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Newer β-Lactamases: Clinical and Laboratory Implications, Part II*
Clinical Microbiology Newsletter
Vol. 30, No. 11
www.cmnewsletter.com
$88 June 1, 2008
Newer β-Lactamases: Clinical and Laboratory Implications, Part II* Ellen Smith Moland, B.S.M.T., Soo-Young Kim, M.D., Seong Geun Hong, M.D., and Kenneth S. Thomson, Ph.D., Center for Research in Antiinfectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska
Abstract
For optimal patient care, clinical laboratories should be capable of detecting clinically significant, novel β-lactamases produced by gram-negative pathogens. However, with over 700 β-lactamases now described, it is a struggle to keep abreast of the various types of β-lactamases, their clinical relevance, and methods for detection. Furthermore, the increasing prevalence of isolates that produce multiple β-lactamases increases the difficulty of accurate detection. Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) recommendations for detection of β-lactamases do not keep pace with this rapidly evolving field. While perfection may not always be possible, it is important that clinical laboratories provide a relevant diagnostic service to ensure appropriate antibiotic therapy and infection control. Part II of this article will provide a discussion of AmpC β-lactamases and other β-lactam resistance mechanisms, along with methods for their laboratory detection.
Plasmid-Mediated AmpC Beta-Lactamases
Imported, transmissible, or plasmidmediated AmpC β-lactamase genes were first reported in Escherichia coli and Klebsiella pneumoniae in 1988 (30,31). Today, there are over 40 known plasmidmediated AmpC β-lactamases (http:// www.lahey.org/Studies/). Molecular data suggest these enzymes are derived from Enterobacter cloacae, Citrobacter freundii, Morganella morganii, Hafnia alvei, and unknown sources. They have been mostly detected in isolates of E. coli, K. pneumoniae, Proteus mirabilis,
*Editor’s Note: Part I of this article was published in the May 15, 2008 issue of CMN (Vol. 30, No. 10). Mailing Address: Ellen Smith Moland, B.S.M.T., Center for Research in Antiinfectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska 68178. Tel.: 402-2802921. Fax: 402-280-1875. E-mail: [email protected] and [email protected] Clinical Microbiology Newsletter 30:11,2008
and Salmonella. Most plasmid-mediated AmpCs are constitutively expressed, but some enzymes, such as DHA-1, DHA-2, ACT-1, CFE-1, and CMY-13, are inducible and may be more clinically dangerous. Inducibility may confer the capability for an organism to become more resistant during β-lactam therapy than indicated by susceptibility tests. Clinical data verify that this happens (32). Therapeutic failures with cefotaxime have been reported with susceptible isolates of plasmid-mediated AmpC-producing K. pneumoniae (22). In most studies, plasmid-mediated AmpC β-lactamases have been less common than extended-spectrum β-lactamases (ESBLs). For example, in a 2001/2002 U.S. study, plasmidmediated AmpCs were detected in 3.3% of K. pneumoniae isolates from 63 U.S. sites, whereas ESBLs were detected in 11.3% of isolates (33). Prevalence can be expected to increase in the absence of detection by clinical laboratories, because lack of detection makes infection control impossible. © 2008 Elsevier
Plasmid-Mediated AmpC BetaLactamase Detection Issues
There are currently no CLSI recommendations for detection of plasmid-mediated AmpC β-lactamases. Klebsiella is a convenient indicator organism for detection of plasmidmediated AmpCs, because it does not produce a chromosomally mediated AmpC. P. mirabilis and Salmonella are also good indicator organisms for the same reason. E. coli is also a likely source of plasmid-mediated AmpCs, but phenotypic detection is complicated, because positive results may also occur with occasional isolates that hyper-
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produce their chromosomal AmpC β-lactamases. Cephamycins (cefoxitin and cefotetan) are good screening agents for detection of most AmpC β-lactamases. As indicated above, screening by cefoxitin insusceptibility (i.e., resistance or intermediate susceptibility) is useful but not specific. Reduced outer membrane permeability may also confer cefoxitin insusceptibility, making differentiation between these two resistance mechanisms necessary. Cefotetan is potentially useful for AmpC screening, but it must be remembered that this agent is more potent than cefoxitin, and a more sensitive screening criterion than insusceptibility will be necessary. Amp-C Class (ACC)-like plasmid-mediated AmpC β-lactamases that do not hydrolyze cephamycins significantly will not be reliably detected by insusceptibility to cefoxitin. A variety of approaches to AmpC detection have been recommended. The AmpC disk test is useful, because it determines if an organism produces an enzyme that hydrolyzes cefoxitin significantly (Fig. 5). This test utilizes Tris/ EDTA disks (BD Diagnostic Systems, Baltimore, MD). Since other enzymes, particularly carbapenem-hydrolyzing enzymes, also hydrolyze cefoxitin, it is important to interpret this test in conjunction with inspection of carbapenem susceptibility test results. This is especially important in locations where Klebsiella pneumoniae (KPC) enzymes have been detected (see below). The AmpC disk test seems to be the most sensitive test for detecting the DHAlike enzymes. It is an indirect test in that an indicator organism is the lawn inoculum on the plate and not the test isolate. For this reason, the test requires a separate plate other than the routine disk diffusion susceptibility plate. Another approach to the detection of AmpC β-lactamases involves using AmpC inhibitors, such as boronic acid,
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Figure 5. AmpC disk test with Tris/EDTA disk inoculated with test organism placed near a cefoxitin disk (FOX 30) showing positive test (distortion of zone surrounding a cefoxitin disk).
