Clinical Microbiology Newsletter

Clinical Microbiology Newsletter $88 Vol. 30, No. 10 www.cmnewsletter.com May 15, 2008 Newer β-lactamases: Clinical and Laboratory Implications, Part I * 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 I of this article will provide a brief discussion of extended-spectrum β-lactamases and methods for their laboratory detection. Introduction β-lactamases, the most important cause of bacterial resistance to β-lactam antibiotics, are enzymes that inactivate β-lactam antibiotics by hydrolysis of the β-lactam bond. These enzymes may differ in substrate profiles, i.e., the drugs they can inactivate. It is vital that clinical laboratories have the capability to detect clinically significant resistance caused by β-lactamases. Despite the discovery of extendedspectrum β-lactamases (ESBLs) in 1983 and plasmid-mediated AmpC β-lactamasesin 1989, asignificantnumber of *Editor s Note: Part II of this article will be published in the June 1, 2008 issue of CMN (Vol. 30, No. 11). 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.: 402-280- 2921. Fax: 402-280-1875. E-mail: esmoland@creighton.eduand kstaac@creighton.edu clinical laboratories still have difficulty in detecting them and are unaware of the relevant reporting guidelines (1,2). There are also other novel β-lactamases, such as carbapenem-hydrolyzing enzymes and inhibitor-resistant variants of common β-lactamases. This article addresses the role of the clinical microbiology laboratory in detecting and reporting the newer β-lactamases. It also covers other issues, such as clinical significance, occurrence, and therapeutic considerations, regarding these resistance mechanisms. The primary focus of this article is to provide some helpful testing approaches that can be used in a variety of laboratory situations, including those where currently there are no Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) recommendations. These tests involve simple materials and a low to medium level of difficulty. Although they can be performed by individuals who have had little training, a microbiologist should interpret the test results. Extended-Spectrum Beta- Lactamases The term, extended-spectrum β-lactamase, refers to the distinctive substrate profile of these enzymes. Most are plasmid mediated: hydrolyze penicillins, cephalosporins, and aztreonam and are inhibited by β-lactamase inhibitors, such as clavulanate, tazobactam, and sulbactam. ESBLs cause clinically significant resistance to penicillins, cephalosporins, and aztreonam. ESBL-producing organisms occur worldwide with varying prevalences and types. They have been most commonly reported in isolates of Klebsiella spp. and Escherichia coli. They have also been reported in Enterobacter, Salmonella, Proteus, Citrobacter, Morganella morganii, Serratia marcescens, Clinical Microbiology Newsletter 30:10,2008 2008 Elsevier 0196-4399/00 (see frontmatter) 71

Shigella dysenteriae, Pseudomonas aeruginosa, Burkholderia cepacia, and Acinetobacter baumannii. It is important for clinical laboratorians to be aware that some ESBL-mediated resistance is not reliably detected by routinesusceptibility tests (3). If laboratories do not test appropriately, susceptibility reports may indicate false susceptibility to some β-lactam antibiotics and mislead clinicians into ineffectively treating infected patients. Carbapenems are the most effective antibiotics for treating infections caused by ESBL-producing Enterobacteriaceae (3).The CLSIreportingrecommendation for ESBL-producing isolates of Klebsiella spp., E. coli, andproteus mirabilis is to report resistance to all penicillins, cephalosporins, and aztreonam but to report cephamycin and β- lactamase inhibitor combinations tests without modification. The latter part of this recommendation is controversial, because cephamycin resistance due to altered porin expression may emerge (4-11) and piperacillin/tazobactam therapy has been reported to be less successful than carbapenem therapy (12-15). For this reason,some laboratories may choose either not to report cephamycins and β-lactamase inhibitor combinations or to report resistance to these