoir
of resistance genes.
Klebiella pneumoniae is the first bacterial species in which blaKPC was found. This organism is a
frequent cause of healthcare-associated infections e.g. pneumonias, bacteraemias, and has a well-
documented propensity to accumulate antibiotic resistance determinants (Paterson and Bonomo,
2005).
Another genus of bacteria, which has recently emerged as an organism of clinical importance, is
Acinetobacter spp., most frequently Acinetobacter baumanni, especially strains that are MDR.
Acinetobacter spp. are typically soil and water organisms. They have also been isolated from food and
the environment; they were previously mostly reported as the cause of infections in tropical climates
and in victims of natural disasters, as well as war casualties and were previously considered colonisers
in hospital environments. Acinetobacter spp. have now emerged as an important cause of healthcare-
associated infections, especially because of their resistance profile and their ability to accumulate
mechanisms of resistance, thereby making them resistant to most, if not all antimicrobials, including
the carbapenems. Such resistance compromises the treatment of patients, since there are often no
effective antibiotics with which to treat them, or may result in delayed appropriate therapy, when such
therapy is possible (Munoz-Price and Weinstein, 2008). This development is extremely worrisome, as
such organisms can survive environmental desiccation for long periods of time, making eradication
from hospitals difficult thus promoting transmission to patients and causing healthcare-associated
infections and outbreaks of infection in hospitals (Maragakis et al., 2008; Munoz-Price and Weinstein,
2008).
A variety of carbapenemases have now been reported in Enterobacteriaceae and non-fermenters such
as P. aeruginosa and Acinetobacter spp. These include the MBL, class A carbapenemases such as
KPC, and carbapenem-hydrolysing class D -lactamases (CHDLs) oxacillinases (OXA) (Souli et al.,
2008; Patel and Bonomo, 2013).
High and increasing rates of ESBL-producing bacteria represent an indirect risk for the spread of
carbapenem resistance mechanisms. This is because carbapenems are the antibiotics of choice with
which to treat patients infected with these types of MDR microorganisms. Uncontrolled use and
misuse of antimicrobial agents in addition to the high rates of MDR microorganisms (e.g. ESBL-
producing Enterobacteriaceae), all drive the large use of carbapenems (ESAC-Net interactive
database5). The result is an interminable cycle of antimicrobial use, antimicrobial selection pressure
and high rates of antimicrobial resistance (AMR).
5
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The emergence and spread of carbapenem-non-susceptible Enterobacteriaceae (CNSE), and CPE has
also been identified as a public health threat for a number of reasons. As discussed in 1.2.1 above,
CPE are commonly susceptible only to the polymyxins (e.g. colistin), fosfomycin and variably
susceptible to tigecycline, although there have been recent reports of fosfomycin-resistant
(Karageorgopoulos et al., 2012) and colistin-resistant Enterobacteriaceae (Marchaim et al., 2011). This
limits the antimicrobial armamentarium to be used to treat an infected patient, since only a few and
sometimes no effective antimicrobials remain, but also causes inadequate choice or delayed
administration of antimicrobial therapy which are associated with poorer patient outcomes, increased
morbidity, mortality, increased length of stay and increased costs in MDR organisms (Ibrahim et al.,
2000; Cosgrove et al., 2003; Anderson et al., 2006; Maragakis et al., 2008; Roberts et al., 2009; de
Kraker et al., 2011).
An additional concern is that these bacteria, and the mobile genetic elements (MGE) therein, are
highly transmissible, rapidly disseminating within hospitals after being introduced. This secondary
transmission can quickly lead to outbreaks, epidemics and, if left unchecked, to endemicity
(Grundmann et al., 2010; Nordmann et al., 2011a).
Lastly, studies performed for CPE and CNSE have shown that infection or colonization has been
associated with in-hospital mortality rates of 40-50% (Patel et al., 2008; Gasink et al., 2009; Marchaim
et al., 2010; Chitnis et al., 2012). The risk posed by infections with these MDR microorganisms
becomes even greater when considering the very limited number of new antimicrobial agents that are
in the developmental pipeline (Boucher et al., 2009; ECDC and EMEA, 2009; Livermore et al., 2011).
Complete knowledge of the geographical distribution and magnitude of CNSE and CPE is unavailable
for many reasons. Heterogeneous processes of clinical culturing, microbiological practices, detection,
surveillance, reporting, notification of CNSE and CPE as well as when and if molecular testing is
performed, likely contribute to the lack of complete data that exist worldwide. Most surveillance data
report carbapenem resistance only for K. pneumoniae. Since molecular testing is not always performed
and CNSE are frequently found to be CP, carbapenem resistance (or CNS) is frequently used as a
surrogate marker for the presence of carbapenemases.
Data from Europe originate from that collected and reported by the European Antibiotic Resistance
Surveillance System (EARS-Net)6. EARS-Net reports annual rates of AMR in bloodstream infections
(BSI) and infections of cerebrospinal fluid (CSF) from hospitals in Europe, and brings together data on
drug-resistant isolates and isolates with intermediate resistance7. These data originate from clinical
microbiological laboratories across Europe, reporting antimicrobial susceptibility results only (no
molecular mechanisms are reported) for seven invasive bacterial pathogens, including K. pneumoniae
and E. coli. EARS-Net classifies carbapenem-resistant and carbapenem-intermediate isolates as
„carbapenem-resistant‟, so carbapenem-resistant Enterobacteriaceae (CRE) will be used with regard to
EARS-Net data. Data from EARS-Net likely represent “the tip of the iceberg” of the total burden of
CRE and CPE, for a number of reasons. EARS-Net collects only data on invasive bacterial isolates
and since these bacteria also cause non-invasive infections, EARS-Net data probably correspond to
only a fraction of the total number of infections.
6
EARS-Net: http://www.ecdc.europa.eu/en/activities/surveillance/EARS-Net/Pages/index.aspx
7
http://www.ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/database/Pages/database.aspx
Twenty-eight countries reported to EARS-Net in 2011, out of which 15 reported one or more
carbapenem-resistant isolates. Twenty-three countries reported percentages of carbapenem-resistant
isolates under 1%, two between 1 and 5%, one between 10-25% and two above 25%, the highest being
Greece with 68.2%. Trends for 2008-2011 for 22 countries showed a significant increase for three
countries, Greece, Hungary and Italy (ECDC, 2012).
Poirel et al., 2012a; Wang et al., 2012; Fischer et al., 2013a; Shaheen et al., 2013; Stolle et al., 2013;
Zhang et al., 2013; Zurfluh et al., 2013).
Bacterial communities from aquatic environments including hospital sewage, wastewater treatment
plants (WWTP), lakes, or rivers may also contribute to the dissemination of carbapenemase-encoding
genes of clinical relevance, such as class A β-lactamase KPC-2, which is increasingly recovered from
Aeromonas and enterobacterial species at hospital sewage in different countries (Valverde et al., 2012;
Picao et al., 2013; Zurfluh et al., 2013), GES-5 identified from a bacterial community of activated
sludge in a WWTP (Girlich et al., 2012a), BIC-1 from P. fluorescens (Girlich et al., 2010a), and IMI-2
from Enterobacter asburiae recovered in rivers of different countries (Aubron et al., 2005); class B β-
lactamases VIM-1, VIM-2, VIM-13 from P. aeruginosa, P. alcaligenes, K. pneumoniae and diverse
environmental species from rivers and lakes (Quinteira and Peixe, 2006; Scotta et al., 2011); IMP-8,
IMP-10, and IMP-13 from Tunisian polluted rivers (Chouchani et al., 2013; Zurfluh et al., 2013),
NDM-1 from diverse Enterobacteriaceae, Pseudomonas spp. and Aeromonas spp., and V. cholerae
from wastewaters, and Achromobacter spp., Kingella denitrificans, and P. aeruginosa from tap water
(Walsh et al., 2011); and class D OXA-23 from A. baumannii isolates recovered from the Seine river
at a hospital wastewater discharge site (Girlich et al., 2010b).
Contamination of vegetables, manure and farm facilities with contaminated water of anthropogenic
origin might facilitate the penetration of highly promiscuous broad-host range plasmids in specific
settings where some of these species may occur (e.g. Salmonella spp. in poultry, E. coli in livestock,
Klebsiella spp. in dairy cattle, Aeromonas spp. and Vibrio spp. in aquaculture)8. Similarly aquaculture
using water from contaminated rivers would result in a risk for the emergence and spread of
carbapenemase producers in such settings.
Aeromonas spp. possess naturally-occurring carbapenemases, the genes for which could, in theory, be
mobilised by genetic elements. The same considerations apply to other environmental species also
possessing intrinsic carbapenemases (Stenotrophomonas spp., Flavobacteriaceae, etc). Aeromonas is
unique in that the species has often been shown to be an intermediate reservoir of acquired clinically-
relevant resistance genes (Girlich et al., 2011); for instance there have been many examples of
plasmid-encoded ESBLs found in Aeromonas spp., and a plasmid-borne NDM-1 carbapenemase-
encoding gene has also been identified in this species (Walsh et al., 2011). This phenomenon has not
been described for other genera. Thus other environmental species (e.g., Vibrio spp., Acinetobacter
spp. and Pseudomonas spp.) or even some Enterobacteriacae may also have to be considered in
screening regimens. In this respect a strain of P. fluorescens with a new carbapenemase (BIC-1), so far
never identified in strains from humans, was reported in 2010 (Girlich et al., 2010a).
Besides the risk of contaminated soil and water, the colonisation of wildlife by CP bacteria through
contact with sewage, human faeces, or animal manure might facilitate the global spread of resistance
genes, with consequences for public health, animal welfare, and ecosystem functions (Pesapane et al.,
2013).
The occurrence, both in animals, including domestic pets and in healthy humans, of CP bacteria might
be underestimated, taking into account the “silent” and successful dissemination of certain species of
carbapenemase producers documented among humans (Gijon et al., 2012; Viau et al., 2012). Also,
only a few studies performed by groups dealing with the environment or animals have taken this topic
in consideration.