FOX
TZB
ATM
Flatting of zone Figure 6. Disk approximation test showing inducible AmpC production, indicated by flattening or truncation of inhibition zones around aztreonam (ATM) and piperacillin/tazobactam (TZP) disks. The cefoxitin (FOX) disk (center) was strategically placed at the location where the aztreonam and piperacillin/tazobactam zone margins were expected to be if no induction occurred.
© 2008 Elsevier
Clinical Microbiology Newsletter 30:11,2008
to potentiate the activity of cephamycins (34-36). This approach may also yield positive results with carbapenemases and also with certain ESBLs and OXA-12. It can be less sensitive than the AmpC disk test in detecting DHA-like plasmid-mediated AmpC β-lactamases. Cloxacillin is also an inhibitor of AmpC β-lactamases, making it possible to use the double-disk test with cloxacillin (instead of clavulanate) as an AmpC inhibitor and cephalosporins, such as cefotaxime and ceftazidime, as substrates. As with any application of the double-disk test, it can sometimes be difficult to achieve optimal disk spacing for this test. Since inducible AmpC β-lactamases are associated with an increased risk of therapeutic failure (see above), the inducibility of confirmed plasmid-mediated AmpC β-lactamases should be investigated. This can be done with the disk approximation test (Fig. 6) (37). Because some enzymes are only weakly inducible, interpretation of this test requires care.
Plasmid-Mediated AmpC BetaLactamases – Final Comments
Our understanding of these enzymes is hampered by the lack of information. This is a consequence of most laboratories not attempting to detect plasmidmediated AmpCs. More clinical data and therapeutic outcome studies are needed. Currently, some investigators recommend that resistance to third-generation cephalosporins and aztreonam should be reported irrespective of the MIC if a plasmid-mediated AmpC βlactamase is detected (32). If such an approach is warranted, it may be necessary to extend it to first- and secondgeneration cephalosporins, aztreonam, and β-lactamase inhibitor combinations, leaving the carbapenems and (providing isolate is ESBL-negative) fourth-generation cephalosporinsas the only β-lactam drugs to which susceptibility should be reported. Another consideration is that, because E. coli and Salmonella isolates are also known to produce plasmidmediated AmpCs, occurrence and prevalence studies focusing on these enzymes should include these organisms.
Carbapenem-Hydrolyzing Beta-Lactamases
The carbapenem-hydrolyzing
Clinical Microbiology Newsletter 30:11,2008
MBLs are so-called because they require one or more divalent cations at the active site for activation (usually zinc). These enzymes are resistant to β-lactamase inhibitors that are used clinically but are inhibited by EDTA. When produced at high levels, MBLs
have a broad spectrum of activity and can cause resistance to all carbapenems, penicillins, cephamycins, cephalosporins, and β-lactamase inhibitor combinations. MBLs do not hydrolyze aztreonam very well, which is different than ESBLs or class A β-lactamases. Chromosomally encoded MBLs are produced by Bacillus cereus, Bacteriodes fragilis, Aeromonas spp., and Stenotrophomonas maltophilia, and also by some members of the former genus Flavobacterium, including Elizabethkingia meningosepticum (formerly Chryseobacterium meningosepticum), Myroides spp. (formerly Flavobacterium odoratum), Legionella gormanii, and Sphingobacterium multivorum. Acquired or transmissible MBLs can be plasmid, integron, or transposon mediated and have been reported in organisms, such as B. fragilis, P. aeruginosa, Psuedomonas putida, Psuedomonas fluorescens, Alcaligenes xylosoxidans, Acinetobacter spp., E. coli, C. freundii, Enterobacter aerogenes, E. cloacae, Proteus vulgaris, Providencia rettgeri, S. marcescens, and K. pneumoniae. The emergence of acquired MBLs among major gram-negative pathogens is a worldwide concern. There are five major families of acquired MBLs (IMP, VIM, SPM, GIM, and SIM). In 1990, IMP-1, the first MBL encoded on a plasmid, was discovered in Japan. Since then, other variants numbering up to IMP-23 have been reported. The other major family of MBLs is the VIM family (VIM-1 to -14). VIM enzymes have been associated with nosocomial outbreaks of P. aeruginosa infection in Italy and Greece. Of the remaining families, SPM was first reported in Brazil, GIM in Germany, and SIM in Korea, all named for the places they were discovered. MBLs are of increasing clinical significance, especially in countries where outbreaks have occurred. There have been reports of outbreaks due to isolates that produce acquired MBLs in Japan, southern Europe, South America, Australia, Canada, and the U.S. Because acquired MBLs are such a dangerous threat, it is vital not to be complacent in areas where these enzymes are not endemic and not to neglect testing for them. The introduction of pathogens possessing MBLs in a previously “clean” population can occur through
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enzymes possess the most extensive substrate profiles of all β-lactamases and are increasingly clinically important β-lactamases (38). Chromosomal enzymes capable of hydrolyzing carbapenems have been recognized without causing undue concern for a long time, but the more promiscuous acquired or imported versions of these enzymes are a major threat. All may cause problems of false susceptibility in routine susceptibility tests.
