Fighting the rising tide of carbapenemases in Enterobacteriaceae

I N T E R N A T I O N A L N E W S L E T T E R / O C T O B E R 2 0 1 2 editorial In the context of multi-drug resistance, the identifi cation of an increasing number of carbapenemase-producing Enterobacteriaceae is worrying because carbapenems represent the last line of effi cient antibiotics for treating many enterobacterial infections. The reservoirs identifi ed for carbapenemase producers lie not only in developing countries. A recent KPC outbreak at the main National Institutes of Health (NIH) hospital in Washington D.C. clearly shows that the problem may arise even in the most advanced hospital facilities*. In this newsletter, Prof. Neil Woodford reviews the situation worldwide, and ponders what can be done to prevent a nightmare that is becoming reality, i.e. pan-drug resistance. STaTe-OF-THe-aRT HIGHlIGHT PRODUCT NeWSFlaSH Fighting the rising tide of carbapenemases in Enterobacteriaceae STaTe-OF-THe-aRT Diagnostic solutions for carbapenemase producing Enterobacteriaceae chromid CARBA LyfoCults Plus Fighting the rising tide of carbapenemases in Enterobacteriaceae Prof. neil Woodford Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Health Protection Agency, Microbiology Services Colindale, London, United Kingdom In the face of this threat, we must focus on the use of early detection techniques, implementation of hygiene control measures to prevent infections and onward transmission, and the development of novel antibiotic molecules that may be marketed soon. Prof. Patrice nordmann Director, research Unit InSerm 914 emerging resistance to antibiotics Director, national reference Center for antibiotic resistance Bicêtre University Hospital, France * http://haicontroversies.blogspot.fr/2012/08/scary-kpcoutbreak-at-nih.html When we started to use antibiotics over 70 years ago, we chose to pick a fight with evolution by exerting new and successive waves of selective pressure on bacteria. These bacteria have, of course, proven themselves only too able to counter the repeated challenges posed by newly introduced antibacterial agents. Resistant bacteria are a daily problem for clinical microbiologists, who require ever more powerful agents to treat infections. In some countries, including the UK, the use of thirdgeneration cephalosporins and fl uoroquinolones has declined in recent years as part of efforts to control Clostridium diffi cile and/or bacteria with ESBLs. This prescribing gap has been fi lled by an increased reliance on carbapenems (our most powerful ß-lactams) and on penicillin/ ß-lactamase inhibitor combinations. This change in practice has coincided with the emergence of acquired carbapenemases, and has provided selective pressure to encourage their spread. These carbapenem-hydrolysing enzymes are a cause of grave concern in all fi ve Gram-negative members of the so-called eskapee pathogens (Table 1), which collectively cause the majority of healthcare-associated infections. This concern is warranted because there are too few active antibiotics left on the formulary shelf, and the developmental pipeline of new agents now offers little more than a trickle. a key challenge for the next decade is to limit the spread of carbapenemase producers and so minimize their impact on public health.

Table 1 High-impact resistances associated with the eskapee pathogens. Gram-negative genera dominate, with acquired carbapenemases an issue in all. Pathogen established resistance problems E. faecium Glycopeptides ; highlevel aminoglycosides ; ampicillin S. aureus Healthcare and community MRSA emerging resistance threats Linezolid ; daptomycin ; tigecycline Vancomycin nonsusceptibility or resistance; linezolid ; daptomycin Klebsiella ESBLs Carbapenemases, colistin Acinetobacter MDR ; Carbapenemases Tigecycline ; colistin Pseudomonas MDR ; except colistin Carbapenemases ; colistin Enterobacter AmpC ; ESBLs Carbapenemases ; other carbapenem resistance, colistin E. coli Fluoroquinolones ; ESBLs Carbapenemases CLaSSIFICaTIOn OF CarBaPenemaSeS acquired carbapenemases are diverse, with representatives in three of the four Ambler ß-lactamase classes, but emerging global problems in the Enterobacteriaceae are associated primarily with the big fi ve (Table 2). Class a carbapenemases are dominated by the KPC family (Klebsiella pneumoniae carbapenemase). Don t be fooled by this misnomer because KPC enzymes can be found in many members of the Enterobacteriaceae and, currently less often and with geographical restriction, also in Acinetobacter and Pseudomonas. By contrast with KPC variants, other class A carbapenemases (IMI, NMC and SME enzymes) are reported only rarely. Class B metallo-carbapenemases include three globallydominant families, the IMP (imipenemase), VIM (either Verona imipenemase or Verona integron-associated metallo) and, most recently, NDM (New Delhi metallo) variants. There