To date, the main ISs associated with the capture of CP genes such as ISKpn7 (blaKPC genes), IS1999
(blaOXA), or ISAba125 and ISAba1 (blaOXA, blaNDM) have also been associated with other bla
determinants in Enterobacteriaceae, Acinetobacter spp. and Pseudomonadaceae, highlighting their
ability to acquire a wide diversity of genes of different origin (Aubert et al., 2006; Hamidian et al.,
2013). Other ISs as IS26, and IS1111-like variants (e.g. IS5045, IS4321) which are widely spread
among plasmids and genomes of species of the above mentioned species, have been frequently
associated with different genetic context-carrying blaIMP-, blaVIM-, blaKPC-, blaOXA- or blaNDM-genes
generating composite transposons, thereby enhancing the possibilities of transfer among diverse
bacterial communities by different mechanisms (Tzouvelekis et al., 2012). Some plasmids of
Enterobacteriaceae (e.g. IncFI/FII, IncI, IncL/M, IncA/C, IncQ), Pseudomonas spp. and
Achromobacter xylosoxidans (pNOR-like), or Acinetobacter spp. are able to spread in a broad host
range of bacterial species, and are abundant among humans and animals, probably as a consequence of
previous selection by antibiotics, heavy metals and other pollutants. They have been implicated
repeatedly in recent independent acquisition events and thus are able to acquire new genes from
environmental bacteria and to spread among humans, animals and the environment (Carattoli, 2009;
Bertini et al., 2010; Girlich et al., 2010b; Andrade et al., 2011; Carattoli, 2011; Zhou et al., 2011; Chen
et al., 2012; Girlich et al., 2012a; Bonnin et al., 2013; Di Pilato et al., 2013; Naas et al., 2013).
Exposure to antibiotics, metals and other environmental pollutants may enhance the mobilization of
resistance genes (Kristiansson et al., 2011). Also, concentrations representing action limits used in
environmental risk assessment may be enough to exert a significant selective pressure on genes of
clinical relevance. Of particular concern is the release of antibiotics used in human and veterinary
medicine into sewage and manure distribution systems as they can be associated with sewage sludge,
contamination of rivers, liquid manure and farm soil (Tello et al., 2012).
Carbapenem MICs may vary widely ranging from full susceptibility to high-level resistance
according to CLSI or EUCAST clinical breakpoints. Low levels of resistance to carbapenems
are more often observed among Enterobacteriacae.
Factors that favour the emergence of carbapenem resistance include the increased
consumption of carbapenems in humans driven in part by the worldwide spread of ESBLs in
Enterobacteriacae, the location of carbapenem-encoding resistance genes on mobile genetic
elements, and positive selection due to co-resistance with other commonly-used antibiotics.
Additional studies in which the isolation of CRE in livestock or food were reported, but presented
inconsistencies in some of the methodologies. These reported: (i) Salmonella serovars Teko,
Weltevreden and Saintpaul, from vegetables in India (Singh et al., 2007); (ii) Salmonella Paratyphi B
variant Java, S. Saintpaul and Salmonella Virchow from buffalo calves and beef in India (Singh et al.,
2012); and (iii) E. coli from oysters, water, shrimps and pond environment in Brazil (Vieira et al.,
2008; Vieira et al., 2010). These studies have therefore not been considered further in this Opinion.
Most Salmonella spp. infections are considered to be directly or indirectly zoonotic in origin, and thus
reports of CP isolates of this species from cases of human infections and other animal sources (i.e.
wildlife) have been included in this Opinion and are discussed below (Miriagou et al., 2003; Savard et
al., 2011; Cabanes et al., 2012; Le Hello et al., 2013; Pfeifer et al., 2013; Rasheed et al., 2013;
Woodford et al., 2013b). It has not been possible to elucidate if the carbapenemase-encoding genes
described above were acquired in animal, environmental or clinical settings. In respect of wildlife, a
CP Salmonella Corvallis isolate from a wild bird in Germany (Fischer et al., 2013c) has also been
reported.
In addition, the isolation of NDM-1-producing E. coli isolates from companion animals (cats and
dogs) in the USA (Shaheen et al., 2013), OXA-48 in E. coli and K. pneumoniae from dogs (Stolle et
al., 2013), and OXA-23-like-producing A. baumannii from human head lice in Senegal (Kempf et al.,
2012) have been described, but isolates from these origins are out of the scope of the Opinion and will
not be discussed further.
2.1. Carbapenemase-producing microorganisms relevant for public health that have been
linked to food-producing animals, foodborne transmission and wildlife
The following CP microorganisms relevant for public health and linked to food-producing animals or
foods have been described.
Nine Gram-negative non-fermentative Acinetobacter bacteria belonging to different species (see 1.4)
resistant to imipenem (MIC>8 µg/mL) were identified in a study in different chicken farms (cloacal
swabs) and one pig slaughterhouse (carcass swabs) in China. Among these isolates, an A. lwoffii
isolate producing NDM-1 (isolate SGC-HZ9) recovered from a chicken anal swab was identified. The
isolate was resistant to all tested β-lactams (excepting aztreonam), ciprofloxacin, tetracyclines,
kanamycin, and chloramphenicol, and susceptible to gentamicin and polymyxin B (CLSI, 2008). No
molecular typing data on the isolate were provided (Wang et al., 2012).
An A. baumannii isolate (GF216) which produced NDM-1 was recovered from a sick pig in a Chinese
swine farm. The isolate was resistant to all tested β-lactams, ciprofloxacin, chloramphenicol,
tetracyclines, kanamycin, tilmicosin and erythromycin, being susceptible to gentamicin and colistin.
No molecular typing data on the isolates were provided (Zhang et al., 2013).
Two isolates of Acinetobacter spp. producing OXA-23 were collected from different hospitalised
horses in a University Faculty in Belgium. One of these horses had been previously treated with
penicillin. These isolates showed MICs for imipenem of 16 µg/ml, and together with other β-lactams,
were also resistant to tetracyclines, sulphonamides, trimethoprim and gentamicin, but susceptible to
colistin). Both isolates showed the same PFGE pattern (Smet et al., 2012).
Three CP Salmonella spp. isolates were obtained from two fattening pig farms and one broiler farm in
Germany (Fischer et al., 2013a). The isolates from the two pig farms were from swine single faeces
(isolates R27) or from the farm environment (isolate R25). The poultry-related isolate (R3) was
collected from a dust sample. The isolates were classified as Salmonella enterica group C, antigenic
formula “6,7:-:-“9 and on this basis are herewith referred to as „putative S. Infantis‟. The multi locus
sequence type ST3210 and the two highly related PFGE-patterns are typical of Salmonella Infantis
(6,7:r:1,5) (Hauser et al., 2012). The Salmonella isolates R3, R25 and R27 showed decreased
susceptibility to carbapenems (non-wild type by the EUCAST ECOFF), but had MIC values below the
clinical breakpoint (CLSI, 2011). The three isolates produced VIM-1 carbapenemase and carried both
the AmpC-encoding gene blaACC-1 and blaVIM-1. When Salmonella R3 was grown in liquid medium
with carbapenems (1 to 16/8 µg/ml imipenem/ertapenem) the isolate was able to grow and showed
clinical resistance (clinical breakpoints CLSI vs. EUCAST, imipenem ≥ 4 vs. >8 mg/L, ertapenem ≥ 1
vs. >1 mg/L). All isolates showed additional resistance to chloramphenicol, streptomycin,
sulphonamides and trimethoprim and susceptibility to colistin, fosfomycin, nitrofurantoin and
tigecycline (CLSI, 2011). S. Infantis is among the top ten Salmonella spp. serovars implicated in
human salmonellosis worldwide (Hendriksen et al., 2011; EFSA and ECDC, 2013). The serovar has
been implicated in several outbreaks, and broilers, layers and pigs are important reservoirs (Hauser et
al., 2012; EFSA and ECDC, 2013). Isolates of this serovar have also caused disease with severe
symptoms (septicaemia, fatal cases) and nosocomial outbreaks.
Several E. coli isolates (31) from pig faeces, flies, manure and bootsocks taken in one of the German
swine farms cited above also produced VIM-1. These isolates showed slightly decreased susceptibility
to carbapenems (above the EUCAST ECCOFFs), together with resistance to streptomycin,
sulphonamides, and in all except one isolate, to tetracycline. Like the Salmonella isolate R27, the E.
coli isolates R178 and R29 were also able to grow in medium with concentrations of carbapenems
larger than or equal to the CLSI breakpoints. All isolates were positive for the blaACC-1 and the blaVIM-1
genes. These isolates were identified as E. coli sequence type (ST) 88 which belong to the E. coli
phylogenetic group A, and showed highly similar XbaI PFGE patterns and three different plasmid
profiles. E. coli ST88 is commonly isolated from chickens, cattle, turkeys and humans in Germany
(Fischer et al., 2012, 2013a; Fischer et al., 2013d; Fischer et al., 2013b).
The first description of a CP Salmonella spp. was a S. Cubana isolate (S. Cubana 4707) collected from
a stool sample taken from a chronically ill 4-year old patient in 1998 in the USA (Miriagou et al.,
2003). The patient had been hospitalised several times, and there was no history of recent travel before
hospitalization. This isolate produced KPC-2 and exhibited resistance to most β-lactam antibiotics,
including oxyimino-cephalosporins and imipenem. The isolate was also resistant to streptomycin,
trimethoprim, and sulphamethoxazole. In the previous hospitalisations (three times in the six months
previous to the isolation), the patient had been treated with ceftriaxone and ceftazidime, but not with
carbapenems.