Molecular Class A Enzymes (Bush Group 2f)
Class A carbapenemases have a serine molecule required for action. These enzymes are functionally different from metallo-β-lactamases (MBLs) (described below), because they hydrolyze ampicillin and some cephalosporins more rapidly than carbapenems and because of their ability to hydrolyze aztreonam. Tazobactam and clavulana te inhibit these enzymes but not as well as ESBLs (3840). Class A carbapenemases have been detected in isolates of E. coli, Klebsiella pneumoniae, K. oxytoca, E. cloacae, C. freundii, Serratia marcescens, Enterobacter hormaechei, Salmonella, and P. aeruginosa (41-45). Currently, described enzymes include KPC-1 to -4, SME-1 to -3, NmcA, IMI-1 and -2, and GES-2, and -4 to- 6. KPC β-lactamases are now endemic in the eastern U.S., with many isolates producing KPC-2 and KPC-3 enzymes (46-48). Isolates producing a KPC enzyme have been detected in New York, Arizona, Arkansas, Maryland, Massachusetts, Michigan, Missouri, North Carolina, New Jersey, Ohio, Pennsylvania, and Virginia, as well as Puerto Rico, China, Israel, Colombia, France, and Scotland. The occurrence of isolates that produce KPCs is likely much higher than is currently known, because screening and detection methods are lacking in many laboratories.
Molecular Class B Enzymes (Metallo-β-Lactamases of Bush group 3)
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the transfer of patients who were previously in areas of endemicity.
Molecular Class D Enzymes
The molecular class D enzymes that have been reported to be capable of hydrolyzing the carbapenems are mainly of the OXA family. The OXAs are so named because of their ability to hydrolyze oxacillin. There is significant amino acid diversity among the members of this family. The OXA family has been known since the late 1970s, but in recent years, it has become recognized as a reason for carbapenem resistance in Acinetobacter baumannii, and P. aeruginosa. Most OXA-type carbapenemases are chromosomal. Some, such as OXA50, OXA-51, and OXA-60, have been reported to be natur ally occurr ing enzymes in the species P. aeruginosa, A. baumannii, and Ralstonia pickettii, respectively (49). In some cases, hydrolysis of carbapenems by OXA enzymes is weak, but the effect can be magnified by porin and efflux mutations, which can result in carbapenem resistance (38,50-52).
Screening and Detection of Carbapenemases
Reduced susceptibility to any carbapenem can be used as a screen for carbapenemases. However, ertapenem has been advocated as the most convenient screening agent for class A carbapenemases, because KPC-producing isolates are usually insusceptible to ertapenem, while some remain susceptible to meropenem and imipenem (53). In Japan, screening with ceftazidime for MBLs has been recommended (54). Because other β-lactamases, especially AmpCs and ESBLs, cause ceftazidime resistance, it is essential that positive screens be followed by a confirmatory test for MBL production. Insusceptibility to ceftazidime may also prove to be a versatile screen that covers other classes of carbapenemases, as well as MBLs, in addition to ESBLs and AmpCs. If this screening approach is adopted, confirmatory methods must be used to distinguish between the various types of enzymes.
Tests for Class A Enzymes and Metallo-β-Lactamases
Although a variety of phenotypic tests have been proposed for the detec-
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Figure 7. A modified Hodge test which is positive for three different types of carbapenemhydrolyzing enzymes.
tion of carbapenemases, none have been recommended by the CLSI. This deters some clinical laboratories from attempting to detect and confirm these important enzymes. The Hodge or clover leaf test is an economical approach for detection and confirmation of carbapenemase activity (55). This test, however, cannot differentiate between a class A carbapenemase and an MBL, making further confirmatory testing necessary. Depending on whether imipene m, meropenem, or ertapenem is utilized in the Hodge test, false-positive results can occur with isolates that produce high levels of AmpC. Imipenem is a more sensitive but less specific carbapenem than meropenem or ertapenem for this test, allowing detection of even OXA carbapenemases, but it is the least specific agent. As shown in Fig. 7, a carbapenem disk is placed on a lawn inoculum of susceptible E. coli, and test colonies on loops are streaked away from the disk. After overnight incubation, a positive test is interpreted to be one in which there is an indentation of the inhibition zone where it meets the streaked growth. MBL detection tests involving inhibitors such as EDTA and 2-mercaptopropionic acid (2-MPA) (thiolac tic acid, Sigma T31003) have been recommended, some of which are commer© 2008 Elsevier
cially available (56-60). Tris/EDTA disks (see Plasmid-Mediated AmpC Beta-Lactamase Detection Issues above.) can also be used in combination with a carbapenem disk to detect carbapenemhydrolyzing enzymes and to differentiate between class A enzymes and MBLs. The MBLs are inhibited by the Tris/ EDTA disk. The inhibition can be amplified by the addition of 20 μl of the chelator, 2-MPA, diluted 1:320 with sterile water or saline (61), to the disk. The inhibitory effect of the two chelating agents on a VIM-2-like enzyme is shown in Fig. 8. Class A carbapenemases cause a distinctly different effect in that there is an indentation in the carbapenem zone (Fig. 9) instead of the extended zone that is produced by inhibition of an MBL (Fig. 8). Imipenem disks are the most sensitive carbapenem disks to use for detection of class A carbapenemases, because the zone is usually larger and indentation is more pronounced. An advantage of the direct method is that it can be included as part of the routine disk diffusion test (Fig. 9). Class A carbapenemase testing can also be done by the indirect method, similar to the AmpC Disk test. A susceptible E. coli isolate is used as the lawn culture (Fig. 10). This method is recommended if there is no inhibition zone around the carbapenem disk or to Clinical Microbiology Newsletter 30:11,2008
confirm a difficult-to-interpret result in the direct Tris/EDTA test. The indirect test using meropenem as a substrate is preferable for detection of class A carbapenemases in P. aeruginosa because tests with imipenem and ertapenem may yield weak false-positive tests with isolates that produce high levels of chromosomal AmpC. In the absence of therapeutic-outcome data to prove otherwise, it seems prudent to report isolates with positive carbapenemase test results as resistant to all carbapenems and cephalosporins. This should protect infected patients from receiving potentially suboptimal or ineffective therapy.