agents. Carbapenems (e.g., imipenem, ertapenem, meropenem, and doripenem) are the most effective therapeutic agents for infections caused by ESBL-producing Enterobacteriaceae.SomeESBLproducing isolates may be susceptible in vitro to α-methoxy β-lactams, such as the cephamycins (e.g., cefoxitin and cefotetan) and moxalactam, as well as the aminoglycosides, fluoroquinolones, β-lactamase inhibitor combinations, or tigecycline; however, peer-reviewed therapeutic-outcome data are either lacking or less convincing than for the carbapenems. Classical ESBLs Classical ESBLs are ESBLs for which there is the most clinical experience in the United States and upon which the CLSI ESBL tests are predominantly based. Mutations in the common broadspectrum, plasmid-mediated β-lactamases TEM-1, TEM-2, and SHV-1 have expanded the hydrolytic spectrum of these enzymes (16). The parental enzymes (TEM-1, TEM-2, and SHV-1) hydrolyze penicillins and early cephalosporins, such as cephalothin, cefaclor, and cefazolin, but do not hydrolyze the later, more stable cephalosporins, like cefuroxime, cefixime, cefotaxime, ceftriaxone, ceftazidime, and cefepime, and the monobactam, aztreonam. The mutations extend the substrate profiles, permitting hydrolysis of at least some of the more stable drugs listed above. Currently, there are over 160 TEMand approximately 100 SHV-derived β-lactamases. Other ESBLs Other types of ESBLs may differ from TEM and SHV ESBLs with respect to detection and therapeutic issues (17). Some phenotypically resemble certain chromosomal β-lactamases, such as the K1 chromosomal β-lactamase of Klebsiella oxytoca and the chromosomal β-lactamases of Citrobacter koseri, Yersinia enterocolitica, Kluyvera spp., and Proteus vulgaris. Some, such as the CTX-M ESBLs, are derived from chromosomal enzymes and are not mutated forms of broadspectrum β-lactamases. The name derives from CTX, a common abbreviation for cefotaxime, which indicates the tendency of many CTX-M enzymes to hydrolyze cefotaxime faster than ceftazidime; however, a few CTX-Ms significantly hydrolyze ceftazidime (e.g., CTX-M-15, -19, -25, and -32). There are currently over 65 CTX-M ESBLs; some were previously designated Toho but now have CTX-M numbers (http://www.lahey.org/studies/).these are now the most common ESBLs worldwide. CTX-Ms have been detected mainly in isolates of Enterobacteriaceae, with E. coli being the predominant host. They have also been reported in isolates of P. aeruginosa and Stenotrophomonas maltophilia(18). Infectionscausedby community-acquired CTX-M ESBLproducing isolates are an increasing problem, and there have been many reports of clonally related outbreaks which have spread within the community and the hospital (19,20). Isolates that produce CTX-M ESBLS may have greatly reduced susceptibility to cefepime, with MICs of 64 μg/ml or higher (21).Thisisinsharpcontrasttothe majority of isolates that produce TEMand SHV-derived ESBLs. PER-1, -2, and -3 comprise a highly clavulanate-sensitive family of ESBLs, with a different epidemiology from the TEM and SHV ESBLs. PER-1, identified in 1991, has been detected in Enterobacteriaceae and nonfermenting gram-negative bacilli. PER-2 was first detected in Argentina and is now reported to be the second most prevalent ESBL in that country, having been found only in other SouthAmerican countries(22). PER-3 was discovered in an isolate of Aeromonas punctata (formerly Aeromonas caviae)infrance. ESBLs of the OXA family are increasing in occurrence. The OXA family of enzymes were given this name because they hydrolyze oxacillin and cloxacillin. These ESBLs occur mainly in P. aeruginosa. Ceftazidime resistance is a phenotypic marker, but the poor inhibition of OXAs by clavulanate can make ESBL phenotypic detection difficult. There are other individual nonclassical ESBLs that do not belong to the previously-mentioned families. These include VEB-, SFO-, and GES-type enzymes. 72 0196-4399/00 (see frontmatter) 2008 Elsevier Clinical Microbiology Newsletter 30:10,2008