are several other acquired class B carbapenemases, and although these have not yet become global issues some are a concern in particular countries, notably SPM (Sao Paulo metallo) enzyme, which is found in Pseudomonas aeruginosa throughout Brazil. The acquired class D carbapenemases are highly diverse, refl ecting the diversity of this entire ß-lactamase class. Four subtypes (OXA-23-like, OXA-40-like, OXA-58-like and OXA-143-like) are restricted almost entirely to Acinetobacter spp., while OXa-48-like enzymes are a growing concern in the Enterobacteriaceae. Carbapenem non-susceptibility in the Enterobacteriaceae is a growing problem across the European region. Although EARS- Net* data for 2010 show that rates in K. pneumoniae exceeded 5% in four countries - Greece (60%), Cyprus (18%), Italy (16%), and Hungary (6%) - non-susceptible isolates were observed in many others (1). In 2010, countries participating in EARS-Net all reported carbapenem non-susceptibility in <1% of Escherichia coli, but reports of carbapenemases in this species are also increasing, which forewarns of their potential to escape from hospital settings and to follow extended-spectrum beta-lactamases (esbls) into the community. Of course these surveillance data do not indicate underlying resistance mechanisms. Many instances of carbapenem resistance will refl ect carbapenemase production, but one must remember that members of the Enterobacteriaceae can also become non-susceptible (or resistant) to carbapenems by combining production of ESBLs or intrinsic / acquired AmpC enzymes with loss of outer membrane porins. The epidemiology of carbapenemases has been extensively documented globally (2) and across europe (1) (Figure 1). In the UK, the Health Protection Agency (HPA) has observed dramatic increases in the numbers of carbapenemase-producing Enterobacteriaceae submitted for reference investigation. In 2003-2007, fewer than fi ve isolates per year were reported (most associated with repatriation from countries with established problems, such as Greece, Israel and Turkey), but by 2011 this fi gure had risen to over 550 confi rmed isolates (not patients) (Figure 2). Most of these isolates were K. pneumoniae, but E. coli accounted for around 10%. The HPA has confi rmed all of the big fi ve carbapenemases in referred isolates of Enterobacteriaceae, with VIM, IMP and NDM metallo-enzymes also found in isolates of Pseudomonas and, less commonly, Acinetobacter; OXA-type carbapenemases, particularly OXA-23, remain the dominant types in Acinetobacter. In terms of absolute numbers, KPC enzymes dominate among UK referrals of Enterobacteriaceae, but the NDM and OXA-48-like enzymes have wider national scatter. Table 2 acquired carbapenemases highlighting (in red) the big five enzyme types. Class Carbapenemase Enterobacteriaceae Nonfermenters A (non-metallo) KPC +++ + IMI, NMC, SME + - B (metallo) IMP, VIM +++ +++ NDM +++ ++ AIM, DIM, KHM, - ++ SIM, SPM, TMB D (non-metallo) OXA-48-like +++ - OXA-23, -40, -58, -143 +/- +++* * Only in Acinetobacter spp. 2

3 Fighting the rising tide of carbapenemases in Enterobacteriaceae FIGURe 1 european situation regarding carbapenemase-producing Enterobacteriaceae, using an epidemiological scale of nationwide expansion and carbapenemase types in different countries or geographical areas known until January 2012. Reproduced from citation (1). Endemic Interregional spread Regional spread Independent hospital outbreaks Single-hospital outbreaks Sporadic occurrence Not reported / no data FIGURe 2 KPC VIM NDM OXA-48 Other countries: Israel Luxembourg Numbers of confi rmed carbapenemase-producing clinical isolates referred to the HPa from UK laboratories. Non-fermenters are Acinetobacter spp. and Pseudomonas spp. Data exclude Acinetobacter spp. with OXa-type carbapenemases. 600 500 400 300 200 100 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Enterobacteriaceae Non-Fermenter non-metallo-carbapenemases The KPC family currently comprises 12 enzymes, which differ from each other by very few amino acids. As mentioned above, the epithet KPC is somewhat misleading since the genes have been found widely in many genera of the Enterobacteriaceae. In parts of the Caribbean, Central America and northern regions of South America, KPC carbapenemases have also been found in Acinetobacter and Pseudomonas spp. The fi rst member, KPC-1, was reported in the mid-1990s, but DNA resequencing subsequently showed identity of its gene with bla KPC-2. The KPC-2 and/or KPC-3 variants are the predominant members of the family in most countries. These two variants differ by a single amino acid, His272 Tyr, but whether there is any biological benefi t or cost associated with either, has not been defi ned. It is striking, however, that some KPC-encoding plasmids differ only by the single nucleotide polymorphism (SNP) that causes this substitution in the KPC enzyme, which