Conclusion
It is crucial that the clinical microbiology laboratory provide early and accurate reports about pathogens producing clinically significant β-lactamases. Bacteria continue to evolve increasingly complex mechanisms of resistance to β-lactam antibiotics (62). Older methods may no longer be accurate. Therefore, it is necessary to regularly review the adequacy of testing methods. For laboratories that encounter ESBLs and potential plasmid-mediated AmpC, carbapenemase, or other novel β-lactamase problems, it is necessary to extend testing beyond the scope of current CLSI recommendations. When this happens, it is advisable to confer with a reference laboratory to determine if non-CLSI test findings of important β-lactamases are real. References 30. Papanicolaou, G.A., A.A. Medeiros, and G.A. Jacoby. 1990. Novel plasmidmediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34:2200-2209. 31. Bauernfeind, A., Y. Chong, and S. Schweighart. 1989. Extended broadspectrum ß-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17:316-321. 32. Pai, H. et al. 2004. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamaseproducing Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48:3720-3728. 33. Moland, E.S. et al. 2006. Prevalence of newer β-lactamases in gram-negative Clinical Microbiology Newsletter 30:11,2008
Figure 8. Direct metallo-β-lactamase (MBL) test. VIM-2-like producing K. pneumoniae is the lawn culture and is inoculated onto a Tris/EDTAdisk that is placed to the right of an imipenem disk (yielding a negative class A carbapenemase test). The extended inhibition zone between the imipenem disk and the Tris/EDTA+ 2-MPA disk (not inoculated with the test organism) and placed further away to the left of the imipenem disk indicates production of an MBL (positive test).
Figure 9. Direct test for class A carbapenemase. KPC-2-producing K. pneumoniae is the lawn culture and is inoculated onto a Tris/EDTA disk that is placed beside an imipenem disk. Note the indentation that indicates production of a carbapenem-hydrolyzing enzyme (positive test). Note also that a second Tris/EDTA+ 2-MPAdisk (not inoculated with the test organism) was placed further away from the imipenem disk to test for MBL production (negative test).
clinical isolates collected in the United States from 2001 to 2002. J. Clin. Microbiol. 44:3318-3324. 34. Yagi, T. et al. 2005. Practical methods © 2008 Elsevier
using boronic acid compounds for identification of class C {beta}-lactamaseproducing Klebsiella pneumoniae and Escherichia coli. J. Clin. Microbiol. 0196-4399/00 (see frontmatter)
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43:2551-2558. Coudron, P.E. 2005. Inhibitor-based methods for detection of plasmidmediated AmpC beta-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. J. Clin. Microbiol. 43:4163-4167. Jacoby, G.A., K.E. Walsh, and V.J. Walker. 2006. Identification of extendedspectrum, AmpC, and carbapenemhydrolyzing beta-lactamases in Escherichia coli and Klebsiella pneumoniae by disk tests. J. Clin. Microbiol. 44:19711976. Sanders, C.C. and W.E. Sanders, Jr. 1979. Emergence of resistance to cefamandole: possible role of cefoxitininducible beta-lactamases. Antimicrob. Agents Chemother. 15:792-797. Queenan, A.M. and K. Bush. 2007. Carbapenemases: the versatile betalactamases. Clin. Microbiol. Rev. 20:440-458 Livermore, D.M. 1997. Acquired carbapenemases. J. Antimicrob. Chemother. 39:673-676. Walther-Rasmussen, J. and N. Hoiby. 2007. Class A carbapenemases. J. Antimicrob. Chemother. 60:470-482. Yigit, H. et al. 2001. Novel carbapenemhydrolyzing beta-lactamase, KPC-1, from a carbapenem- resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45:1151-1161. Miriagou, V. et al. 2003. Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2. Antimicrob. Agents Chemother. 47:1297-1300. Pottumarthy, S. et al. 2003. NmcA carbapenem-hydrolyzing enzyme in Enterobacter cloacae in North America. Emerg. Infect. Dis. 9:999-1002. Hossain, A. et al. 2004. Plasmid-mediated carbapenem-hydrolyzing enzyme KPC2 in an Enterobacter sp. Antimicrob. Agents Chemother. 48:4438-4440. Bratu, S. et al. 2005. Carbapenemaseproducing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents. J. Antimicrob. Chemother. 56:128-132. Bradford, P.A. et al. 2004. Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenemhydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New York City. Clin. Infect. Dis. 39:55-60. Woodford, N. et al. 2004. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in a New York 0196-4399/00 (see frontmatter)
Figure 10. Indirect test for class A carbapenemase with Tris/EDTA disk inoculated with test organism placed near imipenem, ertapenem, and meropenem showing both positive test (distortion of zone surrounding carbapenem disks) and negative results (no distortion).