Detection of ESBLs in E. coli, Klebsiella, andp. mirabilis The two main approaches to ESBL detection are screening tests and specific (or confirmatory) tests. Screening tests detect reduced susceptibility to indicator drugs, such as cefotaxime, ceftriaxone, ceftazidime, cefpodoxime, aztreonam, and cefepime. The CLSI recommended indicator drugs are listed intable1(20).screeningtestsarenot specific because mechanisms other than ESBLs may also cause positive screens. Therefore, positive screens should be followed by confirmatory tests. If a laboratory does not test for all of the screening agents, the likelihood increases that screening tests will be less than 100% sensitive. Most ESBL confirmatory tests indirectly detect hydrolysis of indicator drugs, using the principles of the doubledisktest(23,24).thatis,esblproduction is confirmed if clavulanate significantly enhances the activity of the indicatordrug(fig. 1). Adiskcontaining clavulanate, usually amoxicillin/ clavulanate, is strategically placed on an inoculated Mueller-Hinton agar susceptibility plate, typically 30 mm or less from disks containing indicator drugs. The optimal disk spacing varies from strain to strain. Clavulanate-mediated enhancement of the activity of an indicator drug, seen as a lens of inhibition or a keyhole effect, is indicative of ESBL production. Interpretation of the confirmatory test is subjective, not quantitative. This test may present difficulties, because optimal disk spacing is not always achieved on initial testing and some results require considerable experience to interpret correctly. The CLSI ESBL confirmatory tests involve testing cefotaxime and ceftazidime alone and in combination with clavulanate(table2). Contrary toa common misunderstanding, the CLSI ESBL confirmatory disk tests are not the double disk test. The CLSI ESBL disktest(fig.2)involvesquantitative, as well as qualitative, interpretation, whereas the double-disk test is qualitative. The CLSI test is confirmatory if the inhibition zone of either cephalosporin increases at least 5 mm in the presence of clavulanate. The CLSI also recommends a dilutionmethodconfirmatory testing(table 2)by determiningmicsofceftazidime Table 1. CLSI ESBL screens for E. coli, Klebsiella, andp. mirabilis E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca MIC test 2 μg/ml for cefotaxime, ceftriaxone, ceftazidime, or aztreonam and 8 μg/ml for cefpodoxime Disk test P. mirabilis MIC test Disk test Figure 1. Double-disk test showing keyhole positive results in tests with ceftazidime (CAZ), ceftriaxone (CRO), and cefotaxime (CTX), but not the cephamycin cefoxitin (FOX); the central disk is amoxicillin/clavulanate (AMC). and cefotaxime with and without the presence of clavulanic acid (4 μg/ml). A decrease in the MIC of either antimicrobial agent by 3 twofold dilutions in the presence of clavulanate is indicative of the presence of an ESBL. The Etest ESBL confirmatory test strips (AB Biodisk, Solna, Sweden) are based on the CLSI dilution method. One strip has concentration gradients of cefotaxime (0.25 to 16 μg/ml) and cefotaxime (0.016 to 1 μg/ml) plus 4 μg/ml clavulanic acid gradient, the other has a ceftazidime (0.5 to 32 μg/ml), and another strip has ceftazidime (0.064 to 17 mm for cefpodoxime, 22 mm for ceftazidime, 25 mm for ceftriaxone, and 27 mm for cefotaxime or aztreonam 2 μg/ml for cefpodoxime, cefotaxime, or ceftazidime 22 mm for cefpodoxime, 22 mm for ceftazidime, and 27 mm for cefotaxime Table 2. CLSI ESBL confirmatory tests for E. coli, Klebsiella, andp. mirabilis MIC test 3 two-fold concentration decrease in MIC of cefotaxime/ceftazidime +/- clavulanate 4 μg/ml Disk test 5-mm increase in zone diameter for cefotaxime/ceftazidime +/- clavulanate 10 μg CRO CAZ AMC FOX CTX 4 μg/ml) plus 4 μg/ml clavulanic acid gradients. These are less extensive concentration ranges than those recommended by CLSI, namely, cefotaxime (0.25 to 64 μg/ml) and ceftazidime (0.25 to 128 μg/ml) with and without 4 μg/ml of clavulanic acid. Consequently, the Etest strips may yield more nondeterminable, difficult-to-interpret results. The Etest method provides both quantitative and qualitative interpretations. The presence of an ESBL is confirmed by the appearance of a phantom zone or deformation of the cefotaxime or ceftazidime ellipse or when either Clinical Microbiology Newsletter 30:10,2008 2008 Elsevier 0196-4399/00 (see frontmatter) 73

