Identification and screening of carbapenemase-producing Enterobacteriaceae

REVIEW 10.1111/j.1469-0691.2012.03815.x Identification and screening of carbapenemase-producing Enterobacteriaceae P. Nordmann 1, M. Gniadkowski 2, C. G. Giske 3, L. Poirel 1, N. Woodford 4, V. Miriagou 5 and the European Network on Carbapenemases* 1) Service de Bactériologie-Virologie, INSERM U914 Emerging Resistance to Antibiotics, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine et Université Paris-Sud, K.-Bicêtre, France, 2) Department of Molecular Microbiology National Medicine Institute, Warsaw, Poland, 3) Clinical Microbiology MTC, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 4) Antibiotic Resistance Monitoring and Reference Laboratory Health Protection Agency, London, UK and 5) Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece Abstract Carbapenem-hydrolysing b-lactamases are the most powerful b-lactamases, being able to hydrolyse almost all b-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media. Keywords: Antibiotic, carbapenem, Gram-negatives, resistance, b-lactams Article published online: 21 February 2012 Clin Microbiol Infect 2012; 18: 432 438 Corresponding author: P. Nordmann, Service de Bactériologie-Virologie-Hygiène, Hôpital de Bicêtre, 78 rue de Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France E-mail: nordmann.patrice@bct.aphp.fr *See Appendix. Multidrug resistance is now emerging at an alarming rate among a variety of bacterial species, causing both nosocomial and community-acquired infections. One of the most important emerging traits is resistance to extended-spectrum b-lactams in Gram-negative bacilli. Increasing resistance to carbapenems, which are most often the last line of therapy, is now frequently being observed in many hospital-acquired and several community-acquired Gram-negatives rods. Carbapenem-resistant Enterobacteriaceae have now been reported worldwide. This may be related either to an association of decreased outer membrane permeability with overexpression of b-lactamases possessing very weak carbapenemase activity, or to expression of carbapenemases [1 6]. Their carbapenem resistance trait is not transferable, unlike most of the carbapenemase genes, and porin defect may have a significant fitness cost. This explains why carbapenem-resistant isolates that do not produce carbapenemases are considered to be much less important from a public health perspective than carbapenemase producers. The spread of carbapenemase producers is by far the most important current clinical issue in antibiotic resistance in Gram-negatives, and must be strictly controlled. The first carbapenemase identified in Enterobacteriaceae was the chromosomally encoded NmcA from an Enterobacter cloacae clinical isolate in 1993 [7]. Since then, carbapenem-resistant Enterobacteriaceae have been reported worldwide, mostly as a consequence of acquisition of carbapenemase genes. A large variety of carbapenemases have been identified in Enterobacteriaceae, belonging to three classes of b-lactamase, the Ambler class A, B and D b-lactamases, which have been extensively described elsewhere [6]. In addition, rare chromosomally encoded cephalosporinases (Ambler class C/AmpC) produced by Enterobacteriaceae may possess slightly extended activity towards carbapenems, but their clinical significance remains debatable [6]. Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases

CMI Nordmann et al. Carbapenemase-producing Enterobacteriaceae 433 TABLE 1. Resistance phenotypes resulting from the expression of the main carbapenemases reported in Enterobacteriaceae without or with extended-spectrum b-lactamases (ESBLs) AMX AMC TZP CTX CAZ IMP ETP MER ATM KPC R S/I R R R S/I/R I/R S/I/R R KPC + ESBL R I/R R R R I/R I/R I/R R IMP/VIM/NDM R R I/R R I/R S/I/R I/R S/I/R S IMP/VIM/NDM + ESBL R R I/R R R I/R R S/I/R R OXA-48/OXA-181 R R S/I/R S/I S S/I S/I S/I S OXA-48/OXA-181 + ESBL R R I/R R R I/R I/R I/R R AMX, amoxycillin; AMC, amoxycillin clavulanic acid; TZP, piperacillin tazobactam; CTX, cefotaxime; CAZ, ceftazidime; IMP, imipenem; ETP, ertapenem; MER, meropenem; ATM, aztreonam. True carbapenemases hydrolyse most b-lactams, including the carbapenems (imipenem, ertapenem, meropenem, and doripenem) [6]. The most clinically significant Ambler class A carbapenemases are of the KPC type [6,8,9]. They hydrolyse all b-lactams, and their activity is inhibited by boronic acid and, at least partially, by clavulanic acid and tazobactam (Table 1). The class B b-lactamases are the enzymes possessing the highest carbapenemase activity. They are mostly of the IMP, VIM and NDM types in Enterobacteriaceae [6,10,11]. These enzymes exhibit a broad spectrum of hydrolytic activity, including all penicillins, cephalosporins, and carbapenems, sparing only the monobactam aztreonam (Table 