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The AmpC inhibitor, Syn2190, can be used to reveal extended-spectrum β-lactamases in Escherichia coli
Diagnostic Microbiology and Infectious Disease 58 (2007) 345 – 348 www.elsevier.com/locate/diagmicrobio
Antimicrobial Susceptibility Studies
The AmpC inhibitor, Syn2190, can be used to reveal extended-spectrum h-lactamases in Escherichia coli B Tisha C. Netzela, Irfan Jindanib, Nancy Hansonc, Bradley M. Turnera, L. Smithd, Kenneth H. Randa,4 a
Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, P O Box 100275, Gainesville, FL 32610, USA b Shands Hospital at the University of Florida, Gainesville, FL 32610, USA c Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA d Pharmacy Department, Shands at the University of Florida Hospital, Gainesville, FL 32610, USA Received 11 August 2006; accepted 18 January 2007
Abstract AmpC h-lactamases are not inhibited by clavulanic acid and could potentially mask detection of extended-spectrum h-lactamases (ESBLs) using the Clinical and Laboratory Standards Institute confirmatory test. Syn2190 (1,5-dihydroxy-4-pyridone monobactam) selectively inhibits AmpC, but not ESBLs. Fifty-four MicroScan ESBL screen-positive strains of Escherichia coli and an unrelated group of 20 cefoxitinnonsusceptible E. coli strains were tested with the confirmatory ceftazidime–cefotaxime–clavulanate disk method with or without 4 Ag/mL of Syn2190 in the agar. Without Syn2190, 8 (14.8%) of 54 E. coli isolates and 0 of 20 cefoxitin-nonsusceptible E. coli isolates were confirmed. With Syn2190, an additional 9 (16.6%) of 54 of the MicroScan screen-positive E. coli isolates and 6 (30%) of 20 of the cefoxitinnonsusceptible E. coli isolates were found. Multiplex polymerase chain reaction and sequence analysis confirmed the presence of the plasmidassociated h-lactamase gene bla CMY-2 in the 2 available MicroScan-screened E. coli isolates and in 5 of 6 of the cefoxitin-resistant group. These data suggest that in the presence of AmpC, ESBLs in E. coli may not be detected by the currently recommended confirmatory test. Published by Elsevier Inc. Keywords: Extended-spectrum h-lactamase; ESBL; AmpC; Syn2190
1. Introduction The detection of organisms that produce extendedspectrum h-lactamases (ESBLs) is an important challenge for clinical microbiology laboratories. Failure to detect an ESBL may result in treatment failure if the appropriate antimicrobial drugs are not given (Pai et al., 2004; Paterson et al., 2001; Paterson et al., 2004). Therefore, the Clinical and Laboratory Standards Institute (CLSI) recommends confirmation using both ceftazidime and cefotaxime in the presence and absence of clavulanic acid. In this test, increase in the zone of inhibition by z 5 mm in the presence of clavulanic acid is considered a positive test for the presence of an ESBL (CLSI, 2005). Many automated microbiology analyzers, B
This study was supported in part by the Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, and Shands at the University of Florida Hospital, Gainesville, FL. 4 Corresponding author. Tel.: +1-352-392-5621; fax: +1-352-392-4693. E-mail address: [email protected] (K.H. Rand). 0732-8893/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2007.01.017
including the Dade MicroScanR, include screening tests for ESBL-producing isolates in their Gram-negative identification and sensitivity panels. However, the CLSI confirmatory method is still required. Confounding the detection of ESBLproducing organisms is the coproduction of an AmpC hlactamase. This enzyme is resistant to clavulanic acid, and the presence of an AmpC h-lactamase in Gram-negative pathogens could potentially cause false-negative reporting of ESBL-producing organisms. Syn2190 (1,5-dihydroxy-4pyridone monobactam) is an AmpC h-lactamase inhibitor that does not inhibit ESBL activity (Nishida et al., 1999; Babini and Livermore, 2000). Syn2190 inhibited both class A and D h-lactamases in Pseudomonas aeruginosa and many Enterobacteriaceae, with the exception of Klebsiella sp. Its activity was significantly increased when tested in the presence of the iron chelator conalbumin, although the precise mechanism of action of Syn2190 has not been identified. The addition of this compound to media when performing the CLSI confirmatory test for ESBLs could