the MIC of cefotaxime or ceftazidime is reduced by 3 log 2 dilutions in the presence of clavulanic acid. It should be noted that there is an additional cefepime-based Etest ESBL detection strip. This is discussed below. There are also automated systems, such as the Vitek Legacy and Vitek 2 (BioMérieux, Hazlewood, MO), Micro- Scan (Siemens Medical Solutions Diagnostics, Sacramento, CA), Sensititre (TREK Diagnostic Systems, Cleveland, OH), and Phoenix (BD Diagnostic Systems, Sparks, MD), that can detect ESBLs. These may involve ESBL screening and confirmatory tests, as well as expert system software to analyze susceptibility profiles that infer underlying resistance mechanisms. While helpful for many isolates, sometimes the expert systems provide misleading interpretations, because the software has not been sufficiently updated to cope with new ESBL typesofisolates (25,26). Regardlessofthe ESBL confirmatory methodology, some results may be inconclusive or non-determinable (beyond the limits of detection or offscale results). The laboratory must then use other confirmatory tests (if available) or report the results as inconclusive or non-determinable (with an explanation that ESBL production cannot be excluded). If an ESBL is detected in an isolate of Klebsiella spp., E. coli, orp. mirabilis, the CLSI recommends that the laboratory report resistance to all penicillins, cephalosporins, and aztreonam, regardless of thesusceptibilitytesting result(27). ThecurrentCLSI ESBL guidelines cover K. oxytoca, which produces the chromosomally encoded K1 β-lactamase. When hyperproduced, this enzyme may cause false-positive ESBL confirmatory tests. This enzyme is similar to ESBLs because it hydrolyzes cefpodoxime, ceftriaxone, and aztreonam efficiently but, unlike many ESBLs, does not usually hydrolyze ceftazidime. For therapeutic and infection control purposes, it is helpful to differentiate between ESBL production and hyperproduction of the K1 enzyme. Ceftazidime is, therefore, the most useful of the CLSI-recommended substrates for ESBL screening and confirmatory tests of K. oxytoca isolates. Positive CAZ/ CLA CAZ Figure 2. CLSI ESBL confirmatory disk test with ceftazidime (CAZ), alone and in combination with clavulanate (CAZ/CLA), and cefotaxime (CTX), alone and in combination with clavulanate (CTX/CLA). Although all four disks must be used, only one of the cephalosporin zones needs to be increased by at least 5 mm for a positive interpretation. Depending on the locations of the disks, it is also occasionally possible to get a keyhole result (double-disk test effect, as shown) to also indicate ESBL production. ceftazidime-basedtests are suggestive of ESBL production, and negative ceftazidime-based tests are suggestive of hyperproduction of the K1 enzyme. The value of this generalization is diminished in populations where CTX-M ESBLs are present in K. oxytoca isolates. As indicated above, CTX-M ESBLs may yield negative ceftazidime-based ESBL detection tests. If CTX-Ms are present, reduced susceptibility to cefepime may be a useful indicator for differentiating between K1 and CTX-M ESBLs in this organism. In 2005, P. mirabilis wasaddedto the list of organismsto screen foresbls (28).However, CLSI does not recommend routine screening of P. mirabilis for ESBL production, unless it is deemed clinically relevant, such as a bacteremic isolate. P. mirabilis is the second most common causative organism of urinary tract infections, so it is not clear why the CLSI has this recommendation only for serious infections. It would make sense to screen all P. mirabilis isolates, as they may harbor genes with ESBLs, which may later be transferred to other organisms and/or cause serious infections themselves. CTX/ CLA CTX ESBL Detection in Other Organisms ESBL-producing organisms other than E. coli, Klebsiella, or P. mirabilis also cause infections and may be unsuspected reservoirs of transferable multiple antibiotic resistance. Detection difficulties arise because there are no CLSI screening or