1) [6,11]. Their activity is not inhibited by commercially available b-lactamase inhibitors (clavulanic acid, tazobactam, or sulbactam) [6,11]. The mechanism of hydrolysis of class B enzymes is dependent on the interaction of the b-lactam with zinc ion(s) in their active site, explaining the inhibition of their activity by EDTA, a chelator of divalent cations, or dipicolinic acid [11 13]. The Ambler class D enzymes with carbapenemase activity in Enterobacteriaceae are mostly OXA-48 and OXA-181 [6]. They have a peculiar hydrolysis profile, sparing ceftazidime, hydrolysing cefotaxime at a very low level, and being resistant to inhibition by clavulanic acid tazobactam (Table 1) [6]. Clinical isolates rarely show the phenotype of resistance that is attributable to carbapenemase expression alone, because they often have other co-resident b-lactamases such as extended-spectrum b-lactamases (ESBLs), leading to a broader composite resistance profile (Table 1) [6]. Most carbapenemase producers are Klebsiella pneumoniae or Escherichia coli, and although they are being increasingly identified worldwide, there are some clear endemic areas, such as KPC producers in the USA, Greece, and Israel [9], VIM producers in Greece [6,13], OXA-48 producers in North Africa and Turkey [6], and NDM producers in the Indian subcontinent and possibly the Balkans [9]. Accurate detection of carbapenemase-producing Enterobacteriaceae is required in two situations: detection in clinical specimens; and detection of colonizing strains that have grown on media used to screen for carriers. Detection Detection of carbapenemase producers in Enterobacteriaceae is becoming a major issue, as carbapenemases are usually associated with many other resistance determinants, giving rise to multidrug resistance and even pandrug resistance. The detection of carbapenemase producers in clinical specimens is based first on a careful analysis of susceptibility testing results obtained with automated systems, liquid media, and disk diffusion tests. Automated systems may not reliably detect all types of carbapenemase producer, and discrepancies may arise [14]. The CLSI (USA) breakpoints for carbapenems have been lowered significantly to permit better detection of carbapenem-resistant isolates (Table 2) [15,16]. Ertapenem seems to be a good candidate for detecting most of the carbapenemase producers, as MICs of ertapenem are usually higher than MICs of other carbapenems (Table 3) [17]. However, detection of carbapenemase producers based only on MIC values of ertapenem lacks specificity. According to these CLSI and EUCAST guidelines, breakpoints are all that are needed for making treatment decisions. Special tests for carbapenemase detection are TABLE 2. Breakpoint values for carbapenems according to the US (CLSI) and European (EUCAST) guidelines, as updated June 2010 (MIC values, mg/l) CLSI EUCAST S( ) R ( ) S ( ) R (>) Imipenem 1 4 2 8 Meropenem 1 4 2 8 Ertapenem 0.5 2 0.5 1 Doripenem 1 4 2 8 TABLE 3. Range of MICs of carbapenems for clinical Enterobacteriaceae expressing the main carbapenemases MIC (mg/l) Imipenem Meropenem Ertapenem KPC 0.5 to >32 0.5 to >32 0.5 to >32 IMP/VIM/NDM 0.5 to >32 0.5 to >64 0.38 to >32 OXA-48/OXA-181 0.25 to 64 0.38 to 64 0.38 to >32

434 Clinical Microbiology and Infection, Volume 18 Number 5, May 2012 CMI recommended only for epidemiology and infection control issues. However, intermediate susceptibility and even susceptibility to carbapenems have been observed for producers of all types of carbapenemase (Table 3). This is particularly true for OXA-48/OXA-181 producers that do not co-produce an ESBL [6,18,19]. Therefore, we believe that a search for carbapenemase production should be performed in any enterobacterial isolates with any slight decrease in susceptibility to carbapenems. This belief is supported in part by the current paucity of clinical experience in treating infections caused by carbapenemase producers, the high inoculum of carbapenemase producers in the site of the infection in vivo (e.g. pneumonia), and the possibility of selecting in vivo mutants with increased levels of resistance to carbapenems and possessing additional mechanisms that contribute to carbapenem resistance. In addition, using an experimental model of peritonitis in mice infected with an OXA-48 producer, we showed recently that imipenem and ertapenem failed to cure infected mice despite their low MIC values [20]. There is no consensus on the cut-off value of MICs of carbapenems that should be applied for research into carbapenemase activity. On the basis of the literature and our own experience, we propose that investigations of carbapenemase activity should be performed on enterobacterial isolates with MIC values of 0.5 mg/l for ertapenem or 1 mg/l for imipenem and meropenem. We are aware that these