CP S. Saintpaul (1 isolate) and Salmonella Kentucky (1) from a patient returning to France from Egypt
in 2009 have also been described (Le Hello et al., 2013). The patient was co-infected with S. Kentucky
resistant to ciprofloxacin and 3rd-generation cephalosporins (carrying blaCMY-2), and with S. Saintpaul
which produced OXA-48. One of the subsequent S. Kentucky isolates (obtained from the same patient
more than one year later) also contained the blaOXA-48 carbapenemase gene, although the isolate was
phenotypically susceptible to imipenem (MIC 0.75 mg/L). This isolate was also resistant to
azithromycin but susceptible to colistin and tigecycline. In the same study, S. Kentucky isolates (5)
producing VIM-2 was described. All isolates were recovered from human clinical samples (one blood,
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two urine and two faecal cultures) in 2010 in Morocco. They showed resistance to ciprofloxacin and
3rd-generation cephalosporins, and decreased susceptibility to imipenem (MIC 1-3 mg/L). Also S.
Kentucky carrying blaOXA-48 was isolated from a patient from Morocco in a German hospital (Pfeifer et
al., 2013).
Salmonella Westhampton resistant to imipenem and producing NDM-1 was isolated from urine and
faecal samples from a French patient who was transferred from India to a hospital on Reunion Island
in 2011. This isolate also carried blaCTX-M-1 and blaTEM genes. E. coli and Klebsiella isolates showing
an ESBL phenotype, but no imipenem resistance were obtained from the same patient. No data on the
presence of blaNDM-1 in these isolates were presented (Cabanes et al., 2012).
Although Salmonella spp. is in general regarded as a zoonotic pathogen, to our knowledge there are no
indications that the infections involving CP strains described above were derived from food-producing
animals directly or from foods thereof.
2.2. Carbapenemase-encoding genes relevant for public health that have been linked to
food-producing animals, foodborne transmission and wildlife
As described above, the following CP-encoding genes relevant for public health and which have been
linked to food-producing animals or foods have been described:
The blaOXA-23 gene was found in isolates of Acinetobacter genomospecies 15TU from a dairy cattle
farm in France (Poirel et al., 2012a), and was also detected in Acinetobacter spp. from two horses in
Belgium (Smet et al., 2012).
The blaNDM-1 gene was found in A. lwoffii from a chicken in a broiler farm (isolate SGC-HZ9) (Wang
et al., 2012) and A. baumannii from a pig farm (isolate GF216) (Zhang et al., 2013) in different
regions of China.
The blaVIM-1 gene was found in S. Infantis from German broiler and pig farms, and E. coli from one of
the same pig farms (Fischer et al., 2012, 2013a).
In other Salmonella spp. isolations from humans and other sources, the following genes have also been
found:
The detection of blaKPC-2 gene in a S. Cubana isolate made in 1998 in the USA (Miriagou et al., 2003).
blaVIM-2 in five S. Kentucky isolates from clinical human samples in Morocco (Le Hello et al., 2013).
blaOXA-48 in isolates of S. Saintpaul and S. Kentucky from the same patient, with more than one year
between both isolation dates, supporting the “in vivo” transfer of the gene (Le Hello et al., 2013). This
gene was also found in a S. Kentucky isolate from a patient from Morocco hospitalised in Germany
(Pfeifer et al., 2013).
The blaNDM-1 gene has been also found in Salmonella spp. from human clinical samples in USA and
Reunion Island, in both cases in patients previously hospitalized in India (Savard et al., 2011; Cabanes
et al., 2012; Rasheed et al., 2013), and S. Corvallis from a black kite in Germany – see above (Fischer
et al., 2013c).
2.3. Mobile genetic elements involved in transmission of carbapenem resistance relevant for
public health that have been linked to food-producing animals, foodborne transmission
and wildlife
The following mobile genetic elements involved in the transmission of CP-encoding genes relevant for
public health and linked to food-producing animals or foods have been so far described:
The Acinetobacter genomospecies 15TU isolate BY2, collected from cattle in France carried blaOXA-23
located in the Tn2008 transposon. In other eight isolates (BY-1, BY2-9) the ISAbaI element of
Tn2008 was truncated by the ISAcsp2 IS. The gene appears to be chromosomally-located (Poirel et al.,
2012a).
pAL-01 is a 270 kb self-transferable plasmid which carries the blaNDM-1 gene (partial sequence of
the plasmid, accession number JN616388), isolated from A. lwoffii SGC-HZ9 from a chicken in China.
The incompatibility (Inc) group of the plasmid could not be determined. The gene is attached to an
intact ISAba125 insertion element that seems to play a role in the expression of the carbapenemase.
The gene aphA6 is located upstream the ISAba125 (Wang et al., 2012).
pRHR-27 is a 300 kb incHI2 plasmid from the S. Infantis isolate R27 collected from a German pig
farm (see above). The blaVIM-1 was located in a class 1 integron together with aacA4 (aac (6‟)-Ib) and
aadA1 in its variable region, and qac/sul1 in its 3‟CS conserved segment. This integron forms part of
a Tn402-like transposon. The plasmid also carries the blaACC-1, strA/B, catA1, and a trimethoprim
resistance gene. The pRH27 plasmid was neither self-transferable nor mobilisable under the conditions
used (Fischer et al., 2013a). An indistinguishable plasmid has been found in other S. Infantis isolates
from environmental samples from pig and poultry farms (Fischer et al., 2013a; Fischer et al., 2013d;
Fischer et al., 2013b).
pRHR-178 (HG530658) is a 220 kb IncHI2 plasmid isolated from the E. coli isolate R178, collected in
a German pig farm. Like in pRH27, the blaVIM-1 is located in a class 1 integron forming part of a
Tn402-like transposon (accession number HE663536). The plasmid also carries the blaACC-1, and
strA/B genes, and was not transferable. The same plasmid has been found in other E. coli isolates
collected within the same farm from pig faecal samples, manure and a fly (Fischer et al., 2012, 2013a;
Fischer et al., 2013d; Fischer et al., 2013b).
In other Salmonella spp. isolates obtained from human clinical samples and from non-food producing
animals, the following plasmids have been detected:
pST4707, a self-transferable plasmid of between 36 – 63 kb, in which both blaKPC-2 and blaTEM-1 genes
were co-localized, from the isolate of S. Cubana described above. Genes encoding resistance to
streptomycin, trimethoprim and sulphonamides were also present on the plasmid (Miriagou et al.,
2003).
A 30 kb IncW plasmid carrying blaVIM-2 within the In58 integron (containing also aacC7, aacC1 and
aacA4 genes) in five isolates of S. Kentucky, and a 70 kb IncL/M plasmid in a S. Saintpaul isolate (Le
Hello et al., 2013).
An IncL/M plasmid in a monophasic S. Senftenberg isolate. In this isolate the armA gene
(aminoglycoside resistance) was also located on this IncL/M plasmid (Rasheed et al., 2013).
pRH-1738 is an ∼180 kb IncA/C conjugative plasmid found in a strain of S. Corvallis (from a wild
kite) in which both the blaNDM-1 carbapenemase gene flanked by ISAb125 and the bleBML gene
(accession number HG007972), and a blaCMY-16 AmpC gene were located. On the same plasmid, two
class 1 integrons (carrying dfrA1-aadA5 or aacA4) as well as floR, tet(A), strA/B, and sul1, sul2 were
co-localized (Fischer et al., 2013c).
2.4.2. Data from other published studies designed to detect carbapenem resistance
As stated above, few studies have reported the presence of CP bacteria from food-producing animals
and their environment (Fischer et al., 2012; Poirel et al., 2012a; Wang et al., 2012; Fischer et al.,
2013a; Fischer et al., 2013d; Fischer et al., 2013b; Woodford et al., 2013b; Zhang et al., 2013). The
available data have come from studies conducted within several research projects in different
countries, but not as a result of monitoring programmes. Thus, the methods used to detect the isolates
were different. In some cases the studies focused on the isolation of ESBL/AmpC producers. When
such isolates were subjected to tests for their susceptibility to carbapenems they were recognised as
carbapenemase producers (Fischer et al., 2012, 2013a; Fischer et al., 2013d). In other investigations,
the primary purpose was the identification of carbapenemase producers. The following studies have
been published.
In a study conducted in one dairy farm in France in 2010, samples taken from 50 dairy cows were
tested. The screening of the samples was done using selective Drigalski Agar plates (BioMerieux)
with 1 µg/mL imipenem. Nine of the samples were positive for CPE Acinetobacter; all isolates
produced OXA-23 (Poirel et al., 2012a).
In a survey on chicken farms (8), duck farms (6) and one pig slaughterhouse in Eastern China (Oct-
Dec. 2010) in total 396 samples were collected. The samples were screened with selective brain heart
infusion agar plates with 8 µg/ml imipenem, looking for non-fermenting Gram-negative isolates. In
total, nine isolates (eight from pig samples, one from a chicken) from different bacterial genera
(Stenotrophomonas, Myroides, Chryseobacterium, and Acinetobacter) were non-susceptible against
carbapenems. One A. lwoffii producing NDM-1 was isolated from one of the broiler samples. The
antibiotic usage records for the chicken farm indicated that penicillin, cefotaxime, cefradine,
tilmicosin, doxycycline, and neomycin had been used (Wang et al., 2012).
Within a survey of Gram-negative bacteria from pig (5), broiler (3) and duck (1) farms conducted in
South China (2011-2012), 1293 samples (single animals) were collected. The screening method used
for isolation is not available. Isolates were tested for their susceptibility to carbapenems by growth on
brain-heart infusion agar plates with 2 µg/ml meropenem. Isolates that grew on the media (276) were
tested for the presence of blaNDM-1 and other carbapenemases. As described above one isolate from a
pig with pneumonia and sepsis was positive for blaNDM-1. Treatment records of the farm revealed the
use of ß-lactams including 3rd and 4th generation cephalosporins, aminoglycosides and quinolones.
Data on the remaining isolates have as yet not been published (Zhang et al., 2013).
Although testing for reduced susceptibility to carbapenems among isolates from food and food-
producing animals has as yet not been included as part of the Danish Integrated Antimicrobial
Resistance Monitoring and Research Programme (DANMAP), two recent studies have been conducted
on E. coli subpopulations to test for isolates with reduced susceptibility to carbapenems. In a
collection from 2011, 138 cephalosporin resistant E. coli from pigs and cattle as well as meat from
these animals and from poultry were subjected to phenotypic testing using discs containing
meropenem, ertapenem and imipenem. None of these isolates showed reduced susceptibility to any of
the three antimicrobials. Similar, in a collection from 2012, 249 cephalosporin-resistant E. coli from
pigs and meat from pigs, cattle and poultry were tested and found negative for the presence of
carbapenemase genes by Whole Genome Sequencing (WGS) by the use of the ResFinder Web tool
(see below) (DANMAP, 2012).