confirmatory tests. An experienced microbiologist might be suspicious that a susceptibility pattern is suggestive of ESBL production in these organisms and should try one of the confirmatory tests mentioned previously. Even though it can be difficult to detect ESBLs, it is in the best interests of the patient and the hospital for the laboratory to attempt detection. Detection problems arise especially with organisms that produce an inducible AmpC β-lactamase, as clavulanate can induce high-level production of AmpC, which may obscure recognition ofanesbl(seeexampleintable3). One approach to circumvent this problem is to use cefepime, which is not significantly hydrolyzed by AmpC, as a substrate in clavulanate inhibitionbased ESBLtests(Fig. 3). Anotherapproachisto incorporate 74 0196-4399/00 (see frontmatter) 2008 Elsevier Clinical Microbiology Newsletter 30:10,2008

an inhibitor of AmpC β-lactamases, suchascloxacillin, intheagar(29a) and to utilize the CLSI-recommended disks (ceftazidime and cefotaxime, withandwithoutclavulanate)(fig.4). Thereis no CLSI recommendation to automatically report resistance to all penicillins, cephalosporins, and aztreonam if an ESBL is detected in organisms other than E. coli, Klebsiella, or P. mirabilis. According to the CLSI, susceptibility results should be reported without modification. This is inconsistent with the CLSI recommendation for ESBL-producing E. coli, Klebsiella, and P. mirabilis and implies that ESBLs are not clinically significant in organismsotherthan these.table4shows β-lactam MICs for an SHV-3-producing Citrobacter freundii isolate. According to current CLSI recommendations, a laboratory would report this isolate as susceptible to cefotaxime, ceftriaxone, and cefepime. We advocate extension of the CLSI reporting recommendations to cover all ESBL-producing organisms, i.e., report all ESBL-producing organisms as resistanttoallpenicillins,cephalosporins,and aztreonam(1).this is especially important with organisms that produce an inducible AmpC β- lactamase(table5),becausecefepime may be considered a therapeutic choice. AmpC Beta-lactamases Molecular classes of β-lactamases are based on the amino acid sequence and can be subdivided based on functional characteristics. Molecular class C, or AmpC, β-lactamases primarily hydrolyze the cephems (cephalosporins and cephamycins) but also hydrolyze penicillins and aztreonam. These enzymes are resistant to the currently available β-lactamase inhibitors clavulanate, sulbactam, and tazobactam. With rare exceptions, the hydrolysis of cephamycins, such as cefotetan and cefoxitin, is a property that can be helpfultodistinguishampcs fromesbls (16).The exceptions are the chromosomal AmpC of Hafnia alvei and imported Amp-C Class (ACC) enzymes which evolved from it. Genes encoding inducible chromosomal AmpC β-lactamases are part of the genomes of many gram-negative bacteria(table5).normally,ampcisproduced at a low level and does not cause resistance, but higher-level production of Figure 3. Example of an Etest investigational strip which has cefepime (PM) without and (PML) with clavulanate. The test is both quantitative and qualitative. This test can be quantitative, as the PM MIC is 2 μg/ml and the PML MIC is <0.064 μg/ml, which is >3 doubling dilutions. The zone between the cefepime and cefepime/clavulanate ellipses is referred to as a phantom zone. This provides a qualitative interpretation, in that the manufacturer recommends that isolates yielding a phantom zone be reported as ESBL positive. Table 3. Example of induction of AmpC interfering with CLSI ESBL confirmatory test Isolate Test agent MIC (μg/ml) SHV-2-producing Enterobacter cloacae Ceftazidime alone 4 Ceftazidime + 4 μg/ml clavulanate 16 Table 4. MICs in µg/ml: SHV-3-producing Citrobacter freundii Cefotaxime Ceftazidime Ceftriaxone Cefepime 2 μg/ml 32 μg/ml 4 μg/ml 0.5 μg/ml AmpC may cause resistance to the first-, second-, and third-generation cephalosporins, cephamycins, penicillins, and the β-lactamase inhibitor combinations. Higher-level AmpC production may occur as a consequence of mutation (derepression) or when the organism is exposed to an inducing agent. Cephamycins (e.g., cefoxitin and cefotetan), Cefepime Cefepime/clav ampicillin, and carbapenems are good inducers(table6).inductionistemporary and is reversed by removal of the inducing agent from the vicinity of the organism. Induction of AmpC is not of great clinical consequence, providing patients are not treated with two β-lactam antibiotics, one of which is an inducer and Clinical Microbiology Newsletter 30:10,2008 2008 Elsevier 0196-4399/00 (see frontmatter) 75