suggests positive selection. The bla KPC genes are typically located on Tn4401-like transposable elements and are often found on plasmids belonging to incompatibility groups IncN and also IncFII. The latter group is represented by plasmid pkpqil, sequenced originally in Israel (3), but which has been found widely, including in the UK. There is therefore evidence for the movement of Tn4401-like elements on to diverse plasmid backbones. However, much of the success of KPC enzymes can be attributed to the strong association of KPC-2/-3 variants with a single clonal lineage of K. pneumoniae, multi-locus sequence type (ST), ST258 (4). This clone is internationally disseminated and has caused large hospital outbreaks of infections in the USA, Greece and Israel. Many other countries experiencing a KPC producer for the fi rst time will fi nd it to belong to ST258. KPC-positive isolates of this clone share a multi-drug resistance profi le, but typically remain susceptible to gentamicin and colistin, and have borderline susceptibility to tigecycline. However, colistin-resistant ST258 variants have been described in several countries, further restricting treatment options. There are some notable exceptions to the clonal ST258 nature of the global KPC problem; in Spain KPC is associated with K. pneumoniae ST384 (5), while in north-west England the horizontal transmission of resistance plasmids into multiple K. pneumoniae STs and other species is the dominant feature. OXa-48 carbapenemase was fi rst identifi ed in an isolate of K. pneumoniae collected from Turkey in 2001 and producers have caused hospital outbreaks in that country. Early examples of OXA-48 producers in Europe, including the fi rst isolate detected in the UK, were often associated with repatriations from Turkey, but more recently this carbapenemase has become linked to the Middle East and North Africa. Limited data (since there are no national or regional surveillance programmes) suggest that OXA-48 is widespread throughout North Africa, including Egypt, Libya, Morocco, and Tunisia. During a six-month period (October 2009 March 2010), the bla OXA-48 gene was detected in 14% of cephalosporin-resistant K. pneumoniae isolates (belonging to four clones) in one Tunisian hospital (6). Indeed, OXA-48 producers were detected, either as colonists or causes of infection, in victims of the recent confl icts in North African countries who were treated in European hospitals. Other carbapenemases closely related to OXA-48 include OXAs -181 (which is geographically linked with India), -162, -163, 204 and 232. The ancestral hosts of most of the resistance genes that are detected regularly in clinical isolates remain undefined; the source of TEM-1 penicillinase, for example, is unknown. Among the big fi ve carbapenemases, a source has been identifi ed only for the OXa-48- like enzymes, the genes for which have escaped from the chromosomes

4 of Shewanella spp., environmental organisms from lake and river sediments, but isolated occasionally from clinical specimens. The OXA-48-like carbapenemases have been reported as acquired determinants only in members of the Enterobacteriaceae. ae. OXA-48 carbapenemase has been reported in multiple STs of both K. pneumoniae and E. coli and in other genera. Nevertheless, there is some association of this carbapenemase with a disseminated host strain; K. pneumoniae e isolates with OXA-48 enzyme and belonging to ST395 have been found in several countries (France, Morocco and The Netherlands) and have caused hospital outbreaks. However, this is not comparable in scale or distribution to the much stronger association between K. pneumoniae ST258 and KPC enzymes. The bla OXA-48 gene is typically fl anked by IS1999 elements, which may facilitate its mobilization through transposition. Surprisingly, however, the molecular epidemiology of OXA-48 carbapenemase does not indicate that such events are frequent. At the current time, the prime driver for OXA-48 dissemination is the horizontal spread of a successful plasmid. Highly related IncL/M plasmids of around 62 kb have been found to encode OXA-48 in diverse host strains and species in many countries (7). The potential for signifi cant changes to this current epidemiology is, however, illustrated by a report from Tunisia, where the OXA-48 gene was found on IncA/C plasmids that also encoded CMY-4 acquired AmpC enzyme (6). A major concern with OXA-48 producers is that they typically remain susceptible to oxyimino-cephalosporins unless they also produce an ESBL or AmpC ß-lactamase. Most have these additional enzymes, but this can make inferring the presence of a carbapenemase particularly problematic and prone to subjective error (see Detection below) which means there is potential for OXa-48 carbapenemase to spread unnoticed. metallo-carbapenemases The three major families in this sub-group all have zinc ions as a