From longitudinal studies conducted in swine (n=7) and broiler (n=7) farms in Germany (Laube et al.,
2013a; Laube et al., 2013b), 2 fattening pig farms and 1 broiler farm were found to be positive for CP
Salmonella spp. One of these farms was also positive for CP E. coli. The three farms were distributed
in different locations in the same German Federal Region, with no apparent epidemiological link. The
screening of the samples was done using selective MacConkey agar with 1 µg/mL cefotaxime, looking
for ESBLs/AmpC producing isolates (Fischer et al., 2012, 2013a). When the isolates were tested for
their susceptibility against a broad panel of ß-lactams, the production of carbapenemases was
suspected and confirmed.
In a study carried out on hospitalised horses in Belgium in 2012, faecal samples collected from 20
horses were analysed for the presence of CP bacteria. The samples were screened using MacConkey
agar plates with 1 µg/ml imipenem. In two of the samples Acinetobacter spp. isolates showing
resistance to carbapenems were detected (Smet et al., 2012).
In a recent study, 184 Salmonella isolates belonging to the collection of the German National
Reference Laboratory for Salmonella (mainly food and animal isolates) that showed clinical resistance
to 3rd-generation cephalosporins (MICs ≥ 4mg/L for cefotaxime, and had been collected since 2006)
were tested for their susceptibility to carbapenems (imipenem, meropenem and ertapenem). Only one
of these isolates (S. Corvallis from a wild bird) showed susceptibility values (over the EUCAST cut
off values) that suggested the presence of carbapenemases (in this case the presence of NDM-1 was
confirmed) (Fischer et al., 2013c).
Kempf et al. (2012) performed a study in 2010 in Senegal, in which fecal samples from human head
lice (354 samples), human faecal samples (717) and animal faecal samples (118) were screened for the
presence of A. baumannii by means of real-time Polymerase Chain Reaction targeting the blaOXA51-like
gene. Samples positive for A. baumannii were further screened for blaOXA23-like and blaOXA24-like genes.
A. baumannii was detected in the head lice samples (4%), human stool samples (5.4%) and the animal
stool samples (5%). No blaOXA24 gene was detected in the isolates (cultured from 14 head lice, 39
human and seven animal stool samples). Six of the isolates from the human faecal samples and three
from the lice were positive for the blaOXA23-like gene. None of the A. baumannii from the animal
samples was positive for any of the bla genes tested.
In a study in Switzerland (Stephan et al., 2013), investigations to determine the occurrence of CPE in
food-producing animals in Switzerland were presented. Faecal samples from pigs (200), cattle (150)
and sheep (110), collected from individual healthy food-producing animals at slaughter (one animal
per farm), as well as pooled faecal samples from poultry flocks (99 herd-level pooled faecal samples
of chicken at the entry of a large slaughterhouse from the crates of 99 poultry flocks) were collected
and investigated. In 16 of the samples (three sheep, five cattle and eight poultry) there was growth on
Brilliance CRE agar. The isolates recovered were Stenotrophomonas maltophilia, A. baumannii, E.
coli and Citrobacter freundii. The E. coli (4) and the C. freundii (4) isolates showed imipenem MICs
between 0.19 and 1.5 μg/ml. None of the genes screened (genes encoding for VIM, GIM, Sim, NDM-
1, IMI, KPC, OXA-48, OXA-40-like OXA-51-like and OXA-58-like) were present in these
Enterobacteriacae.
Within another study conducted in Ethiopia (Kumsa et al., 2012) to determine the presence of
Acinetobacter species in lice and Melophagus ovinus (sheep ked) of animals in 2011, cattle (207),
sheep (85), dogs (47) and cats (16) were examined for ectoparasites. DNA from different
Acinetobacter species was detected in lice (11.1%) and keds (86.4%): Acinetobacter soli in
Linognathus vituli of cattle, Acinetobacter lowffii in M. ovinus of sheep, Acinetobacter pittii in
Heterodoxus spiniger of dogs, one new Acinetobacter spp. in M. ovinus and two new Acinetobacter
spp. in H. spiniger of dogs were detected. None of the carbapenemase resistance-encoding genes
tested for blaOXA-23, blaOXA-24, blaOXA-58, blaNDM-1 and blaOXA-51 were found in any of the lice and sheep
keds.
A classic example of the rapidity of the spread, in the farm environment, of a plasmid encoding
resistance to ESBL and other β-lactam antibiotics was that initially described by Liebana et al. (2006).
In this study both the conjugative IncK, CTX-M-carrying plasmid, and strains carrying this pCT-like
plasmid, were shown to spread rapidly among cattle on the farm. Later, they have also been reported
among other farm animals and hospitals in different countries (Valverde et al., 2009; Cottell et al.,
2011; Dhanji et al., 2012; Stokes et al., 2012). Results indicated that horizontal plasmid transfer
between strains as well as horizontal gene transfer between plasmids contributed to the spread of
resistance. Furthermore, some ESBL clones were shown to persist for several months, suggesting that
clonal spread also contributes to the perpetuation of resistance. Although there is no suggestion that
strains on this farm were resistant to carbapenem antibiotics, the study demonstrates how rapidly such
ESBL-producing strains and plasmids coding for ESBL production can spread in a farm environment,
and how unrelated antibiotic selection pressure can contribute to the transmission and establishment of
such strains.
Carbapenemase-producing isolates from companion animals and from a wild bird have also
been reported. Genes identified have included blaOXA-48 and blaNDM-1 in Enterobacteriaceae and
blaOXA-23 in Acinetobacter spp.
Active surveillance of cultures along with point prevalence surveys has been shown to be the most
effective approach (Ben-David et al., 2010; Ciobotaro et al., 2011). Various versions of screening
culture-based or molecular-based methodologies have been proposed (Landman et al., 2005;
Schechner et al., 2009; Nordmann and Poirel, 2013). In-house prepared agar media (e.g. MacConkey,
Isolates obtained from the initial screening are considered as non-susceptible to carbapenems and then
are further investigated by phenotypic, biochemical and molecular assays (Nordmann et al., 2012c;
Nordmann and Poirel, 2013).
One of the in-house prepared selective agar media for the screening of carbapenemase producers is
McConkey agar supplemented with meropenem (0.5 g/ml). Although the diagnostic values for this
screening medium have not been determined it has been successfully used and exhibited high
sensitivity during active surveillance (Sypsa et al., 2012; Adler et al., 2013). Colonies obtained on
meropenem-containing MacConkey agar plates are visualized macroscopically as members of
Enterobacteriaceae, or as non-fermenting Gram-negative bacilli; further identification is needed to the
species level. One disadvantage of this medium is that the shelf-life of the plates is limited (one-two
weeks) due to the instability of meropenem, and thus they should be prepared regularly.
CHROMagar KPC screening medium (CHROMagar company, Paris, France), which contains a
carbapenem as the selective agent for resistance, was specifically designed for screening KPC
producers. Using this medium, CP isolates with MIC values >16 µg/mL can be detected (Samra et al.,
2008). The main problem associated with the use of this medium is that many carbapenemase
producers, including those producing KPC-type enzymes, do not exhibit high MICs to carbapenems.
Therefore the sensitivity of CHROMagar KPC screening medium appears to be quite low (Carrer et
al., 2010).
SUPERCARBA medium has been developed to overpass the two main shortcomings of ChromID
ESBL and CHROMagar KPC screening medium listed above, these being the lack of detection of
isolates fully susceptible to broad-spectrum cephalosporins and of those exhibiting low MICs of
carbapenems (Nordmann et al., 2012a). Hence, selection is based on a carbapenem (ertapenem) and
not a cephalosporin. Furthermore the concentration of ertapenem is low (0.25 µg/ml), thus
significantly enhancing sensitivity. Nevertheless, the specificity remains good, since SUPERCARBA
medium has been supplemented with cloxacillin, which inhibits production of class C
cephalosporinases, and that may confer reduced susceptibility to ertapenem when combined with some
permeability defects. In addition, the SUPERCARBA medium contains zinc, thereby enhancing the
production of MBLs and therefore constituting a significant advantage compared to the other media in
order to detect those MBL producers showing low MICs to carbapenems.
Brilliance CRE (Oxoid, Basingstoke, U.K) is a chromogenic medium that contains a modified
carbapenem and it is designed to detect carbapenem-non-susceptible bacteria. Results from several
studies have shown that Brilliance CRE agar is a reliable selective medium, since it allows growth of
the vast majority of the CP enterobacterial isolates, even those exhibiting low levels of resistance to
carbapenems (Wilkinson et al., 2012; Girlich et al., 2013b; Kotsakis et al., 2013; Stuart et al., 2013).
The specificity of Brilliance CRE is considered adequate and comparable to that of the
SUPERCARBA. It should be noted that one of the advantages of this screening medium is its
chromogenic ability to differentiate microorganisms to the genus or species level by colony color and
morphology.
As reported by several investigators, the sensitivity of all three chromogenic media is affected by the
difficulties in detecting OXA-48 producers which exhibit low carbapenem MICs. Therefore, a novel
chromogenic medium, chromID OXA-48 (bioMerieux), has been developed for the efficient detection
of OXA-48 producers. Indeed, this medium exhibits high sensitivity for the detection of OXA-48
positive micro-organisms. Because growth of isolates producing either class A or B carbapenemases is
inhibited (Girlich et al., 2013a), in most instances chromID OXA-48 should be used in combination
with an additional selective medium.