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theoretically permit detection of an ESBL in the presence of an AmpC h-lactamase. In this study, we tested 193 isolates of E. coli and 80 isolates of Klebsiella pneumoniae for the presence of ESBLs using the CLSI confirmatory disk diffusion test in the presence and absence of Syn2190. 2. Materials and methods 2.1. Isolates Escherichia coli and K. pneumoniae isolates were collected from specimens processed in the clinical microbiology laboratory at Shands Hospital at the University of Florida (UF), Gainesville, FL, between June 2002 and July 2005. These isolates were identified by the Dade MicroScanR automated microbiology analyzer. A possible ESBLpositive E. coli or K. pneumoniae isolate is flagged by the MicroScan Gram-negative breakpoint combo 30 panels with one or more of the following: cefpodoxime z 4 Ag/mL, aztreonam z 16 Ag/mL, cefotaxime z 32 Ag/mL, ceftazidime z 8 Ag/mL, or ceftriaxone z 32 Ag/mL. The criteria are the same for the Gram-negative breakpoint Combo 11 panels, with the exception that the cefpodoxime MIC must be N1 Ag/mL to be considered a positive screen for ESBLs. One hundred seventy-three isolates of E. coli and 80 isolates of K. pneumoniae were tested with either the MicroScan Gram-negative breakpoint combo 30 panel or Gram-negative breakpoint combo 11 panel and subsequently tested for the presence of ESBLs by the CLSI confirmatory method (UF group). In addition, 20 strains of cefoxitin-
nonsusceptible E. coli were provided by Creighton University Medical Center (CUMC group), Omaha, NE. 2.2. Syn2190 Syn2190 was generously provided by Naeja Pharmaceuticals, Edmonton, Alberta, Canada. As of January 2007, Naeja Pharmaceuticals has sold this compound to an undisclosed company and no longer supplies it. 2.3. CLSI confirmatory test in the presence and absence of Syn2190 The diameters of the zones of inhibition for ceftazidime and cefotaxime without clavulanic acid were compared with those for ceftazidime and cefotaxime with clavulanic acid. A positive result was obtained when the zone with clavulanic acid was at least 5 mm larger than the corresponding zone without clavulanic acid for either ceftazidime or cefotaxime (CLSI, 2005). Mueller–Hinton agar was prepared in 90-mm Petri dishes (30 mL each) according to the instructions of the manufacturer (Difco, Sparks, MD). Syn2190 was added to the agar to give a final concentration of 4 Ag/mL. The coproduction of both an ESBL and AmpC h-lactamase was predicted when the zone with clavulanic acid was at least 5 mm larger than the corresponding zone without clavulanic acid for either ceftazidime or cefotaxime in the presence of Syn2190, but not in the absence of Syn2190 (Fig. 1). 2.4. Molecular analysis Multiplex ampC polymerase chain reaction (PCR) was performed on DNA template obtained from the 2 available
Fig. 1. A positive test for ESBL detection in the presence of AmpC interference. An E. coli isolate known to produce the CMY-2 AmpC h-lactamase was tested for the presence of an ESBL in the presence and absence of the AmpC inhibitor Syn2190. Without Syn2190, the plate on the left shows zones of inhibition for ceftazidime and ceftazidime + clavulanic acid (top left and top right) of 7 and 10 mm, respectively, whereas the zones of inhibition for cefotaxime and cefotaxime + clavulanic acid (bottom left and bottom right) are 11 and 12 mm, respectively. The plate on the right contains 4 Ag/mL of Syn2190. The zones of inhibition for ceftazidime and ceftazidime + clavulanic acid (top left and top right) are 16 and 24 mm, respectively, whereas those of cefotaxime and cefotaxime + clavulanic acid (bottom left and bottom right) are 17 and 24 mm. The appearance of a 5-mm zone size increase in the presence of Syn2190, without a corresponding increase in its absence, indicates the presence of the AmpC h-lactamase, CMY-2, which would otherwise inhibit detection of the ESBL.