critical part of their active sites and so they are collectively called metallo-carbapenemases, metallo-ß-lactamases, or MBLs. The zinc ions are essential for enzyme activity, which is why these carbapenemases can be inhibited by EDTA and other metal ion chelators. ImP-1 carbapenemase was the fi rst carbapenemase reported in the Enterobacteriaceae. It was found fi rst in Japan, in Pseudomonas aeruginosa in the late 1980s and in an isolate of Serratia marcescens in 1991. There are now at least 39 known sequence variants in the IMP family (www.lahey.org/studies), but these carbapenemases rank fi fth in prevalence among the big fi ve in many European countries (though they are more dominant in the Far East). VIm-1 enzyme was fi rst reported in 1997 in Italy in an isolate of Pseudomonas aeruginosa. The VIM family of carbapenemases includes at least 37 variants and these are more numerous that IMP-types in most countries. The genes that encode the VIM and IMP carbapenemases exist as gene cassettes and so can be captured by integrons. This feature contributes much to the success of these two families, which are widely distributed geographically, and are the dominant acquired carbapenemases in Pseudomonas spp. VIM and IMP enzymes have also been found in Acinetobacter spp., but MBL-producers are outnumbered vastly by the clones of A. baumannii with intrinsic or acquired OXA-type carbapenemases (though OXA-48 has not been reported in the genus). ndm-1 carbapenemase was fi rst detected in 2008 in Sweden in isolates from a patient who had been hospitalized ized in India. Many of the NDM producers reported in other countries were also isolated from patients with healthcare contact ct and/ or travel to the Indian subcontinent. The oldest known n NDMpositive isolates, dating from 2005/6, also come from India. A number of affected patients have links only to the Balkan an region, which suggests a second epicentre. Worryingly, a third group of patients have no history of foreign travel or contact with healthcare settings, suggesting autochthonous acquisition of the NDM producer, and implying the existence e of unrecognized reservoirs in some countries, possibly involving the community. The bla NDM genes (there are at least 7 variants) are not integron cassettes, but have been detected ected on a range of plasmids, in a huge number of species and genera. They are often fl anked upstream (and sometimes also downstream) by an intact copy or remnant of an ISAba125 element and usually have a bleomycin resistance gene immediately downstream. It has been proposed that bla NDM and the bleomycin resistance genes were mobilized together from an undefi ned host species into Acinetobacter where they became associated with the insertion sequence(s), s), which then facilitated wider dissemination, including into the Enterobacteriaceae.

5 Fighting the rising tide of carbapenemases in Enterobacteriaceae ndm-1 FOCUSeS InTernaTIOnaL attention On CarBaPenemaSeS NDM enzymes have achieved greater publicity and notoriety in political and mass media arenas that any other carbapenemase... even though NDM-1 was fi rst identifi ed only in 2008. A Lancet Infectious Disease article on NDM-1 (8) caused a global media storm in September and October 2010, by highlighting the key role of overseas hospitalization and/or international travel as a risk factor for colonization or infection by carbapenemase producers and other multi-drug-resistant bacteria. NDM-1 had been reported a full year previously, but had passed unnoticed except by microbiologists. Travel as a risk factor had, of course, been widely recognised for several years, not least in the context of ESBL producers; countries with high prevalence serve as reservoirs. For example, the fi rst UK cases of K. pneumoniae with VIM enzymes were in patients repatriated from Greece, those with KPC were from Greece or Israel, the UK s fi rst OXA-48 producer was a road traffi c accident victim repatriated from Turkey, and many NDM producers were from patients linked to the subcontinent. Nevertheless, the intense attention of the world s media in 2010 prompted demands by national bodies and the governments of many countries for screening initiatives. As a result, many countries have introduced guidelines, recommendations or advice on the detection of carbapenemases. There is no consensus methodology, nor is there a uniform approach to deployment. Some countries went so far as to make NDM producers notifi able (though, curiously, did not always do so for other carbapenemases). Most regard patients with a history of hospitalization overseas as high risk, and either advise (e.g. UK, USA) (9, 10) or mandate (e.g. France) (11) that such patients should be screened for multi-drug-resistant bacteria on admission to hospital. The defi nition of high risk must, of course, refl ect local, regional or national carbapenemase problems in addition to