ECOFFs are determined from the MICs or inhibition zone diameter distributions of bacterial
populations. They are defined as the values identifying the upper limit of the wild type population (i.e.
characterized by the absence of acquired and mutational mechanism of resistance to a specific
antimicrobial agent). The screening cut-off values are the lowest MICs or inhibition zone diameters
identified in the non-wild type population possessing acquired mechanisms of resistance to a specific
agent. For Enterobacteriaceae, the screening cut-off value is of 0.125 mg/l for meropenem, 1 mg/l for
imipenem and 0.125 mg/l for ertapenem. If disk diffusion is used, applying zone diameters of 25 mm
for meropenem, 23 mm for imipenem, and 25 mm for ertapenem would be indicative of non-
susceptibility to carbapenems. Screening cut-off values for imipenem and meropenem have not yet
been defined for Acinetobacter spp. Based on expert opinion, a decision has been made by the WG
that values above the ECOFFs should be used as screening cut-offs for the latter micro-organism.
The use of ertapenem for screening confers high sensitivity but suffers from low specificity. On the
other hand, for imipenem the MICs of wild-type population for several bacteria, such as Proteae,
overlaps with the bacterial population bearing a carbapenem resistance mechanism. Screening with
meropenem offers a good balance between sensitivity and specificity. Stability of carbapenem
antimicrobials might be a problem because of their short active life.
A similar approach can be adopted for the monitoring of carbapenem resistance and identification of
carbapenemase producers in animals and the environment. Specifically designed surveys for the
detection of carbapenemase producers should be conducted. Pre-enrichment by incubation of samples
in selective broth containing a carbapenem at a low concentration (e.g. meropenem 0.125 mg/L) may
increase sensitivity (see also workflow in Figure 1). Cephalosporin-based pre-enrichment of the
samples has been already recommended for the screening of ESBL-producers (EFSA Panel on
Biological Hazards (BIOHAZ), 2011). A similar pre-enrichment approach using a carbapenem has not
been validated for the screening of carbapenemase producers of animal origin, and any method
proposed would have to be subjected to thorough experimental verification. In that respect, the
proposed methodology is based on the CDC guidelines for screening of carbapenem resistance in
Enterobacteriaceae (CDC, 2012) and on expert opinion.
Methods for application for two different uses are presented below:
(i) investigation of resistance for any given isolate from monitoring and/or surveillance schemes,
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In 2010, following a report from the US SENTRY Programme and analysing results from a study of
27,415 Enterobacteriaceae collected world-wide from a wide variety of infections, CLSI reduced
breakpoint levels to ertapenem, imipenem and meropenem to levels similar to those of EUCAST
(CLSI, 2010). The current EUCAST13 and CLSI (2013) carbapenem breakpoints for
Enterobactericaeae for imipenem, ertapenem and meropenem are shown in Tables 5 and 6 below.
Table 5: EUCAST12 and CLSI (2013) MIC and zone diameters (ZD) for testing for phenotypic
resistance to imipenem, ertapenem and meropenem in Enterobacteriaceae.
CLSI EUCAST
MIC Breakpoints Breakpoints ECOFF Screening cut-off
S I R S R (WT ≤ X mg/l)
Imipenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 1.0 mg/l > 1 mg/l
Meropenem ≤ 1 mg/l 2 ≥ 4 mg/l ≤ 2 mg/l > 8 mg/l ≤ 0.125 mg/l > 0.125 mg/l
Ertapenem ≤ 0.5 mg/l 1 ≥ 2 mg/l ≤ 0.5 mg/l > 1 mg/l ≤ 0.064 mg/l > 0.125 mg/l
Table 6: EUCAST12 and CLSI (2013) MIC and zone diameters (ZD) for testing for phenotypic
resistance to imipenem, and meropenem in Acinetobacter spp.
CLSI EUCAST
MIC Breakpoints Breakpoints ECOFF Screening cut-off
S I R S R (WT ≤ X mg/l)
Imipenem ≤ 4 mg/l 8 ≥ 16 mg/l ≤ 2 mg/l > 8 mg/l ≤ 1.0 mg/l not defined*
Meropenem ≤ 4 mg/l 8 ≥ 16 mg/l ≤ 2 mg/l > 8 mg/l ≤ 2.0 mg/l not defined*
12
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The recently published technical specifications on the harmonised monitoring and reporting of AMR
in Salmonella spp., Campylobacter spp., indicator E. coli and Enterococcus spp. transmitted through
food (EFSA, 2012a) made a recommendation to include a carbapenem as a complementary
antimicrobial to be inserted into the harmonised panel of antimicrobials used for test. Because of
methodological difficulties a suggestion was made to include meropenem in the harmonised
antimicrobial panel and then further test isolates which are non-susceptible to meropenem against
imipenem and ertapenem in secondary panel. The point was also made that ertapenem is not advised
as indicator of carbapenem resistance (Cohen Stuart et al., 2010).
Modified Hodge test: The cloverleaf or modified Hodge test (MHT) has been extensively used for the
detection of carbapenemase activity (Lee et al., 2001). The assay is based on the inactivation of
imipenem or meropenem by whole bacterial cells or crude cell extracts.
MHT performs well for detection of KPC and OXA-48 producers (Pasteran et al., 2009; Nordmann et
al., 2012c). However, i) its specificity is low against high-level AmpC producers or CTX-M producers
accompanied also with porin loss (Pasteran et al., 2009) and ii) it exhibits relatively low sensitivity for
producers of MBLs, especially NDM (though supplementing culture media with zinc may improve
performance) (Girlich et al., 2012b). The assay is time-consuming and cannot distinguish the type of
carbapenemase involved. MHT is still the only carbapenemase-detecting method proposed by the
CLSI for screening purposes, although according to EUCAST the method is not recommended due to
the aforementioned reliability problems and interpretation difficulties14.
Detection of carbapenemase producers based on specific inhibitors: The relevant tests are based
on inhibition of MBLs by various chelating agents capable of depriving the enzymes from the
hydrolytically essential zinc ions. EDTA, dipicolinic acid, 2-mercaptopropionic acid and
mercaptoacetic acid have been used as inhibitors, the former two being the most common (reviewed in
(Miriagou et al., 2010; Nordmann et al., 2012c)). Phenotypic detection of class A carbapenemases
(KPC) production is based on the inhibitory effect of tazobactam, boronic acid derivatives
phenylboronic (PBA) and 3-aminophenylboronic acid (APBA) (reviewed by Miriagou et al.(2010) and
Nordmann et al. (2012c)). Performance of tests utilizing PBA is considered superior to those with
APBA (Tsakris et al., 2011; Miriagou et al., 2013).
The MBL-detecting assays are actually diffusion tests using disks containing a hydrolyzable -lactam
(typically a carbapenem, imipenem or meropenem) and/or a MBL inhibitor (Arakawa et al., 2000;
Yong et al., 2002; Lee et al., 2003; Kimura et al., 2005). The double disk synergy test (DDST) is
similar to that used for ESBL detection. Two disks containing predefined amounts of the -lactam and
the inhibitor are placed close to each other. Formation of a synergy image is indicative of MBL
production. The method is considered highly sensitive even with isolates with low carbapenem
resistance levels. The main drawback is that interpretation is subjective and results cannot be
quantified. Therefore, the combined disk test (CDT) is currently preferred by most laboratories. In the
CDT format, the -lactam disk is potentiated with an inhibitor and the diameter of the inhibition zone
is compared with that of the -lactam disk alone. Increase of the inhibition zone diameter above a pre-
defined cut-off value denotes MBL activity (Tsakris et al., 2010; Giske et al., 2011).
Detection of class A carbapenemases by the CDT synergy test using boronic derivatives combined
with imipenem or meropenem exhibits high sensitivity for K. pneumoniae and E. coli (Tsakris et al.,
14
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2010; Giske et al., 2011). A major drawback of these methodologies is that boronates also inhibit
cephalosporinases and in that respect the CDT performance is suboptimal (false positives), especially
with AmpC hyperproducing enterobacterial isolates. This problem can be overcome by the use of
cloxacillin-containing plates or disks (Giske et al., 2011; Nordmann et al., 2012c).
Generally, the inhibitor-based assays are considered reliable for the identification of MBL-producers,
although there have been studies reporting failures to detect VIM production among A. baumannii
strains (Ikonomidis et al., 2008; Loli et al., 2008; Picao et al., 2008). False positive results have also
been reported in P. aeruginosa due to the strong antimicrobial activity of EDTA against the species
(Chu et al., 2005) and A. baumannii isolates due to the presence of OXA-type carbapenemases (Segal
and Elisha, 2005). In addition, double carbapenemase producers (e.g. MBL plus KPC) may “deceive”
the conventional CD tests frequently appearing negative for one or even both carbapenemases due to a
masking effect. Inclusion of a carbapenem disk (imipenem or meropenem), potentiated with both
inhibitors for class A (boronate) and class B (EDTA or DPA) carbapenemases, may facilitate the
correct identification of carbapenemases in such isolates (Miriagou et al., 2003; Tsakris et al., 2011).
An additional shortcoming of this type of methods is that there is no inhibitor-based method for the
detection of class D CP bacteria.
A similar method, the Blue-CARBA, also based on in-vitro hydrolysis of imipenem, has been
proposed by Pires et al. (Pires et al., 2013). In this case, the carbapenemase activity can be determined
directly from bacterial culture without bacterial extraction. As described by the authors, sensitivity and
specificity of the method was also 100% for Enterobacteriaceae, Pseudomonas spp., and
Acinetobacter spp.
Recently the use of mass spectrometry for the detection of carbapenemase activity has been proposed,
based on the analysis of the degradation of a carbapenem molecule (Burckhardt and Zimmermann,
2011; Hrabak et al., 2011). This method was shown to have a 97% sensitivity and 98% specificity
(Hrabak et al., 2011). Although this technique has to be further evaluated, matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-Tof) equipment is increasingly used
in diagnostic bacteriology laboratory.
The recent EFSA report with technical specifications (EFSA, 2012a) further recommended that
isolates with a carbapenemase phenotype (i.e. with apparent resistance to meropenem or imipenem)
should be re-tested phenotypically against these antimicrobials to confirm such resistance using discs
or gradient strips for synergy between carbapenems and EDTA (indicating a probable MBL) and for
inhibition by boronic discs (indicating the possible presence of KPC enzymes).