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Medical Center. Antimicrob. Agents Chemother. 48:4793-4799. Lomaestro, B.M. et al. 2006. The spread of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae to upstate New York. Clin. Infect. Dis. 43:e26-e28. Walther-Rasmussen, J. and N. Hoiby. 2006. OXA-type carbapenemases. J. Antimicrob. Chemother. 57:373-383. Poirel, L. and P. Nordmann. 2002. Acquired carbapenem-hydrolyzing betalactamases and their genetic support. Curr. Pharm. Biotechnol. 3:117-127. Heritier, C. et al. 2005. Contribution of acquired carbapenem-hydrolyzing oxacillinases to carbapenem resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 49:3198-3202. Donald, H.M. et al. 2000. Sequence analysis of ARI-1, a novel OXA betalactamase, responsible for imipenem resistance in Acinetobacter baumannii 6B92. Antimicrob. Agents Chemother. 44:196-199. Anderson, K.F. et al. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45:2723-2725. Hirakata, Y. et al. 1998. Rapid detection and evaluation of clinical characteristics of emerging multiple-drug-resistant gram-negative rods carrying the metallo© 2008 Elsevier
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beta-lactamase gene blaIMP. Antimicrob. Agents Chemother. 42:2006-2011. Lee, K. et al. 2001. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clin. Microbiol. Infect. 7:88-91. Walsh, T.R. et al. 2002. Evaluation of a new Etest for detecting metallo-betalactamases in routine clinical testing. J. Clin. Microbiol. 40:2755-2759. Migliavacca, R. et al. 2002. Simple microdilution test for detection of metallobeta-lactamase production in Pseudomonas aeruginosa. J. Clin. Microbiol. 40:4388-4390. Arakawa, Y. et al. 2000. Convenient test for screening metallo-beta-lactamaseproducing gram-negative bacteria by using thiol compounds. J. Clin. Microbiol. 38:40-43. Yong, D. et al. 2002. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol. 40:3798-3801. Livermore, D.M., M. Warner, and S. Mushtaq. 2007. Evaluation of the chromogenic Cica-(beta)-test for detecting extended-spectrum, AmpC and metallo(beta)-lactamases. J. Antimicrob. Chemother. 60:1375-1379. Kim, S.Y. et al. 2007. Convenient test Clinical Microbiology Newsletter 30:11,2008
using a combination of chelating agents for detection of metallo-(beta)-lactamases in the clinical laboratory. J. Clin.
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Microbiol. 45:2798-801. 62. Medeiros, A.A. 1997. Evolution and dissemination of β-lactamases acceler-
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ated by generations of β-lactam antibiotics. Clin. Infect. Dis. 24(Suppl. 1): S19-S45.
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Newer β-Lactamases: Clinical and Laboratory Implications, Part II* Ellen Smith Moland, B.S.M.T., Soo-Young Kim, M.D., Seong Geun Hong, M.D., and Kenneth S. Thomson, Ph.D., Center for Research in Antiinfectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska
Abstract
For optimal patient care, clinical laboratories should be capable of detecting clinically significant, novel β-lactamases produced by gram-negative pathogens. However, with over 700 β-lactamases now described, it is a struggle to keep abreast of the various types of β-lactamases, their clinical relevance, and methods for detection. Furthermore, the increasing prevalence of isolates that produce multiple β-lactamases increases the difficulty of accurate detection. Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) recommendations for detection of β-lactamases do not keep pace with this rapidly evolving field. While perfection may not always be possible, it is important that clinical laboratories provide a relevant diagnostic service to ensure appropriate antibiotic therapy and infection control. Part II of this article will provide a discussion of AmpC β-lactamases and other β-lactam resistance mechanisms, along with methods for their laboratory detection.
Plasmid-Mediated AmpC Beta-Lactamases
Imported, transmissible, or plasmidmediated AmpC β-lactamase genes were first reported in Escherichia coli and Klebsiella pneumoniae in 1988 (30,31). Today, there are over 40 known plasmidmediated AmpC β-lactamases (http:// www.lahey.org/Studies/). Molecular data suggest these enzymes are derived from Enterobacter cloacae, Citrobacter freundii, Morganella morganii, Hafnia alvei, and unknown sources. They have been mostly detected in isolates of E. coli, K. pneumoniae, Proteus mirabilis,
*Editor’s Note: Part I of this article was published in the May 15, 2008 issue of CMN (Vol. 30, No. 10). Mailing Address: Ellen Smith Moland, B.S.M.T., Center for Research in Antiinfectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska 68178. Tel.: 402-2802921. Fax: 402-280-1875. E-mail: [email protected] and [email protected] Clinical Microbiology Newsletter 30:11,2008
and Salmonella. Most plasmid-mediated AmpCs are constitutively expressed, but some enzymes, such as DHA-1, DHA-2, ACT-1, CFE-1, and CMY-13, are inducible and may be more clinically dangerous. Inducibility may confer the capability for an organism to become more resistant during β-lactam therapy than indicated by susceptibility tests. Clinical data verify that this happens (32). Therapeutic failures with cefotaxime have been reported with susceptible isolates of plasmid-mediated AmpC-producing K. pneumoniae (22). In most studies, plasmid-mediated AmpC β-lactamases have been less common than extended-spectrum β-lactamases (ESBLs). For example, in a 2001/2002 U.S. study, plasmidmediated AmpCs were detected in 3.3% of K. pneumoniae isolates from 63 U.S. sites, whereas ESBLs were detected in 11.3% of isolates (33). Prevalence can be expected to increase in the absence of detection by clinical laboratories, because lack of detection makes infection control impossible. © 2008 Elsevier