global distribution and should be reviewed regularly. DeTeCTIOn A simple Carbapenemase: Yes or No result would be suffi cient for most diagnostic laboratories and infection control teams, with positive isolates saved or referred for further investigation. However, the ranges of carbapenem MICs for Enterobacteriaceae producing the big five Table 3 carbapenemases span from below the susceptible breakpoints to high-level resistance and, when enzyme combined with the diversity of carbapenemase produced types, this means that few, if any, strategies will reliably detect all producers (12). When trying to detect these enzymes, clinical laboratories should therefore be aware of two confounders: (i) not all carbapenem-resistant isolates produce a carbapenemase (resistance can be mediated by the combination of ESBL / AmpC plus impermeability), (ii) not all carbapenemase producers are resistant to carbapenems. Oxyiminocephalosporins + clavulanic acid The level (or lack) of carbapenem resistance displayed by some carbapenemase producers is a genuine cause for concern. Higher MICs are observed when producers also lack major porins, but the transfer of carbapenemase-encoding plasmids into laboratory strains of E. coli often does not result in MICs above the clinical breakpoints for all carbapenems. This indicates potential for some genes to spread undetected among normally-permeable strains. In reality this concern is probably greatest for OXA-48-like enzymes, since KPC enzymes and MBLs would be expected to have broader effects on the resistance profi le of the host strain. Concerns about carbapenemases justify that all clinically-signifi cant Gram-negative bacteria should be screened routinely for susceptibility to at least one carbapenem. Although ertapenem appears to be the most sensitive indicator, it is most affected by porin-mediated mechanisms and so is not specifi c for carbapenemases in Enterobacteriaceae; it is also inappropriate for use vs. non-fermenters. Phenotypic methods for screening for carbapenem-resistant Enterobacteriaceae (e.g. gut colonisation) range from carbapenem discs placed on to MacConkey or CLED agar plates to commercial chromogenic agars, but do not distinguish carbapenemase producers from other resistant isolates. To do so, diagnostic laboratories might consider using the Modifi ed Hodge Test, but this lacks specifi city and there are also concerns over its sensitivity. automated methods are often able to detect carbapenem resistance, but the ability of their software to infer and warn correctly of the presence of a carbapenemase is more variable, especially for OXA-48-like enzymes (13). Spectrophotometry offers perhaps the ultimate traditional method for detecting carbapenemase activity phenotypically, but is research-oriented and not practicable for use in most diagnostic laboratories. The increasing acceptance of MalDI-TOF technology presents a good opportunity for more laboratories to investigate carbapenem hydrolysis rapidly and to distinguish carbapenemase producers from carbapenem-resistant isolates with other resistance mechanisms (14). Most recently, a rapid (less than 2 hours) chromogenic test for carbapenemase production in 96-well format has been reported and this merits further investigation (15). Whichever method is chosen, the prime advice to diagnostic laboratories must be for a high index of suspicion. Arguably it is better for them to deploy highly sensitive frontline methods at the expense of decreased specifi city. A second line of supplemental tests may then be used to investigate suspected isolates further. Some of these supplemental tests are shown in Table 3. Classic phenotypic profiles associated with ß-lactamases of public health importance. Synergy between aztreonam Temocillin MIC > 64 mg/l Oxyiminocephalosporins + cloxacillin Oxyiminocephalosporins or carbapenems + boronic acid Carbapenem + EDTA or dipicolinic acid ESBL +++ - - - R - AmpC - +++ +++ - R - + porin loss +/- +/- +/- - R - MBL - - - +++ S a + + / - KPC + - +++ - R - OXA-48 - a - - - S a +++ a Note that many clinical isolates fail to show these classic phenotypes because they co-produce other ß-lactamases. Hence many MBL producers are resistant to aztreonam, and many OXA-48 producers are highly resistant to oxyimino-cephalosporins with strong clavulanate synergy owing to ESBL production.