the other a substrate of AmpC. However, the selection of derepressed mutants can be a major concern. Mutants typically occur spontaneously at frequencies of about 1 in every 10 6 to 10 9 cells, are often present as subpopulations in infections, and may emerge to cause therapeutic failures, especially with secondand third-generation cephalosporins and aztreonam. There have been reports of resistance to these agents developing during treatment at rates from less than 20% to over 70%, depending on the site of infection(29b).becauseofthis,some laboratories automatically report resistance to the first-, second-, or thirdgeneration cephalosporins and aztreonam fortheorganismslistedintable5. Some β-lactam antibiotics that are poor inducers of AmpC tend to be good selectorsofmutants(table7). Susceptibility tests vary in reliability for detecting resistance in these organisms, but identification of the organisms listed intable5indicatesthe presenceof this resistance mechanism. Editor s Note: Part II of this article will be published in the June 1, 2008 issue of CMN (Vol. 30, No. 11). References 1. Livermore, D.M. et al. 2002. Multicentre evaluation of the VITEK 2 Advanced Expert System for interpretive reading of antimicrobial resistance tests. J. Antimicrob. Chemother. 49:289-300. 2. Tenover, F.C. et al. 1999. Detection and reporting of organisms producing extended-spectrum ß-lactamases: survey of laboratories in Connecticut. J. Clin. Microbiol. 37:4065-4070. 3. Paterson, D.L. and R.A. Bonomo. 2005. Extended-spectrum beta-lactamases: a clinical update. Clin. Microbiol. Rev. 18:657-686. 4. Pangon, B. et al. 1989. In vivo selection of a cephamycin resistant porin mutant of a CTX-1 ß-lactamase producing strain of Klebsiella pneumoniae. J. Infect. Dis. 159:1005-1006. 5. Martinez-Martinez, L. et al. 1996. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expandedspectrum-cephalosporins. Antimicrob. Agents Chemother. 40:342-348. 6. Ardanuy, C. et al. Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrob. Agents Chemother. 1998. 42:1636-1640. Mueller-Hinton agar Table 5. Common organisms that produce inducible AmpC beta-lactamases Enterobacter spp. Citrobacter freundii Hafnia alvei Serratia marcescens Morganella morganii Providencia spp. Pseudomonas aeruginosa Aeromonas spp. Table 6. Inducer potentials of beta-lactam agents Good Variable Poor Cefoxitin Clavulanate Sulbactam Cefmetazole Desacetyl cefotaxime Tazobactam Imipenem Cefamandole Aztreonam Meropenem Cephalothin Third-generation cephalosporins Ampicillin Cefonicid Fourth-generation cephalosporins Table 7. Mutant selection Good selectors Second- and third-generation cephalosporins Aztreonam Mueller-Hinton agar with cloxacillin 200 µg/ml Figure 4. CLSI ESBL confirmatory disk test results for an E. coli isolate producing both an ESBL (CTX-M-14) and an AmpC β-lactamase (CMY-2). (Left) CLSI ESBL disks on Mueller- Hinton agar. (Right) Same disks on Mueller-Hinton agar supplemented with cloxacillin (200 μg/ml). The ESBL confirmation is only visible with cloxacillin-supplemented agar which has inhibited the AmpC produced by this isolate. Poor selectors Carbapenems Cephamycins First- and fourth-generation cephalosporins Penicillins 7. Hernandez-Alles, S. et al. 1999. Development of resistance during antimicrobial therapy caused by insertion sequence interruption of porin genes. Antimicrob. Agents Chemother. 43:937-939. 8. Crowley, B., V.J. Benedi, and A. Domenech-Sanchez. 2002. Expression of SHV-2 beta-lactamase and of reduced amounts of OmpK36 porin in Klebsiella pneumoniae results in increased resistance to cephalosporins and carbapenems. Antimicrob. Agents Chemother. 46:3679-3682. 9. Nelson, E.C., H. Segal, and B.G. Elisha. 2003. Outer membrane protein alterations and blatem-1 variants: their role in {beta}-lactam resistance in Klebsiella pneumoniae. J. Antimicrob. Chemother. 52:899-903. 10. Skopkova-Zarnayova, M. et al. 2005. Outer membrane protein profiles of clonally related Klebsiella pneumoniae 76 0196-4399/00 (see frontmatter) 2008 Elsevier Clinical Microbiology Newsletter 30:10,2008