values may lead to the inclusion of several enterobacterial species for which the natural distribution of MICs of imipenem is around 1 mg/l (e.g. Proteus species). However, this seems to be the best compromise in order to maximize detection sensitivity. The use of meropenem instead may be proposed, as there is a broader corridor between the wild-type and clinical breakpoints. A series of non-molecular-based tests have been proposed for detection of carbapenemase activity. None of the currently available tests has 100% specificity or 100% sensitivity. The modified Hodge test (MHT) based on in vivo production of a carbapenemase by a carbapenemase-producing strain has been suggested, in particularly in the USA [21 24]. This technique is, however, time-consuming, as it requires at least 24 48 h. It often lacks specificity (e.g. false-positive results for high-level AmpC producers or CTX-M-type ESBL producers, Enterobacter species) and sensitivity (e.g. weak detection of NDM producers), but works well for the detection of KPC and OXA-48 producers. The sensitivity of the MHT in detecting NDM producers is significantly improved if zinc is included in the culture medium (Bicêtre MHT) [25]. This test can be used as the first step in detecting the carbapenemase activity of candidate isolates. In addition, it is also useful for checking carbapenemase activity as part of the infection control process for outbreaks caused by carbapenemase producers. Concomitantly with performance of the MHT, inhibition studies can be performed in liquid or solid culture media with molecules that inhibit the activity of several types of carbapenemase and/or other types of b-lactamase. Inhibition by EDTA or dipicolinic acid may be used for the detection of MBL activity [12,13,22]. The Etest MBL strip (AB bio- Mérieux, Solna, Sweden) is one of the methods advocated for this purpose [6,11]. This latter method, using imipenem and imipenem/edta, is efficient in detecting MBL producers exhibiting high-level resistance but may fail to detect MBL producers exhibiting low-level resistance to imipenem. Novel Etest strips containing other concentrations of inhibitors or other carbapenem molecules (meropenem) will be available soon, and may facilitate this detection. Boronic acid-based inhibition testing has been reported to be specific for KPC detection in K. pneumoniae when performed with imipenem or meropenem [26]. Although these phenotypic tests are adequate for the detection of carbapenemases in AmpC-negative enterobacterial species such as K. pneumoniae, they have some limitations for the identification of carbapenemase producers among AmpC-positive species such as Enterobacter species. Inhibition of cephalosporinase activity by plating these strains on cloxacillin-containing plates or using cloxacillin-containing tablets may help to differentiate those strains that will recover a susceptibility to carbapenems (most hyperproducers of cephalosporinases) from those that exhibit low-level carbapenem resistance owing to the production of a carbapenemase [12]. Boronic acid also inhibits the activity of chromosome-encoded or plasmid-encoded AmpC-like cephalosporinases. Therefore, it is useful to compare boronic acid inhibition with that obtained in presence of cloxacillin (cloxacillin-containing plates or disk). To this end, an algorithm has been proposed for the accurate detection of carbapenemase producers in enterobacteria, including the use of cloxacillin for the discrimination of AmpC hyperproducers [12], and there are also commercially available methodologies based on the same concept (Rosco Diagnostica, Copenhagen, Denmark). None of these inhibition tests is suitable for detecting OXA-48/OXA-181 producers, because the activity of these enzymes is not inhibited by clavulanic acid, tazobactam, sulbactam, or any zinc chelators. High-level resistance to both temocillin (MICs of >64 mg/l) and piperacillin tazobactam in Enterobacteriaceae showing reduced susceptibility or resistance to at least one carbapenem may be used as a first step towards identifying possible OXA-48 producers [27]. Carbapenemase detection by spectrophotometric assay is the most accurate approach advocated for the detection of