The utility of inhibitors to define carbapenemase producers is most obvious for producers of metallo-enzymes where good synergy is seen between EDTA or dipicolinic acid and carbapenems, but the specificity of this test falls when used with non-fermenters. KPC enzymes can be inhibited by boronic acid, which provides a diagnostic aid when combined with a carbapenem. AmpC enzymes are also inhibited by boronic acid, but producers can be distinguished from KPC producers since they also show potentiation of carbapenems by cloxacillin. There are currently no good inhibitors of OXA-48-like carbapenemases, but these enzymes should be suspected in isolates that show reduced susceptibility to any carbapenem and are also highly resistant to both piperacillin/tazobactam and temocillin, irrespective of their degree of (non-)susceptibility to oxyimino-cephalosporins. Of course, genotypic methods offer the only definitive means of further characterizing a suspected carbapenemase producer. Myriad block-based and real-time PCR assays have been described, including multiplexes. There are a growing number of commercial PCR- or array-based systems for detecting carbapenemase genes. These vary in their coverage of the big five and the extent to which users can customize the assays (e.g. by selecting genes to be sought). Several systems will detect all of the dominant types in a single test, and many can be used to seek carbapenemases directly in clinical material to identify infected or colonised patients rapidly. Treatment Therapy of patients infected by carbapenemase producers must be guided by the susceptibility profile of the causative organism and the site of infection. Both EUCAST** and CLSI*** advocate using carbapenems against carbapenemase producers if MICs fall in the susceptible range. There are similar recommendations for use of oxyimino-cephalosporins vs. ESBL producers. These recommendations are much debated, not least because they require accurate MIC determination in clinical laboratories. Recent data from UKNEQAS****, which distributed a KPC-producing ST258 K. pneumoniae isolate, are not encouraging in this regard. None of the participating laboratories could be scored on their ability to detect carbapenem resistance owing to the scatter of results obtained (16). There are clear issues with laboratory detection when consensus cannot be reached on the carbapenem susceptibility or resistance of a single isolate that belongs to an internationally-recognized lineage. If a carbapenem is to be considered as a treatment option, it seems prudent to combine it with another agent to which the isolate remains susceptible (17, 18). Only colistin remained active against 90% of all carbapenemase producers from the UK (Table 4). Many carbapenemase-producing bacteria are multi-resistant owing to the presence of other resistance genes either directly linked on the carbapenemase-encoding plasmid or co-resident on other plasmids or in the chromosomes of the host strains. Nevertheless, some producers have only the carbapenemase gene and remain susceptible to non-ß-lactams (as illustrated in Table 4 by the activity of aminoglycosides and ciprofloxacin vs. >50% isolates with non-metallo-carbapenemases), but unfortunately these isolates are not typical of the carbapenemase producers seen in most hospitals. Temocillin may retain activity against some strains with KPC enzymes, but not those with other carbapenemases. Older or less commonly used agents (e.g. chloramphenicol, fosfomycin, nitrofurantoin, tigecycline) also may retain activity against some multi-resistant carbapenemase-producing strains (19), but individual isolates must be tested, and resistance can emerge during therapy even to these final straws (20). Table 4 Activity of antibiotics vs. carbapenemase-producing Enterobacteriaceae in the UK. Reproduced from citation (22). 6 Antibiotic a Metallo-enzyme Producers (IMP, NDM or VIM) E. coli Klebsiella Enterobacter / Citrobacter Non-metallo-enzyme Producers (KPC or OXA-48-like) E. coli Klebsiella Enterobacter / Citrobacter Imipenem 9% 1% 3% 10% 5% 18% IPM+EDTA b 100% 99% 100% 27% 8% 27% Meropenem 9% 5% 3% 47% 8% 27% Ertapenem 0% 0% 0% 0% 0% 0% Ampicillin 0% 0% 0% 0% 0% 0% Co-amoxiclav 0% 0% 0% 0% 0% 0% Piperacillin 0% 0% 3% 0% 0% 0% PIP + tazobactam 4% 0% 7% 0% 0% 0% Cefotaxime 0% 0% 0% 3% 2% 0% Ceftazidime 0% 0% 0% 17% 6% 0% Aztreonam 4% 18% 13% 13% 6% 0% Ciprofloxacin 9% 10% 17% 53% 49% 50% Gentamicin 0% 12% 27% 70% 65% 41% Tobramycin 0% 1% 0% 50% 58% 50% Amikacin 17% 32% 50% 90% 85% 91% Colistin 100% 97% 93% 100% 92% 100% Tigecycline 100% 47% 47% 100% 74% 68% a. Susceptibility defined using BSAC v. 10.1 breakpoints. b. Diagnostic test to distinguish metallo- from non-metallo- enzymes; not for therapeutic use. Active vs. 90% producers Active vs. >75-89% producers Active vs. 50-74% producers Active vs. <50% producers