isolates that differ in cefoxitin resistance. FEMS Microbiol. Lett. 243:197-203. 11. Lee, C.H. et al. 2006. In vivo selection of OmpK35-deficient mutant after cefuroxime therapy for primary liver abscess caused by Klebsiella pneumoniae. J. Antimicrob. Chemother. 58:857-860. 12. Burgess, D.S. et al. 2003. Clinical and microbiologic analysis of a hospital s extended-spectrum beta-lactamaseproducing isolates over a 2-year period. Pharmacotherapy 23:1232-1237. 13. Paterson, D.L. et al. 1999. Fatal infection due to extended-spectrum betalactamase-producing Escherichia coli: implications for antibiotic choice for spontaneous bacterial peritonitis. Clin. Infect. Dis. 28:683-684. 14. Pillay, T. et al. 1998. Piperacillin/tazobactam in the treatment of Klebsiella pneumoniae infections in neonates. Am. J. Perinatol. 15:47-51. 15. Karadenizli, A. et al. 2001. Piperacillin with and without tazobactam against extended-spectrum beta-lactamaseproducing Pseudomonas aeruginosa in a rat thigh abscess model. Chemotherapy 47:292-296. 16. Bush, K., G.A. Jacoby, and A.A. Medeiros. 1995. A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233. 17. Thomson, K.S. and E. Smith Moland. 2000. Version 2000: the new beta-lactamases of Gram-negative bacteria at the dawn of the new millennium. Microbes Infect. 2:1225-1235. 18. al Naiemi, N., B. Duim, and A. Bart. 2006. A CTX-M extended-spectrum beta-lactamase in Pseudomonas aeruginosa and Stenotrophomonas maltophilia. J. Med. Microbiol. 55:1607-1608. 19. Lewis, J.S., II et al. 2007. First report of the emergence of CTX-M-type extendedspectrum beta-lactamases (ESBLs) as the predominant ESBL isolated in a U.S. health care system. Antimicrob. Agents Chemother. 51:4015-4021. 20. Moubareck, C. et al. 2005. Countrywide spread of community- and hospitalacquired extended-spectrum betalactamase (CTX-M-15)-producing Enterobacteriaceae in Lebanon. J. Clin. Microbiol. 43:3309-3313. 21. Moland, E.S. et al. 2003. Discovery of CTX-M-like extended-spectrum betalactamases in Escherichia coli isolates from five U.S. States. Antimicrob. Agents Chemother. 47:2382-2383. 22. Power, P. et al. 2007. Biochemical characterization of PER-2 and genetic environment of blaper-2. Antimicrob. Agents Chemother. 51:2359-2365. 23. Brun-Buisson, C. et al. 1987. Transferable enzymatic resistance to thirdgeneration cephalosporins during nosocomial outbreak of multiresistant Klebsiella pneumoniae. Lancet ii:302-306. 24. Jarlier, V. et al. 1988. Extended broadspectrum ß-lactamases conferring transferable resistance to newer ß-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10:867-878. 25. Espedido, B.A., L.C. Thomas, and J.R. Iredell. 2007. Metallo-beta-lactamase or extended-spectrum beta-lactamase: a wolf in sheep s clothing. J. Clin. Microbiol. 45:2034-2036. 26. Thomson, K.S. et al. 2007. Comparison of Phoenix and VITEK 2 extendedspectrum-beta-lactamase detection tests for analysis of Escherichia coli and Klebsiella isolates with well-characterized beta-lactamases. J. Clin. Microbiol. 45:2380-2384. 27. Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; 17th informational supplement M100- S17. Clinical and Laboratory Standards Institute, Wayne, PA. 28. Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing; 15th informational supplement M100- S15. Clinical and Laboratory Standards Institute, Wayne, PA. 29a. Poirel, L. et al. 2003. Outbreak of extended-spectrum beta-lactamase VEB-1-producing isolates of Acinetobacter baumannii in a French hospital. J. Clin. Microbiol. 41:3542-3547. 29b. Sanders Jr., W.E. and C.C. Sanders. 1997. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin. Microbiol. Rev. 10:220-241. Clinical Microbiology Newsletter 30:10,2008 2008 Elsevier 0196-4399/00 (see frontmatter) 77