Plasmid-Mediated AmpC BetaLactamase Detection Issues
There are currently no CLSI recommendations for detection of plasmid-mediated AmpC β-lactamases. Klebsiella is a convenient indicator organism for detection of plasmidmediated AmpCs, because it does not produce a chromosomally mediated AmpC. P. mirabilis and Salmonella are also good indicator organisms for the same reason. E. coli is also a likely source of plasmid-mediated AmpCs, but phenotypic detection is complicated, because positive results may also occur with occasional isolates that hyper-
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produce their chromosomal AmpC β-lactamases. Cephamycins (cefoxitin and cefotetan) are good screening agents for detection of most AmpC β-lactamases. As indicated above, screening by cefoxitin insusceptibility (i.e., resistance or intermediate susceptibility) is useful but not specific. Reduced outer membrane permeability may also confer cefoxitin insusceptibility, making differentiation between these two resistance mechanisms necessary. Cefotetan is potentially useful for AmpC screening, but it must be remembered that this agent is more potent than cefoxitin, and a more sensitive screening criterion than insusceptibility will be necessary. Amp-C Class (ACC)-like plasmid-mediated AmpC β-lactamases that do not hydrolyze cephamycins significantly will not be reliably detected by insusceptibility to cefoxitin. A variety of approaches to AmpC detection have been recommended. The AmpC disk test is useful, because it determines if an organism produces an enzyme that hydrolyzes cefoxitin significantly (Fig. 5). This test utilizes Tris/ EDTA disks (BD Diagnostic Systems, Baltimore, MD). Since other enzymes, particularly carbapenem-hydrolyzing enzymes, also hydrolyze cefoxitin, it is important to interpret this test in conjunction with inspection of carbapenem susceptibility test results. This is especially important in locations where Klebsiella pneumoniae (KPC) enzymes have been detected (see below). The AmpC disk test seems to be the most sensitive test for detecting the DHAlike enzymes. It is an indirect test in that an indicator organism is the lawn inoculum on the plate and not the test isolate. For this reason, the test requires a separate plate other than the routine disk diffusion susceptibility plate. Another approach to the detection of AmpC β-lactamases involves using AmpC inhibitors, such as boronic acid,
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Figure 5. AmpC disk test with Tris/EDTA disk inoculated with test organism placed near a cefoxitin disk (FOX 30) showing positive test (distortion of zone surrounding a cefoxitin disk).
FOX
TZB
ATM
Flatting of zone Figure 6. Disk approximation test showing inducible AmpC production, indicated by flattening or truncation of inhibition zones around aztreonam (ATM) and piperacillin/tazobactam (TZP) disks. The cefoxitin (FOX) disk (center) was strategically placed at the location where the aztreonam and piperacillin/tazobactam zone margins were expected to be if no induction occurred.
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to potentiate the activity of cephamycins (34-36). This approach may also yield positive results with carbapenemases and also with certain ESBLs and OXA-12. It can be less sensitive than the AmpC disk test in detecting DHA-like plasmid-mediated AmpC β-lactamases. Cloxacillin is also an inhibitor of AmpC β-lactamases, making it possible to use the double-disk test with cloxacillin (instead of clavulanate) as an AmpC inhibitor and cephalosporins, such as cefotaxime and ceftazidime, as substrates. As with any application of the double-disk test, it can sometimes be difficult to achieve optimal disk spacing for this test. Since inducible AmpC β-lactamases are associated with an increased risk of therapeutic failure (see above), the inducibility of confirmed plasmid-mediated AmpC β-lactamases should be investigated. This can be done with the disk approximation test (Fig. 6) (37). Because some enzymes are only weakly inducible, interpretation of this test requires care.
Plasmid-Mediated AmpC BetaLactamases – Final Comments
Our understanding of these enzymes is hampered by the lack of information. This is a consequence of most laboratories not attempting to detect plasmidmediated AmpCs. More clinical data and therapeutic outcome studies are needed. Currently, some investigators recommend that resistance to third-generation cephalosporins and aztreonam should be reported irrespective of the MIC if a plasmid-mediated AmpC βlactamase is detected (32). If such an approach is warranted, it may be necessary to extend it to first- and secondgeneration cephalosporins, aztreonam, and β-lactamase inhibitor combinations, leaving the carbapenems and (providing isolate is ESBL-negative) fourth-generation cephalosporinsas the only β-lactam drugs to which susceptibility should be reported. Another consideration is that, because E. coli and Salmonella isolates are also known to produce plasmidmediated AmpCs, occurrence and prevalence studies focusing on these enzymes should include these organisms.