7 Fighting the rising tide of carbapenemases in Enterobacteriaceae...BUT IT S not all DOOm and GLOOm Rising numbers (of both producers and enzyme types), geographic spread, outbreaks and multi-resistance all illustrate the growing threat of carbapenemases. However, experience in Israel clearly demonstrates that even national outbreaks can be controlled and reversed, in this case, by intensive adherence to infection control guidance (21). True, this related to a single bacterial clone with KPC (ST258), but it demonstrates that we can at least delay the rising tide. Preparedness plans to deal with carbapenemase producers are of paramount importance, and must be tailored to consider the extent of local, regional or national problems (9, 10, 17, 18). although there aren t enough anti-gram-negative agents in the development pipeline, there are some! The novel ß-lactamase inhibitor avibactam (NXL-104) is able to increase the activity of cephalosporins against Enterobacteriaceae with non-metallo-carbapenemases, and also makes monobactams more effective against those with MBLs. The activity of developmental agents belonging to other antibiotic classes does not relate directly to the type of carbapenemase produced, but linked resistance genes can cause problems. For example, plazomicin (the neoglycoside ACHN-490) has activity against most carbapenemase producers, but not vs. many with NDM enzymes because most of these produce 16S methyltransferases, which confer broad aminoglycoside resistance.?box 1. Ten UnanSWereD QUeSTIOnS 1. How do the clones that acquire carbapenemases refl ect the total population of a given species? 2. Are they the dominant clones within a species with acquisition refl ecting prevalence, or does acquisition serve to enhance the potential of minor lineages, leading to selective amplifi cation? 3. Why hasn t ST258 acquired carbapenemases other than KPC? It clearly has an ability to colonize and cause infections on a national and international scale. 4. How should we explain occurrence of the same strains or clones in widely separated countries in the absence of epidemiological evidence for direct links? 5. What are the environmental reservoirs of the big fi ve carbapenemases and do these pose any continuing public health threat? 6. Do repeated escape events from these reservoirs provide part of the answer to Q4? 7. What biological features of the genes, plasmids and host strains drive or contribute to the diverse epidemiologies observed for the big fi ve carbapenemases, namely clonal expansion, horizontal plasmid transfer and gene mobilisation? 8. Can we prevent carbapenemases becoming established in E. coli in community settings and rising to prevalences currently seen worldwide with ESBLs? And of course we mustn t forget: 9. What is the best treatment for my patient? 10. What new antibiotics are there? COnCLUDInG remarks acquired carbapenemases pose one of the greatest current challenges to the successful treatment of infections caused by multiresistant Gram-negative bacteria. Producers of these ultimate ß-lactamases are typically multi-resistant to many antibiotic classes, leaving few therapeutic options. The carbapenemases are diverse, but are dominated internationally in the Enterobacteriaceae by the big fi ve, which are the KPC and OXa-48-like non-metalloenzymes and the IMP, NDM and VIM metallo-carbapenemases. The epidemiologies associated with these types are similarly diverse and are variously dominated by the spread of an internationallysuccessful clone (KPC), wide horizontal transfer of a successful plasmid (OXa-48-like), or spread of a resistance gene on to multiple plasmids in multiple bacterial species (Mbls). There can, however, be important local departure from these generalizations and numerous questions remain to be answered (see box 1). Rapid detection of carbapenemase-mediated resistance is essential for patient management, to prevent onward transmission and to limit the public health impact of the resistant strains. We must make every effort to prevent carbapenemases becoming as everyday as esbls. Professor Neil Woodford is Head of the Health Protection Agency s Antimicrobial Resistance and Healthcare Associated Infections Reference Unit (AMRHAI), which was created in July 2012 by the merger of the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) and the Laboratory of Healthcare Associated Infections (LHCAI). Prof Woodford has worked on antibiotic resistance in medically-important bacteria for over 25 years, and has co-authored >250 scientifi c papers and edited three books on the subject. His BSc (Microbiology, 1985) was from the University of Bristol and his PhD (Medical Microbiology, 1990) from Imperial College, London. He is a Fellow of the Royal College of Pathologists and an Honorary Professor at Queen Mary, University of London. Neil Woodford Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, Health Protection Agency, Microbiology Services Colindale, London NW9 5EQ, United Kingdom Email : neil.woodford@hpa.org.uk * EARS-Net: web-based European Antimicrobial Resistance Surveillance system. ** EUCAST: European Committee on Antimicrobial Susceptibility Testing. *** CLSI: Clinical and Laboratory Standards Institute. **** UKNEQAS: United Kingdom National External Quality Assessment Service. ReFeReNCeS 1. Canton, R., et al. Clin. Microbiol. Infect. 2012;18:413-431 2. Walsh, T. R. Int. J. Antimicrob. Agents. 2010;36:S8-S14 3. leavitt, a., et al. Antimicrob. Agents Chemother. 