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UF E. coli isolates that had positive CLSI confirmatory tests only in the presence of Syn2190 and on the entire CUMC group (Perez-Perez and Hanson 2002). Sequencing was carried out by automated-cycle sequencing using an ABI Prism 3100-Avant Genetic analyzer. Sequence analysis of ampC was performed by computer-generated amino acid analysis using the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST/). 3. Results One hundred seventy-three E. coli isolates were tested using the Dade MicroScanR panels NBP 11 or NBP 30. Fifty-four of these isolates tested positive for a possible ESBL. Eight of these isolates were shown to produce an ESBL using the CLSI confirmatory method in the absence of Syn2190; 9 additional isolates met CLSI criteria for ESBL production when tested in the presence of Syn2190 (Fig. 1). Multiplex ampC PCR confirmed the presence of an imported ampC of Citrobacter origin in the 2 available isolates from this group. From one strain, the ampC gene was sequenced and found to be bla CMY-2. The other isolates were not available for molecular characterization. None of the 119 screen-negative E. coli isolates tested were shown to produce ESBLs using the CLSI confirmatory disk test with or without the addition of Syn2190. Twenty cefoxitin-nonsusceptible E. coli isolates from CUMC were also screened using the CLSI confirmatory test with and without 4 Ag/mL of Syn2190. All tested negative for the presence of an ESBL without Syn2190 in the agar, but 6 isolates tested positive by the CLSI confirmatory method in the presence of 4 Ag/mL Syn2190. Fourteen of these isolates were confirmed to carry an imported ampC gene of Citrobacter origin by ampC multiplex PCR. In this group, 5 of the 6 that tested positive for an ESBL only in the presence of Syn2190 were positive for a bla CMY-2–like gene by multiplex PCR. Among the CUMC isolates, we observed a marked increase in the mean F SD for ceftazidime and cefotaxime zone sizes without Syn2190 in the agar from 14.3 F 6.1 and 16 F 6.1 mm, respectively, to 27.7 F 4.0 and 29.3 F 4.4 mm with 4 Ag/mL Syn2190 in the agar. Only one strain showed no such increase in zone size. In contrast, only 14% of the MicroScan screen-positive isolates from the UF group showed a comparable increase in zone size, although 40% of the isolates did exhibit a z 5- to V 10-mm increase. Nine of 80 isolates of K. pneumoniae tested positive in the Dade MicroScanR for a possible ESBL; 2 tested positive for ESBLs without Syn2190, but no additional isolates were found to produce ESBLs in the presence of Syn2190. 4. Discussion Eight of 173 strains of E. coli collected from Shands Hospital at the UF and no strains tested from CUMC were positive in this study for ESBLs using the
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CLSI-recommended confirmatory method. However, when Syn2190 was added to the test agar, an additional of 9 strains collected in Shands Hospital at the UF and 6 strains collected from CUMC met the CLSI criteria, indicating the presence of an ESBL. These data indicate that a significant proportion of the UF and CUMC isolates (617% and 30%, respectively) were false negative for ESBL production by the CLSI-recommended methodology. These findings suggest that ESBL production was masked by the presence of an AmpC h-lactamase. MIC results were recorded for 4 of the 9 UF strains. Three of the 4 were reported susceptible (V 8 Ag/mL) to ceftazidime or cefotaxime or both using the MicroScan panel. Several disk-based methods have been described to detect ESBLs in the presence of AmpC h-lactamases. Thomson and Sanders (1992) originally described the 3dimensional test that detected ESBLs by distortion of individual antibiotic disk zones using a direct inoculum of the test organism. Subsequent studies by this group have used disks impregnated with EDTA to release bound hlactamases from an inoculum of the test organism also applied to the disk (Black et al., 2005). Coudron (2005) has reported using boronic acid to inhibit AmpC permitting detection of ESBLs using CLSI-recommended disks with and without clavulanic acid. Others have used agar containing cloxacillin to inhibit AmpC, but this was only useful for isolates lacking chromosomal AmpC (Poirel et al., 2003). With the exception of the boronic acid method, these procedures are time consuming and require subjective interpretation of zone distortion. Both the boronic acid and Syn2190 methods make use of the more objective measurement of a 5-mm increase in zone diameter to identify the presence of an ESBL. A recent study by Tenover et al. (2003) identified 41 (31.3%) of 131 E. coli isolates that were screen positive for ESBLs but did not demonstrate a clavulanic acid (CA) effect. These isolates produced either a TEM extendedspectrum cephalosporinase (n = 36) or an OXA extendedspectrum cephalosporinase (n = 5) in addition to enhanced production of AmpC chromosomal h-lactamase. The relatively high proportion of isolates containing ESBLs despite a negative CA effect may have been due to selection of these strains as part of project ICARE, rather than from isolates from routine clinical care. Although the ESBL screening methods such as those in the MicroScanR negative breakpoint combo panels may overestimate the number of ESBL-containing strains, the current confirmatory CLSI method appears to underestimate ESBL-producing E. coli in the presence of AmpC production. These data suggest that a reservoir of ESBL-producing strains could go undetected, resulting in potential nosocomial dissemination of these resistant organisms. ESBL false-negative strains may be reported as susceptible to 3rd-generation cephalosporins and semisynthetic penicillins. These types of errors have the potential to result in a poor therapeutic outcome or even treatment failure for the
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patient if a cephalosporin is used for therapy (Pai et al., 2004; Paterson et al., 2001; Paterson et al., 2004). The data from this report indicate that further laboratory studies are needed to optimize and simplify detection of AmpC and ESBLcoproducing strains. Once such methods are developed, the clinical significance of these strains can be determined. Acknowledgment The authors gratefully acknowledge the generous support of the staff of the Clinical Microbiology laboratory of the Shands at the University of Florida Hospital, Gainesville, FL. They would also like to acknowledge Drs. Ashfaque Hossain and Mark Reisbig for molecular analysis. Appendix We have identified another 8 (6.2%) of 129 strains of E. coli that were ESBL screen positive by MicroScan, CLSI confirmatory ESBL test negative, but were CLSI ESBL confirmatory test positive in the presence of Syn2190. These 8 strains were tested for the presence of AmpC using the EDTA disk test (Black et al., 2005), and 6 of 8 were positive. When cloxacillin 200 Ag/mL was incorporated into Mueller–Hinton agar (Poirel et al., 2003), zone diameters of cefotaxime and ceftazidime increased by 14.8 F 5.2 mm ( P b 0.001, paired differences t test; range, 6.5–20 mm) and 16.1 F 6.5 mm ( P b .001, paired differences t test; range, 3–23 mm), respectively, for the 8 strains, consistent with inhibition of AmpC. References Babini GS, Livermore DM (2000) Effect of conalbumin on the activity of Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC beta-lactamases. J Antimicrob Chemother 45:105 – 109.