Carbapenem-Hydrolyzing Beta-Lactamases
The carbapenem-hydrolyzing
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MBLs are so-called because they require one or more divalent cations at the active site for activation (usually zinc). These enzymes are resistant to β-lactamase inhibitors that are used clinically but are inhibited by EDTA. When produced at high levels, MBLs
have a broad spectrum of activity and can cause resistance to all carbapenems, penicillins, cephamycins, cephalosporins, and β-lactamase inhibitor combinations. MBLs do not hydrolyze aztreonam very well, which is different than ESBLs or class A β-lactamases. Chromosomally encoded MBLs are produced by Bacillus cereus, Bacteriodes fragilis, Aeromonas spp., and Stenotrophomonas maltophilia, and also by some members of the former genus Flavobacterium, including Elizabethkingia meningosepticum (formerly Chryseobacterium meningosepticum), Myroides spp. (formerly Flavobacterium odoratum), Legionella gormanii, and Sphingobacterium multivorum. Acquired or transmissible MBLs can be plasmid, integron, or transposon mediated and have been reported in organisms, such as B. fragilis, P. aeruginosa, Psuedomonas putida, Psuedomonas fluorescens, Alcaligenes xylosoxidans, Acinetobacter spp., E. coli, C. freundii, Enterobacter aerogenes, E. cloacae, Proteus vulgaris, Providencia rettgeri, S. marcescens, and K. pneumoniae. The emergence of acquired MBLs among major gram-negative pathogens is a worldwide concern. There are five major families of acquired MBLs (IMP, VIM, SPM, GIM, and SIM). In 1990, IMP-1, the first MBL encoded on a plasmid, was discovered in Japan. Since then, other variants numbering up to IMP-23 have been reported. The other major family of MBLs is the VIM family (VIM-1 to -14). VIM enzymes have been associated with nosocomial outbreaks of P. aeruginosa infection in Italy and Greece. Of the remaining families, SPM was first reported in Brazil, GIM in Germany, and SIM in Korea, all named for the places they were discovered. MBLs are of increasing clinical significance, especially in countries where outbreaks have occurred. There have been reports of outbreaks due to isolates that produce acquired MBLs in Japan, southern Europe, South America, Australia, Canada, and the U.S. Because acquired MBLs are such a dangerous threat, it is vital not to be complacent in areas where these enzymes are not endemic and not to neglect testing for them. The introduction of pathogens possessing MBLs in a previously “clean” population can occur through
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enzymes possess the most extensive substrate profiles of all β-lactamases and are increasingly clinically important β-lactamases (38). Chromosomal enzymes capable of hydrolyzing carbapenems have been recognized without causing undue concern for a long time, but the more promiscuous acquired or imported versions of these enzymes are a major threat. All may cause problems of false susceptibility in routine susceptibility tests.
Molecular Class A Enzymes (Bush Group 2f)
Class A carbapenemases have a serine molecule required for action. These enzymes are functionally different from metallo-β-lactamases (MBLs) (described below), because they hydrolyze ampicillin and some cephalosporins more rapidly than carbapenems and because of their ability to hydrolyze aztreonam. Tazobactam and clavulana te inhibit these enzymes but not as well as ESBLs (3840). Class A carbapenemases have been detected in isolates of E. coli, Klebsiella pneumoniae, K. oxytoca, E. cloacae, C. freundii, Serratia marcescens, Enterobacter hormaechei, Salmonella, and P. aeruginosa (41-45). Currently, described enzymes include KPC-1 to -4, SME-1 to -3, NmcA, IMI-1 and -2, and GES-2, and -4 to- 6. KPC β-lactamases are now endemic in the eastern U.S., with many isolates producing KPC-2 and KPC-3 enzymes (46-48). Isolates producing a KPC enzyme have been detected in New York, Arizona, Arkansas, Maryland, Massachusetts, Michigan, Missouri, North Carolina, New Jersey, Ohio, Pennsylvania, and Virginia, as well as Puerto Rico, China, Israel, Colombia, France, and Scotland. The occurrence of isolates that produce KPCs is likely much higher than is currently known, because screening and detection methods are lacking in many laboratories.
Molecular Class B Enzymes (Metallo-β-Lactamases of Bush group 3)
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the transfer of patients who were previously in areas of endemicity.
Molecular Class D Enzymes
The molecular class D enzymes that have been reported to be capable of hydrolyzing the carbapenems are mainly of the OXA family. The OXAs are so named because of their ability to hydrolyze oxacillin. There is significant amino acid diversity among the members of this family. The OXA family has been known since the late 1970s, but in recent years, it has become recognized as a reason for carbapenem resistance in Acinetobacter baumannii, and P. aeruginosa. Most OXA-type carbapenemases are chromosomal. Some, such as OXA50, OXA-51, and OXA-60, have been reported to be natur ally occurr ing enzymes in the species P. aeruginosa, A. baumannii, and Ralstonia pickettii, respectively (49). In some cases, hydrolysis of carbapenems by OXA enzymes is weak, but the effect can be magnified by porin and efflux mutations, which can result in carbapenem resistance (38,50-52).
Screening and Detection of Carbapenemases
Reduced susceptibility to any carbapenem can be used as a screen for carbapenemases. However, ertapenem has been advocated as the most convenient screening agent for class A carbapenemases, because KPC-producing isolates are usually insusceptible to ertapenem, while some remain susceptible to meropenem and imipenem (53). In Japan, screening with ceftazidime for MBLs has been recommended (54). Because other β-lactamases, especially AmpCs and ESBLs, cause ceftazidime resistance, it is essential that positive screens be followed by a confirmatory test for MBL production. Insusceptibility to ceftazidime may also prove to be a versatile screen that covers other classes of carbapenemases, as well as MBLs, in addition to ESBLs and AmpCs. If this screening approach is adopted, confirmatory methods must be used to distinguish between the various types of enzymes.