Regardless of which technique is chosen for detection and characterisation of carbapenemase genes,
there will be both advantages and disadvantages in relation to costs, accuracy, speed and availability.
Such methods are listed in Table 7 and further elaborated upon below.
Microarray, Real-Time PCR and multiplex PCR analysis are based on oligo or primer hybridization to
conserved parts of a given carbapenemase gene group and will often only identify (sub-)groups of
genes, rather than specific gene variants. In addition, these three methods rarely detect all known
carbapenemase gene groups at the same time. An advantage of PCR-based methods is that they are
relatively cheap and fast to perform, even on larger strain collections. Also, the instruments required to
perform these analyses is often available in many microbial laboratories or can be acquired at
relatively low costs. Identification of specific gene variants beyond (sub-group) level requires
individual PCR protocols covering the complete sequence of the relevant genes followed by Sanger
sequencing and bioinformatic sequence analysis as especially multiplex PCR protocols are prone to
create false positive reactions. As opposed to this, WGS holds the possibility to detect and perform
bioinformatic analysis of all genes present in the isolate, including CP genes. Web-based tools such as
ResFinder15 have been developed in order to allow easy detection of resistance genes including
carbapenemase genes based on WGS data. Bench top sequencers are not common in routine microbial
laboratories, and the cost of sequencing equipment and WGS is currently higher than is the case for
the other three methods, but the combined cost of the various phenotypic and genotypic tests that are
usually required to fully characterise the resistance genes (and other relevant genes) present in the
organism is usually greater than the cost of WGS, so investment in this technology is likely to lead to
reduced longer-term costs.
Multiplex PCR Several different Often only the gene related to (Dallenne et al., 2010;
multiplex PCR assays the carbapenemase phenotype is Poirel et al., 2011;
have been developed to detected at the group level. Hong et al., 2012;
detect carbapenemases. Further analysis using methods Kaase et al., 2012;
Multiplexing allows for such as PCR and sequencing Bogaerts et al., 2013)a
parallel screening for (see below) are also required.
several genes at the same Multiplex PCR assays are often
time. not as robust as singular PCR
and can in some instances
produce unspecific DNA
products.
Multiplex PCR is relatively
cheap and fast to run.
15
http://cge.cbs.dtu.dk/services/ResFinder
Whole genome Genomic DNA WGS generates data ideally (Zankari et al., 2012)
sequencing (WGS) sequences can be containing information on all
generated on various genes present in an isolate,
sequencing platforms. including carbapenemase
Benchtop sequencers production genes.
exist where smaller Bioinformatic analysis is
number of isolates can be required to identify the relevant
sequenced within a few carbapenemase production
days. genes.
Phenotypic tests are relatively easy to perform, but lack discriminatory power. Within serovars, and
phage types of, e.g., Salmonella spp., many different genetically-related clusters of isolates can occur
that can be identified by genotyping methods. Macrorestriction DNA analyses followed by PFGE
16
See also http://pubmlst.org/plasmid/ and http://cge.cbs.dtu.dk/services/PlasmidFinder/
With the recent emergence of WGS, the full genetic information of a set of strains can be extracted
and gene content (including MLST genes) or variations in chromosomal mutations (Single Nucleotide
Polymorphisms, SNP) can be compared, which may be relevant for typing purposes (Didelot et al.,
2012). In principle, SNP analysis allows for bacterial typing with high typeability and discriminative
power, but also presents a challenge in relation to defining criteria for detecting outbreaks and
clonality.
The choice of the molecular typing method to be used is determined by knowledge of the
epidemiological relatedness of the isolates and the main purpose of typing – outbreak recognition and
investigation, source attribution or surveillance. Next to phenotypic methods such as serotyping and
phage typing, PFGE, MLVA and WGS can be used to identify clonal clusters of isolates that are
related to a certain „outbreak‟ in a restricted timeframe. MLST is the method of choice to identify
relatedness of isolates of the same species from different backgrounds (e.g. animal versus human) and
their evolutionary history.
Finally, new approaches allowing high-throughput bacterial typing as Fourier transform infrared
spectroscopy with attenuated total reflectance (FTIR-ATR) coupled with chemometrics have been
recently suggested as a reliable and alternative method to accurately discriminate particular E. coli
clones (Sousa et al., 2013).
A variety of in-house and commercially-available selective media has been used for the active
surveillance of carbapenem resistance in hospitals. The choice of the media for testing animal
and food samples needs to be experimentally evaluated and validated.
Meropenem offers a good balance between sensitivity and specificity and has been
recommended to be included in the harmonised antimicrobial panel for the surveillance of
AMR in isolates from food-producing animals, food thereof and environmental samples.
The identity of the genes responsible for the carbapenemase production should be determined
by molecular methods.
Plasmid and strain typing should be undertaken to acquire better knowledge on the
epidemiology of genes encoding carbapenemase production among bacteria from food-
producing animal populations, food thereof and environmental samples.
As yet only a few findings of resistance to carbapenems in different livestock populations have been
reported (See Chapter 2.1). The situation in both animals and humans is most likely underestimated,
taking into account the “silent” and successful dissemination of certain species of CP bacteria reported
among humans (Gijon et al., 2012; Viau et al., 2012). Therefore, monitoring of all sectors is
considered important, especially given the considerable impact that CP bacterial infections may have
on human health. This emphasises the need to ensure that methods are sufficiently sensitive to allow
detection of low numbers of CP bacteria in the sample. Recently, the CDC has made recommendations
for the control of the spread of CRE in hospitals (CDC, 2012). Selective pre-enrichment may be
required to detect very low numbers of these types of organisms. A harmonized monitoring
programme will deliver important baseline information on the presence and prevalence of resistance.
Based on repeated monitoring, trends over time can be assessed. The basic general principles of
harmonized monitoring of AMR in Salmonella spp. and E. coli, as laid down in the Community
legislation (Directive 2033/99/EC17 and implementing Decision 2013/652/EU18) and EFSA‟s
recommendations (EFSA, 2012a), also apply to monitoring resistance caused by carbapenemases.
Some additional considerations are important for this type of resistance, as currently knowledge is
very limited and emergence of this new hazard needs to be adequately addressed. The following
sections address these specific considerations.
Besides E. coli and Salmonella spp., which are the main target species recommended by EFSA for
monitoring AMR, other micro-organisms should also be considered. Spread of MDR Klebsiella spp.
in farms can cause animal health problems, as reported in the USA and Canada (Kim et al., 2005).
This bacterial species can be isolated from faeces of cows and other sites in farms. Besides problems
with mastitis, an increase in the antibiotic resistance rates of Klebsiella spp in cattle has been
reported19. Other members of the family of Enterobacteriaceae (e.g. Morganella spp., Proteus spp.,
Enterobacter spp., Providencia spp.) belonging to the normal flora of the gastro-intestinal tract should
be considered in monitoring activities as they may carry (multidrug) resistance genes and can cause
severe nosocomial infections. Similarly, Acinetobacter spp. has been isolated from several reservoirs,
and has recently emerged as a pathogen of clinical importance due to its ability to accumulate
resistance mechanisms, including to the carbapenems.
17
OJ L 325, 12.12.2003, p.31-40.
18
OJ L 303, 14.11.2013, p.26-39.
19
http://www.nnyagdev.org/index.php/dairy/research-projects/klebsiella-sources-transmission-control-points/ and
http://www.vet.cornell.edu/news/Klebsiella.cfm
to detect the existence, and determine the prevalence of CNS bacteria in food-producing
animal populations and derived food, including imported from outside the EU;
These objectives may complement the general aims for monitoring AMR in bacteria of animal origin
in the EU. In this document the drafted monitoring is restricted to livestock and food of animal origin.
Other sources, like companion animals, environmental contamination and food of plant origin might
be relevant, but out of the scope of this document.
Decision 2013/652/EU foresees, among other requirements, compulsory testing of randomly selected
isolates of Salmonella spp. and E. coli for resistance to meropenem and further characterization and
classification of those isolates showing resistance to meropenem. Whereas specific monitoring of
ESBL- or AmpC- producing E. coli using more sensitive selective methods will be compulsory
according to this Decision, specific monitoring for CP microorganisms, using more sensitive detection
methods (e.g. using selective pre-enrichment), is up to the individual Member State (MS).
Due to the high public health relevance of carbapenem resistance, several approaches may be
combined to monitor the emergence of such resistance in livestock and food. The importance of the
implementation of current recommendations needs to be highlighted and additional specific
approaches need to be defined in order to overcome the problems of detection due to the
heterogeneous expression of different enzymes coding for CP. EUCAST is in the process of
developing some guidelines to improve the detection of different mechanisms of resistance, including
those conferring CP20.
In current monitoring activities, a sample size of 170 randomly selected isolates is regarded as suitable
for detecting trends in changes of prevalence over time. This sample size is not sufficient for the
timely detection of the emergence of new resistance patterns in the case of no reports or only sporadic
20
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Targeted surveys can either be included into active monitoring programs, where additionally more
sensitive methods are applied to screen samples taken for CP isolates, and/or running surveys in
populations not covered by routine monitoring. For the latter, a combination of routine methods and
more sensitive methods should be envisaged.
antimicrobials prior to sample collection, these samples could also be indicative of the emergence of
carbapenemase-mediated resistance.
Furthermore, Salmonella spp. isolates collected within official control programs not subject to active
monitoring for AMR as required by legislation (e.g. food, feed) may be tested for carbapenem
resistance. Of special interest may be isolates from imported products or food types (e.g. seafood and
meat) considered of higher risk and not covered by routine monitoring or specific surveys (Lavilla et
al., 2008; Gundogan et al., 2011; Nawaz et al., 2012).
In a next step, targeted surveys using more sensitive methods for detection of CNS bacteria should be
run in those populations where active monitoring is required by legislation. Finally, similar targeted
surveys should be started to investigate the presence of CNS bacteria within other relevant
populations. This should include imported foods from countries where carbapenem resistance is
known to be prevalent in bacterial isolates from cases of human infection.