2010;54:4493-4496 4. Nordmann, P., et al. Lancet Infect. Dis. 2009;9:228-236 5. Curiao, T., et al. J. Antimicrob. Chemother. 2010;65:1608-1614 6. Ktari, S., et al J. Antimicrob. Chemother. 2011;66:1644-1646 7. Poirel, l., et al. Antimicrob. Agents Chemother. 2012;56:559-562 8. Kumarasamy, K. K., et al Lancet Infect. Dis. 2010;10:597-602 9. arhai-hpa. 2011. http://www.hpa.org.uk/web/hpawebfile/hpaweb_c/1294740725984 10. CDC. 2012. http://www.cdc.gov/hai/organisms/cre/cre-toolkit/index.html 11. lepelletier, D., et al. J. Travel. Med. 2011;18:344-351 12. Nordmann, P., et al Clin. Microbiol. Infect. 2012;18:432-438 13. Woodford, N., et al. JCM. 2012;48:2999-3002 14. burckhardt, I. and S. Zimmermann. JCM. July 2011, doi:10.1128/jcm.00287-11 15. Nordmann, P., et al. Emerg. Infect. Dis. 2012;18:1503-1507 16. livermore, D. M., et al. J. Antimicrob. Chemother. 2012;67:1569-1577 17. Carmeli, Y., et al. Clin. Microbiol. Infect. 2010;16:102-111 18. akova, M., et al. Clin. Microbiol. Infect. 2012;18:439-448 19. livermore, D. M., et al. Int. J. Antimicrob. Agents. 2011;37:415-419 20. Stone, N. R. H., et al. J. Antimicrob. Chemother. 2011;66:2677-2678 21. Schwaber, M. J., et al. Clin. Infect. Dis. 2011;52:848-855 22. anonymous. Health Protection Report 5: issue 24 (17 th June 2011)

HIGHLIGHT Diagnostic solutions for carbapenemase producing Enterobacteriaceae Screening biomérieux Carbapenemases Solutions Detection biomérieux is actively committed to the fight against bacterial resistance and offers a large range of products for the detection, screening and susceptibility testing of carbapenemase-producing Enterobacteriaceae. a Contact your local representative for availability. b Etest MBL IP/IPI and MBL MP/MPI are Research Use Only (RUO) products in USA. NeW! chromid CARBA a VITEK 2 AST cards Etest MBL (IP/IPI) strip b Etest MBL (MP/MPI) strip b 10-12 / 9304435/002/GB/E / This document is not legally binding. biomérieux S.A. reserves the right to modify specifi cations without notice. BIOMERIEUX, the blue logo, API, ATB, chromid, Etest, LyfoCults and VITEK are used, pending and/or registered trademark belonging to biomérieux or one of its subsidiaries, or one of its companies / Figure 1 reproduced with permission from John Wiley and Sons / biomérieux S.A. RCS Lyon 673 620 399 / Photos: biomérieux, N. Bouchut / Printed in France / THeRa Conseil / RCS Lyon B 398 160 242 PrODUCT newsflash SPeCIFICITY SeNSITIVITY - RaPIDITY > chromid CarBa Chromogenic medium for the screening of carbapenemase-producing Enterobacteriaceae Very rapid screening of carbapenemase-producing Enterobacteriaceae in 18-24 hours (1) with high sensitivity 97.4% [93.4-99.3] high specifi city 99.7% [98.9-100.0] (2) (3) (4) defi ned on clinical specimens (stools or rectal swabs) compared to conventional method Product ref. 43861 To find out more about chromid CARBA: www.biomerieux.com/chromid-carba (1) Centers for Disease Control and Prevention - Guidance for Control of infections with carbapenem resistant or carbapenemase-producing Enterobacteriaceae in acute care facilities - MMWR, 2009, 58(10): 256-260. (2) Perry J. et al. Journal of Antimicrobial Chemotherapy, 2011, ISSN:1460-2091. (3) Vrioni G. et al. J. Clin. Microbiol. 2012, 50:1841-1846. (4) Bereksi N. et al. Poster 1718 London 2012 22 nd ECCMID. a faster, easier, safer approach to Quality Control! LyfoCults are high quality, disposable vials containing freeze dried ATCC microorganism cultures. Easy-to-use, all-in-one design Built-in inoculation wand From refrigerator to incubator in less than 5 minutes No need for pipettes, loops or other materials Available as individual strains or pre-packaged sets of organisms for use with VITEK 2, API and ATB QC strains available for ID, AST, PPM, blood culture, reagents, etc. Introducing 68 new QC strains, including the following for carbapenemase testing: Klebsiella pneumoniae ATCC 700603 (ref. 301247) for QC of chromid CARBA agar (ref. 43861) and Etest MBL MP/MPI b (ref. 411361 or 411362) Pseudomonas aeruginosa ATCC 27853 (ref. 301480) for QC of Etest MBL IP/IPI b (ref. 534200 or 534208) For a full listing of available LyfoCults PLUS strains visit www.biomerieux.com/lyfocultsplus or ask your biomérieux representative * The ATCC Licensed Derivative Emblem, the ATCC Licensed Derivative word mark, and the ATCC catalog marks are trademarks of ATCC. biomérieux is licensed to use these trademarks and to sell products derived from ATCC cultures. biomérieux S.A. 69280 Marcy l Etoile France Tel. (33) 04 78 87 20 00 Fax (33) 04 78 87 20 90 www.biomerieux.com www.biomerieux-diagnostics.com * Scientifi c advisor: Prof. Patrice nordmann Chief Dept Bacteriology-Virology, Director Research Unit INSERM 914, Emerging Resistance to Antibiotics, Bicêtre Hospital, South Paris Medical School, University Paris XI, France Director of publications: Thierry Bernard Editor: marie Françoise Gros, md