CMI Nordmann et al. Carbapenemase-producing Enterobacteriaceae 435 carbapenemases. This technique, which is detailed elsewhere (S. Bernabeu, L. Poirel, P. Nordmann, unpublished data), is based on the measurement of imipenem hydrolysis at a wavelength of 297 nm by a carbapenemase-containing extract obtained after 18 h of culture in broth. It can accurately differentiate between carbapenemase producers and carbapenem-resistant bacteria with non-carbapenemase-mediated mechanisms of resistance (i.e. outer membrane permeability defect, or overproduction of cephalosporinases and/or ESBLs). In addition, it is cheap in comparison with any other available molecular technique. However, it does not discriminate between the different types of carbapenemase, and requires a preliminary step of at least 12 h of bacterial culture and specific training. Despite this, it has excellent sensitivity and specificity for detecting carbapenemase activity in Enterobacteriaceae. It should be implemented in any national reference laboratory. Recently, the use of mass spectrometry to detect carbapenemase activity has been proposed, based on analysis of the degradation spectrum of a carbapenem molecule [28,29]. Although this technique must be further evaluated, matrix-assisted laser desorption ionization time-of-flight mass spectrometry equipment is increasingly being used in both reference and diagnostic bacteriology laboratories. Molecular techniques remain the reference standard for the identification and differentiation of carbapenemases. Most are based on PCR, and may be followed by sequencing if needed for precise identification of a carbapenemase, rather than just its group (e.g. VIM-type, KPC-type, NDM-type, and OXA-48-type). They are either single PCR techniques or multiplex PCR techniques [30 32]. The PCR technique performed on colonies can give results within 4 6 h (or less when real-time PCR technology is used), with excellent sensitivity and specificity. Similarly, other molecular techniques are useful for this purpose [33]. Sequencing of PCR products is interesting mostly for epidemiological purposes. The main disadvantages of the molecular-based technologies for detection of carbapenemase genes are their cost, the requirement for trained technicians, and the inability to detect novel carbapenemase genes. Screening of Carriage The prevention of spread of carbapenemase producers relies on early and accurate detection of carriers in hospital units or on admission/discharge either to the hospital or to a specific unit. But who must be screened? Several countries have introduced policies or have recommendations and guidance on which patients should be screened, although the details differ considerably between countries, as does the degree to which the screening is mandated [34,35]. Screening should include at least at-risk patients, such as those in intensivecare units, and transplantation and immunocompromised patients. If a patient is confirmed as being infected or colonized by a carbapenemase producer, the screening programme should be extended to neighbouring patients on the hospital ward. Screening shall be done at least to patients transferred from a foreign hospital on addition to any hospital unit. As the reservoir of Enterobacteriaceae is mostly the intestinal flora, stools and rectal swabs are the most suitable specimens for performing this screening process. These specimens may be plated on screening medium (see below), either directly or after an 18-h enrichment in broth containing imipenem 0.5 1 mg/l or ertapenem 0.5 mg/l [36,37]. The benefits of this enrichment step have not been extensively evaluated, although it has been shown to improve the detection of KPC producers [36]. It may be useful when managing an outbreak or searching for additional carriers when a carbapenemase producer is found in stools. The main disadvantage of this culture step is that it delays results by 18 24 h. Thus, the time needed to confirm or refute carbapenemase activity may be up to 72 h after the sample is taken from the patient. Stools or rectal swabs (with or without enrichment in the presence of a carbapenem) should be plated on selective media. The problem is that the level of resistance to carbapenems displayed by carbapenemase producers varies significantly, making their detection difficult unless they show highlevel carbapenem resistance [38,39]. Several studies have reported using media containing imipenem at 1 2 mg/l, which may be too high a concentration for efficient detection of carbapenemase producers with low-level resistance (e.g. the OXA-48/OXA-181 producers). One would therefore expect the specificity to be quite good but the sensitivity to be poor. A culture medium initially designed to screen for ESBL producers that contains cefpodoxime (ChromID ESBL; biomérieux, La Balme-les-Grottes, France) and a carbapenem-containing medium (CHROMagar KPC; CHROMagar, Paris, France) have been evaluated for screening carbapenemase producers [38,39]. Both media contain chromogenic molecules that may contribute to enterobacterial species recognition. The ChromID ESBL medium has excellent sensitivity, its main disadvantage being the lack of detection of OXA-48- like producers that are susceptible to cefpodoxime, i.e. in the absence of co-production of an ESBL. In addition, it lacks specificity for carbapenemase producers, as it was formulated to support the growth of ESBL producers [40].