4.3. Bacterial species, food animal species and/or food products to be considered for
monitoring of resistance caused by carbapenemase-producing bacteria from a public
health perspective
In defining combinations of bacteria/animal/food to become subject to mandatory AMR monitoring,
the approach followed by EFSA (2012a) was to prioritise potential consumers‟ exposure. Routine
AMR monitoring focuses on domestic productions, covering Salmonella spp. and E. coli in poultry
(separated by production type), cattle and pigs and products thereof on a regular basis. Complementary
monitoring AMR in poultry meat imported from third countries at the EU level has also been proposed
(EFSA, 2012b, a). For those food-producing animal species and their derived fresh meat (e.g. lamb,
duck, geese, goats) for which consumption is more specific to certain MSs, a threshold mechanism,
calculated on the basis of the animals slaughtered, has been introduced for the monitoring to become
performed consistently in a given MS (EFSA, 2012a).
As a priority, animal species already covered by active monitoring for AMR due to current EU
legislation, such as broilers, fattening turkeys, fattening pigs, veal calves, and the derived fresh meat of
domestic origin, preferably sampled at retail, should be addressed in active monitoring and/or
additional targeted surveys for CP bacteria. The inclusion of the breeding level is considered of special
relevance for carbapenemase monitoring as occurrence of CP bacteria in the top level of the animal
pyramid may contribute to a rapid and wide spread introduction into the production level, as observed
for ESBL/AmpC-producing bacteria. Dairy cattle and raw milk samples should also be addressed in
targeted surveys, due to the specific use of cephalosporins in dairy cattle, and to the risk posed by
potential consumption of raw milk. Aquaculture products may also be included in targeted surveys,
due to the possible presence of resistant bacteria in the aquatic environment. Although not the primary
focus of this document, as carbapenem resistance genes may be spread via effluents (Picao et al.,
2013; Zurfluh et al., 2013) the inclusion of vegetables in targeted surveys could be considered.
EFSA (2012a) recommended additional AMR monitoring in imported food from countries outside the
EU. In the absence of specific data on CNS bacteria, and because of the high prevalence of
cephalosporin resistance, priority should be given to poultry meat imported from countries outside the
EU (EFSA, 2012a). Other types of imported food which may be of special relevance are fishery and
aquaculture products, as well as vegetables and fruits, usually consumed raw. Finally, with regard to
passive monitoring, isolates of Enterobacteriaceae and Acinetobacter spp. from all animal and food
isolates should be included into the carbapenem resistance monitoring.
More specific methods involving pre-enrichment and selective plating should be used in targeted
surveys to increase sensitivity for populations with a very low prevalence of CP microorganisms.
Current knowledge on the available methods has been summarised in Chapter 3.
As regards CP, additionally a risk-based approach should be used involving all isolates from
diagnostic submissions showing cephalosporin resistance. Isolates from animals, previously treated
with antimicrobials should not be excluded from testing but information should be recorded.
Furthermore, animal pathogens should be continuously screened for cephalosporin resistance and
those isolates showing results above the ECOFF value should be subject to further testing for CP and
more detailed characterisation and classification.
As regards frequency of sampling, annual sampling would be optimal, but more intensive sampling
every second or third year was considered an option (EFSA, 2012a). As foreseen in Community
legislation, active monitoring should be performed in the major food producing species every second
year involving phenotypic testing for meropenem resistance. This should be complemented by a
targeted survey searching with sensitive methods for CPE.
Targeted studies in other populations of special interest, including for example the breeding level for
broilers and turkeys, should be run at least once and repeated if new information becomes available,
e.g. from the production level, passive monitoring or other research activities.
As regards carbapenem resistance, sample size calculation has to focus on the detection of this new
resistance pattern and has to take into account the impact of imperfect tests. Two scenarios are
discussed below:
collection of a reasonable number of random isolates for routine screening for phenotypic
resistance;
testing of samples with selective media for the detection of the resistance above a certain
prevalence level within an epidemiological unit (e.g. a flock) or population (e.g. broilers).
4.6.1. Sample size for detecting a certain prevalence level within the isolates collected
The number of biological samples to be collected from each animal population in order to achieve 170
isolates depends on the prevalence of the bacteria species monitored. Whereas for commensal bacteria
this will be achieved by active monitoring of random samples, for other bacterial species targeted
investigations are necessary. For feasibility reasons, a passive surveillance scheme may be
implemented using isolates deriving from diagnostic testing or surveillance activities. At least 170
isolates from each E. coli, Salmonella spp. (if available) and other members of the Enterobacteriaceae
family should be collected from each animal population and production type.
No screening system is available which will detect all CP isolates, and most probably those showing
non-susceptibility to meropenem will be detected. Testing of 170 isolates would allow detection of at
least one positive isolate, given a prevalence of around 2% within the isolates tested (95% C.I.).
Testing of more isolates would be desirable, since it would allow the detection of positive isolates in
conditions of a lower within-isolates prevalence level.
4.6.2. Sample size for the detecting a hazard in a population with a certain prevalence level
As currently only few findings of CP bacteria in livestock have been reported, targeted surveys may be
designed with the purpose of confirming that the prevalence of CP bacteria is likely not above a
certain level.
For example, when using a test with 90% sensitivity and 100% specificity, and assuming an
underlying prevalence of 2%, the detection of at least one positive sample would require testing of 165
samples. In a similar scenario, using a test with a reduced sensitivity of 80% would require increasing
the number of samples tested to 186 samples.
If the prevalence was lower (1% or 0.5%), using a test with 90% sensitivity and 100% specificity
would require testing of respectively 332 or 665 samples in order to detect one positive sample. The
number of samples required would further increase to 665 or 748 samples if the sensitivity of the test
was reduced to 80%.
Similar considerations can be applied to detect the presence of a CP isolate within a farm or flock (as
we assume that the within flock prevalence may be low), which highlights that testing of several
pooled samples from the same epidemiological unit would increase the overall sensitivity.
This type of reporting has to be developed further to cover results of testing of animal pathogens from
passive monitoring activities as this is currently not covered by community legislation or EFSA
recommendations. For isolates from diagnostic submissions, information on antimicrobial treatment
before sampling should be provided.
All isolates with phenotypic CNS should be further analysed in the National Reference Laboratory and
reporting of data should include these confirmatory results.
As regards reporting on CNS, for correct interpretation of the results it is important that the sampling
strategy (active vs. passive monitoring) and the selection procedure for each isolate (randomly selected
isolate vs. isolate from selective media) applied are reported. In cases where selective methods have
been used, such methods should be described in detail. Results of phenotypic resistance testing for
cephalosporins and carbapenems, synergy testing as well as further characterisation of resistance
genes should be reported for each isolate.
Since some carbapenemase-producing strains have been identified in food animals and their
environment, more detailed investigation is now required to determine the extent and
distribution of such strains in the food animal ecosystem.
For active monitoring, all isolates of Salmonella spp. and E. coli collected within the
compulsory monitoring programme, as required by European legislation, should be screened
for meropenem resistance using standardized microdilution methods.
Specific targeted surveys for the detection of carbapenemase-producing organisms in the food
animal ecosystem should be implemented at the EU level.
For passive monitoring, diagnostic isolates of veterinary origin (at least those classified as
microbiologically resistant to 3rd- or 4th-generation cephalosporins on the basis of
epidemiological cut-off values) should be subjected to phenotypic testing for carbapenem
resistance and carbapenemase production, and subsequent molecular identification and
characterization of the carbapenemase production genes present.
For correct interpretation of results relating to carbapenem resistance, the sampling strategy
(active vs. passive monitoring) and the selection procedure applied for each isolate (randomly
selected isolate vs. isolate from selective media) should be reported. Results of phenotypic
methods used for testing for carbapenemase production as well as the results from further
characterisation of resistance genes should be reported for each isolate.
Methods involving pre-enrichment and selective plating should be used in specific surveys to
increase sensitivity for populations with a low prevalence of carbapenemase-producing
microorganisms.
5. Possible control options for preventing or minimising the further emergence and
spread of carbapenemase-producing bacterial strains transmitted via the food chain.
5.1. Introduction
Public health risks caused by CP bacteria are primarily determined by (i) the frequency of the
occurrence (prevalence) and the quantity of these organisms in food-producing animals and food, (ii)
the genetic characteristics of the carbapenemase genes involved, and (iii) the frequency and magnitude
of transmission from animals/foods to humans. In the first instance proposed mitigation measures must
therefore be targeted at preventing the introduction of such strains into food-producing animals,
secondly, at reducing the prevalence and quantity of such organisms in food-producing animals and
foods thereof, and thirdly, in reducing their transmission from contaminated animals/foods to humans.
In contrast to possible control options to reduce the public health risk caused by the transmission
through the food chain or via the food-producing animal environment of ESBL- and/or AmpC-
producing bacterial strains to humans (EFSA Panel on Biological Hazards (BIOHAZ), 2011; Liebana
et al., 2013), the impact of similar measures for CP bacteria are difficult, if not impossible, to assess.
This is primarily because, as discussed above, CP microorganisms are only just emerging in food-
producing animals and their environment, and the prevalence of such strains in the food chain is
supposedly very low.
In view of the importance afforded to carbapenems in the treatment of human infections with multiple
drug-resistant or pan drug-resistant pathogens in health care settings or the community (see above), the
control options discussed below should be considered.
5.2. Use of carbapenems and other antimicrobials in food-producing animals and human
medicine
All efforts should be made to continue to regard carbapenems as Critically-Important Antimicrobials
(CIAs) (WHO, 2005, 2007, 2011, 2012), that should be reserved specifically for the treatment of
severe MDR disease in humans, and not used in food-producing animals.
At present carbapenem antibiotics are not licensed for use in food-producing animals in the EU, North
America, and Australasia. Information on the permitted use of such antibiotics in food-producing
animals in third countries is not readily available. In any event, the use of such antibiotics in food
animals globally should be actively discouraged.