Tests for Class A Enzymes and Metallo-β-Lactamases
Although a variety of phenotypic tests have been proposed for the detec-
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Figure 7. A modified Hodge test which is positive for three different types of carbapenemhydrolyzing enzymes.
tion of carbapenemases, none have been recommended by the CLSI. This deters some clinical laboratories from attempting to detect and confirm these important enzymes. The Hodge or clover leaf test is an economical approach for detection and confirmation of carbapenemase activity (55). This test, however, cannot differentiate between a class A carbapenemase and an MBL, making further confirmatory testing necessary. Depending on whether imipene m, meropenem, or ertapenem is utilized in the Hodge test, false-positive results can occur with isolates that produce high levels of AmpC. Imipenem is a more sensitive but less specific carbapenem than meropenem or ertapenem for this test, allowing detection of even OXA carbapenemases, but it is the least specific agent. As shown in Fig. 7, a carbapenem disk is placed on a lawn inoculum of susceptible E. coli, and test colonies on loops are streaked away from the disk. After overnight incubation, a positive test is interpreted to be one in which there is an indentation of the inhibition zone where it meets the streaked growth. MBL detection tests involving inhibitors such as EDTA and 2-mercaptopropionic acid (2-MPA) (thiolac tic acid, Sigma T31003) have been recommended, some of which are commer© 2008 Elsevier
cially available (56-60). Tris/EDTA disks (see Plasmid-Mediated AmpC Beta-Lactamase Detection Issues above.) can also be used in combination with a carbapenem disk to detect carbapenemhydrolyzing enzymes and to differentiate between class A enzymes and MBLs. The MBLs are inhibited by the Tris/ EDTA disk. The inhibition can be amplified by the addition of 20 μl of the chelator, 2-MPA, diluted 1:320 with sterile water or saline (61), to the disk. The inhibitory effect of the two chelating agents on a VIM-2-like enzyme is shown in Fig. 8. Class A carbapenemases cause a distinctly different effect in that there is an indentation in the carbapenem zone (Fig. 9) instead of the extended zone that is produced by inhibition of an MBL (Fig. 8). Imipenem disks are the most sensitive carbapenem disks to use for detection of class A carbapenemases, because the zone is usually larger and indentation is more pronounced. An advantage of the direct method is that it can be included as part of the routine disk diffusion test (Fig. 9). Class A carbapenemase testing can also be done by the indirect method, similar to the AmpC Disk test. A susceptible E. coli isolate is used as the lawn culture (Fig. 10). This method is recommended if there is no inhibition zone around the carbapenem disk or to Clinical Microbiology Newsletter 30:11,2008
confirm a difficult-to-interpret result in the direct Tris/EDTA test. The indirect test using meropenem as a substrate is preferable for detection of class A carbapenemases in P. aeruginosa because tests with imipenem and ertapenem may yield weak false-positive tests with isolates that produce high levels of chromosomal AmpC. In the absence of therapeutic-outcome data to prove otherwise, it seems prudent to report isolates with positive carbapenemase test results as resistant to all carbapenems and cephalosporins. This should protect infected patients from receiving potentially suboptimal or ineffective therapy.
Conclusion
It is crucial that the clinical microbiology laboratory provide early and accurate reports about pathogens producing clinically significant β-lactamases. Bacteria continue to evolve increasingly complex mechanisms of resistance to β-lactam antibiotics (62). Older methods may no longer be accurate. Therefore, it is necessary to regularly review the adequacy of testing methods. For laboratories that encounter ESBLs and potential plasmid-mediated AmpC, carbapenemase, or other novel β-lactamase problems, it is necessary to extend testing beyond the scope of current CLSI recommendations. When this happens, it is advisable to confer with a reference laboratory to determine if non-CLSI test findings of important β-lactamases are real. References 30. Papanicolaou, G.A., A.A. Medeiros, and G.A. Jacoby. 1990. Novel plasmidmediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34:2200-2209. 31. Bauernfeind, A., Y. Chong, and S. Schweighart. 1989. Extended broadspectrum ß-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17:316-321. 32. Pai, H. et al. 2004. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamaseproducing Klebsiella pneumoniae. Antimicrob. Agents Chemother. 48:3720-3728. 33. Moland, E.S. et al. 2006. Prevalence of newer β-lactamases in gram-negative Clinical Microbiology Newsletter 30:11,2008
Figure 8. Direct metallo-β-lactamase (MBL) test. VIM-2-like producing K. pneumoniae is the lawn culture and is inoculated onto a Tris/EDTAdisk that is placed to the right of an imipenem disk (yielding a negative class A carbapenemase test). The extended inhibition zone between the imipenem disk and the Tris/EDTA+ 2-MPA disk (not inoculated with the test organism) and placed further away to the left of the imipenem disk indicates production of an MBL (positive test).
Figure 9. Direct test for class A carbapenemase. KPC-2-producing K. pneumoniae is the lawn culture and is inoculated onto a Tris/EDTA disk that is placed beside an imipenem disk. Note the indentation that indicates production of a carbapenem-hydrolyzing enzyme (positive test). Note also that a second Tris/EDTA+ 2-MPAdisk (not inoculated with the test organism) was placed further away from the imipenem disk to test for MBL production (negative test).
clinical isolates collected in the United States from 2001 to 2002. J. Clin. Microbiol. 44:3318-3324. 34. Yagi, T. et al. 2005. Practical methods © 2008 Elsevier
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Figure 10. Indirect test for class A carbapenemase with Tris/EDTA disk inoculated with test organism placed near imipenem, ertapenem, and meropenem showing both positive test (distortion of zone surrounding carbapenem disks) and negative results (no distortion).
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