436 Clinical Microbiology and Infection, Volume 18 Number 5, May 2012 CMI This is an important consideration, because of the widespread carriage of ESBL producers worldwide, and necessitates additional testing of colonies for carbapenem resistance. The CHROMagar KPC medium detects carbapenemase producers only if they exhibit higher-level resistance to carbapenems [41,42]. Its main disadvantage is therefore its lack of sensitivity, as it does not detect carbapenemase producers with low levels of carbapenem resistance. Although it has not been extensively evaluated, the use of two parallel plates for detection of a carbapenemase producer may be possible with an ESBL screening plate and a Drigalski plate on which an ertapenem disk or an Etest strip is placed [43,44]. A novel and patented medium (SUPERCARBA medium) containing cloxacillin, zinc and a carbapenem molecule that has improved sensitivity and specificity for detecting all types of carbapenemase producer (including OXA-48 producers) has been recently developed [45]. None of these culture-based approaches will identify the type of carbapenemase. The screening process requires patients to be kept in strict isolation prior to results being obtained (at least 48 h). Actually, after this screening step, carbapenemase producers must then be identified with to the techniques described above (antibiotic susceptibility testing and molecular techniques). Recently, PCR-based techniques performed directly on stool specimens have been proposed for the rapid detection of KPC and NDM-1 producers [46,47]. Such methods will speed up detection, and may be especially useful in outbreak investigations. However, the cost-effectiveness of such approaches and the predictive value of a positive test result in low-prevalence settings needs to be considered; as yet, little is known about the occurrence of enterobacterial carbapenemase genes in the normal, and perhaps non-culturable, bowel flora, so there is a potential for molecular tests to have lower specificity than might be intuitively predicted. It is imperative to point out that the screening process on admission still requires the patients to be kept in strict isolation prior to results being obtained (at least for 48 h). Conclusion The worldwide spread of Enterobacteriaceae expressing carbapenemases represents a major significant threat of public health concern. Significant efforts are needed to ensure prompt and accurate detection, and the implementation of effective infection control strategies. Screening procedures should be implemented worldwide for at-risk patients. These procedures represent an essential component of the efforts needed to prevent the further development of infections caused by multidrugresistant or even pandrug-resistant bacteria in hospitals. Acknowledgements This work was partially funded by a grant from INSERM (U914), the European Society for Clinical Microbiology and Infectious Diseases (ESCMID), sponsored the first meeting of the European Network on Carbapenemases held in Paris, September 2011. Transparency Declaration The authors declare no conflicts of interest. Appendix The members of the European Network on Carbapenemases are as follows: M. Akova (Ankara); V. Miriagou (Athens); T. Naas, P. Nordmann, and L. Poirel (Bicêtre); H. Seifert (Köln); D. Livermore and N. Woodford (London); P. Bogaerts and Y. Glupczynski (Louvain); R. Canton (Madrid); G. M. Rossolini (Sienna); C. Giske (Stockholm); A. Adler, Y. Carmeli, and S. Navon-Venezia (Tel-Aviv); O. Samuelsen (TromsØ); G. Cornaglia (Verona); and M. Gniadkowski (Warsaw). References 1. Doumith M, Ellington MJ, Livermore DM et al. Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 2009; 63: 659 667. 2. Lartigue MF, Poirel L, Poyart C et al. Ertapenem resistance of Escherichia coli. Emerg Infect Dis 2007; 13: 315 317. 3. Martinez-Martinez L. Extended-spectrum b-lactamases and the permeability barrier. Clin Microbiol Infect 2008; 14 (suppl 1): 82 89. 4. Girlich D, Poirel L, Nordmann P. Do CTX-M b-lactamases hydrolyze carbapenems? J Antimicrob Chemother 2008; 62: 1155 1156. 5. Lee EH, Nicolas MH, Kitzis MD et al. Assocation of two resistance mechanisms in a clinical isolate of Enterobacter cloacae with high level resistance to imipenem. Antimicrob Agents Chemother 1991; 35: 1093 1098. 6. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791 1798. 7. Naas T, Nordmann P. Analysis of a carbapenem-hydrolyzing class A b-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein. Proc Natl Acad Sci USA 1994; 91: 7693 7697. 8. Baraniak A, Grabowska A, Izdebski R et al. Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008 2009. Antimicrob Agents Chemother 2011; 55: 5493 5499.

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438 Clinical Microbiology and Infection, Volume 18 Number 5, May 2012 CMI 46. Schechner V, Straus-Robinson K, Schwartz D et al. Evaluation of PCR-based testing for surveillance of KPC-producing carbapenemresistant members of Enterobacteriaceae family. J Clin Microbiol 2009; 47: 3261 3265. 47. Naas T, Ergani A, Carrër A et al. Real-time PCR for detection of NDM-1 carbapenemase genes from spiked samples. Antimicrob Agents Chemother 2011; 55: 4038 4043.