Mirroring what has been observed with ESBL-encoding genes, carbapenemase genes are often located
on genetic structures (plasmids, transposons, integrons) bearing genes conferring resistance to other
antibiotics, co-selection both in animals and in the environment is a real possibility. In addition, co-
resistance is an important issue in CP microorganisms, and carbapenemases are mostly plasmid-
encoded (with several antibiotic resistance genes co-located on such plasmids).
The importance of the prudent use of antibiotics in combating the development of resistance to
antimicrobials in food-producing animals has been well-documented (Anthony et al., 2003; ECDC et
al., 2009; EFSA Panel on Biological Hazards (BIOHAZ), 2011). Decreasing the frequency of use of
antibiotics in animals is currently being afforded high priority in numerous fora. Efforts to minimise
overall antibiotic usage, in particular of cephalosporins, in food-producing animals must be actively
continued. This is also important in relation to carbapenem resistance in light of the co-resistance
issues documented above.
Recommendations for reducing total antimicrobial use in animals have been made previously (EFSA
Panel on Biological Hazards (BIOHAZ), 2011), and should be followed in accordance with prudent
use guidelines. Furthermore, since carbapenemase production genes and genes encoding resistance to
certain heavy metals such as zinc are sometimes linked, the use of compounds containing such
elements should also be minimised.
As an example, containment plans were formulated in several countries in the EU in the early 2000s to
combat the possible introduction of AmpC-producing Salmonella Newport into the EU from the USA.
Such plans included restrictions on the index case farm, intensive investigations of all trading links
and contiguous premises, and risk management decisions on compensation. If necessary such plans
could be revisited in anticipation of the appearance of CP strains in livestock.
Measures for the containment of CP organisms at the farm level may range from identification and
isolation of carriers, animal quarantine through to destruction of infected flocks / herds. Restrictions in
the movement of personnel (farm staff, veterinarians, produce delivery staff, etc.) between farms,
implementing increased farm biosecurity, controls on animal trade (of CP carriers) and trade of animal
by-products, or by improving hygiene throughout the food chain (directing products to heat treatment;
specific harvesting requirements; implementing general post-harvest controls for food-borne
pathogens) may also be considered.
targeted at identifying the point of introduction of the CP strain, the level of spread which has occurred
within a farm, between farms, their environment and the staff. Farms from which animals have been
received should be included in the analysis. In case of findings in foodstuffs, the farms from which
these had originated should be included into the targeted search.
The most sensitive methods should be used for all investigations and isolates showing CP, and should
be targeted for detailed genotypic comparison.
In cases where containment measures are taken as recommended in chapter 5.3., targeted surveys
should be started to verify the efficiency of the measures taken.
Because of the importance of carbapenems in human medicine, should resistance to such antibiotics be
identified in bacteria from food-producing animals in targeted surveys, then specific containment
measures, such as the ones described in Chapter 5.3 above, may be considered.
As carbapenems are not licensed for use in food-producing animals in the EU and other parts
of the world, one simple and effective control option to minimise the further emergence and
possible spread of such strains transmitted via the food chain would be to continue to prohibit
the use of carbapenems in food-producing animals.
The effectiveness of any control measures should be monitored on a regular basis by targeted
surveys of food-producing animals and foods for carbapenemase-producing bacteria, using
selective isolation methods and pre-enrichment of samples as necessary.
CONCLUSIONS
General conclusions
Carbapenems are broad-spectrum beta (β)-lactam antimicrobials mostly used for the treatment
of serious infections in humans, frequently in hospitalised patients, and are considered last-
line therapy for infections caused by multidrug-resistant Gram-negative bacteria.
Carbapenem MICs may vary widely ranging from full susceptibility to high-level resistance
according to CLSI or EUCAST clinical breakpoints. Low levels of resistance to carbapenems
are more often observed among Enterobacteriacae.
Factors that favour the emergence of carbapenem resistance include the increased
consumption of carbapenems in humans driven in part by the worldwide spread of ESBLs in
Enterobacteriacae, the location of carbapenem-encoding resistance genes on mobile genetic
elements, and positive selection due to co-resistance with other commonly-used antibiotics.
Answer to ToR1 and ToR2. Define the carbapenemase-producing bacterial strains and genes
relevant for public health and linked to food-producing animals or food-borne transmission.
Review the information on the epidemiology of acquired resistance to carbapenems, including
the genes coding for such resistance, in food-producing animals and food.
Carbapenemase-producing isolates from companion animals and from a wild bird have also
been reported. Genes identified have included blaOXA-48 and blaNDM-1 in Enterobacteriaceae and
blaOXA-23 in Acinetobacter spp.
Answer to ToR3. Perform a critical analysis of the methods (phenotypic and genotypic) and the
interpretive criteria currently used for detection (isolation and identification) and
characterisation of carbapenemase-producing bacterial strains.
A variety of in-house and commercially-available selective media has been used for the active
surveillance of carbapenem resistance in hospitals. The choice of the media for testing animal
and food samples needs to be experimentally evaluated and validated.
Meropenem offers a good balance between sensitivity and specificity and has been
recommended to be included in the harmonised antimicrobial panel for the surveillance of
AMR in isolates from food-producing animals, food thereof and environmental samples.
The identity of the genes responsible for the carbapenemase production should be determined
by molecular methods.
Plasmid and strain typing should be undertaken to acquire better knowledge on the
epidemiology of genes encoding carbapenemase production among bacteria from food-
producing animal populations, food thereof and environmental samples.
Answer to ToR4. Make recommendations for the harmonised monitoring and reporting of
resistance (phenotypic and genotypic) caused by carbapenemases in food and food-producing
animals in the EU.
Requirements for the collection and reporting of antimicrobial resistance data, including
resistance to carbapenems, are laid down in European legislation.
Since some carbapenemase-producing strains have been identified in food animals and their
environment, more detailed investigation is now required to determine the extent and
distribution of such strains in the food animal ecosystem.
For active monitoring, all isolates of Salmonella spp. and E. coli collected within the
compulsory monitoring programme, as required by European legislation, should be screened
for meropenem resistance using standardized microdilution methods.
Specific targeted surveys for the detection of carbapenemase-producing organisms in the food
animal ecosystem should be implemented at the EU level.
For passive monitoring, diagnostic isolates of veterinary origin (at least those classified as
microbiologically resistant to 3rd- or 4th-generation cephalosporins on the basis of
epidemiological cut-off values) should be subjected to phenotypic testing for carbapenem
resistance and carbapenemase production, and subsequent molecular identification and
characterization of the carbapenemase production genes present.
For correct interpretation of results relating to carbapenem resistance, the sampling strategy
(active vs. passive monitoring) and the selection procedure applied for each isolate (randomly
selected isolate vs. isolate from selective media) should be reported. Results of phenotypic
methods used for testing for carbapenemase production as well as the results from further
characterisation of resistance genes should be reported for each isolate.
Methods involving pre-enrichment and selective plating should be used in specific surveys to
increase sensitivity for populations with a low prevalence of carbapenemase-producing
microorganisms.
more detailed analysis of all isolates showing non-susceptibility to carbapenems above the
ECOFF value.
Answer to ToR5. Identify possible means of preventing or minimising the further emergence
and spread of carbapenemase-producing bacterial strains transmitted via the food chain,
including consideration of the advantages and disadvantages of different options.
Because there are no data on the comparative efficacy of individual control options in
reducing the potential public health risks caused by carbapenemase-producing bacteria related
to food-producing animals, prioritisation is complex.
As carbapenems are not licensed for use in food-producing animals in the EU and other parts
of the world, one simple and effective control option to minimise the further emergence and
possible spread of such strains transmitted via the food chain would be to continue to prohibit
the use of carbapenems in food-producing animals.
The effectiveness of any control measures should be monitored on a regular basis by targeted
surveys of food-producing animals and foods for carbapenemase-producing bacteria, using
selective isolation methods and pre-enrichment of samples as necessary.
ADDITIONAL RECOMMENDATION
Fruit and vegetables, particularly those which are more prone to bacterial contamination and
are usually consumed raw, should be assessed for contamination with bacteria with acquired
carbapenemases.
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ABBREVIATIONS
AMR Antimicrobial drug resistance
BIOHAZ Biological Hazards (EFSA Panel on)
BIOMO Biological Monitoring (EFSA Unit on)
BSI Bloodstream infections
CIAs Critically-Important Antimicrobials
CDC Centers for Disease Control and Prevention (USA)
CDT Combined Disk Test
CHDLs Carbapenem hydrolysing class D -lactamases
CLSI Clinical Laboratory Standards Institute (USA)
CNS Carbapenem-non-susceptible / carbapenem non-susceptibility
CNSE Carbapenem-non-susceptible Enterobacteriaceae
CP Carbapenemase-producing / carbapenemase production
CPE Carbapenemase-producing Enterobacteriaceae
CRE Carbapenem-resistant Enterobacteriaceae
CSF Cerebrospinal fluid
EARS-Net European Antibiotic Resistance Surveillance System
ECDC European Centre for Disease Prevention and Control
ECOFF Epidemiological cut-off value
EFSA European Food Safety Authority
EMA European Medicines Agency
ESBL Extended Spectrum Beta (β)-Lactamase
EU European Union
EUCAST European Union Committee for Antimicrobial Susceptibility Testing
FTIR-ATR Fourier transform infrared spectroscopy with attenuated total reflectance
Inc Incompatibility group
IS Insertion sequence
KPC „Klebsiella pneumoniae carbapenemase‟
MALDI-Tof Matrix-assisted laser desorption/ionization (MALDI)- Time-of-flight mass
spectrometry (Tof)
MBL Metallo β-lactamase
MIC Minimal Inhibitory Concentration
MDR Multidrug-resistant
MGE Mobile genetic elements
MLST Multi locus sequence typing
MLVA Multiple-locus variable number tandem repeat analysis
MS(s) Member State(s)
MYSTIC Meropenem Yearly Susceptibility Test Information Collection Program
(USA)
NHSN National Nosocomial National Healthcare Safety Network
NNIS National Nosocomial Infection Surveillance System
OXA Oxacillinases
PBP Penicillin binding protein
PDR Pan drug-resistant