Black JA, Moland ES, Thomson KS (2005) AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 43:3110 – 3113. Clinical and Laboratory Standards Institute (CLSI) (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement. 25[1]. Coudron PE (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. Nishida K, Kunugita C, Uji T, Higashitani F, Hyodo A, Unemi N, Maiti SN, Phillips OA, Spevak P, Atchison KP, Salama SM, Atwal H, Micetich RG (1999) In vitro and in vivo activities of Syn2190, a novel betalactamase inhibitor. Antimicrob Agents Chemother 43:1895 – 1900. Pai H, Kang CI, Byeon JH, Lee KD, Park WB, Kim HB, Kim EC, Oh MD, Choe KW (2004) Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamase–producing Klebsiella pneumoniae. Antimicrob Agents Chemother 48:3720 – 3728. Paterson DL, Ko WC, Von GA, Casellas JM, Mulazimoglu L, Klugman KP, Bonomo RA, Rice LB, McCormack JG, Yu VL (2001) Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory. J Clin Microbiol 39:2206 – 2212. Paterson DL, Ko WC, Von GA, Mohapatra S, Casellas JM, Goossens H, Mulazimoglu L, Trenholme G, Klugman KP, Bonomo RA, Rice LB, Wagener MM, McCormack JG, Yu VL (2004) Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. Clin Infect Dis 39:31 – 37. Perez-Perez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153 – 2162. Poirel L, Menuteau O, Agoli N, Cattoen C, Nordmann P (2003) Outbreak of extended-spectrum beta-lactamase VEB-1–producing isolates of Acinetobacter baumannii in a French hospital. J Clin Microbiol 41:3542 – 3547. Tenover FC, Raney PM, Williams PP, Rasheed JK, Biddle JW, Oliver A, Fridkin SK, Jevitt L, McGowan Jr JE (2003) Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE. J Clin Microbiol 41:3142 – 3146. Thomson KS, Sanders CC (1992) Detection of extended-spectrum betalactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. Antimicrob Agents Chemother, 36:1877 – 1882.
Antimicrobial Susceptibility Studies
The AmpC inhibitor, Syn2190, can be used to reveal extended-spectrum h-lactamases in Escherichia coli B Tisha C. Netzela, Irfan Jindanib, Nancy Hansonc, Bradley M. Turnera, L. Smithd, Kenneth H. Randa,4 a
Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, P O Box 100275, Gainesville, FL 32610, USA b Shands Hospital at the University of Florida, Gainesville, FL 32610, USA c Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178, USA d Pharmacy Department, Shands at the University of Florida Hospital, Gainesville, FL 32610, USA Received 11 August 2006; accepted 18 January 2007
Abstract AmpC h-lactamases are not inhibited by clavulanic acid and could potentially mask detection of extended-spectrum h-lactamases (ESBLs) using the Clinical and Laboratory Standards Institute confirmatory test. Syn2190 (1,5-dihydroxy-4-pyridone monobactam) selectively inhibits AmpC, but not ESBLs. Fifty-four MicroScan ESBL screen-positive strains of Escherichia coli and an unrelated group of 20 cefoxitinnonsusceptible E. coli strains were tested with the confirmatory ceftazidime–cefotaxime–clavulanate disk method with or without 4 Ag/mL of Syn2190 in the agar. Without Syn2190, 8 (14.8%) of 54 E. coli isolates and 0 of 20 cefoxitin-nonsusceptible E. coli isolates were confirmed. With Syn2190, an additional 9 (16.6%) of 54 of the MicroScan screen-positive E. coli isolates and 6 (30%) of 20 of the cefoxitinnonsusceptible E. coli isolates were found. Multiplex polymerase chain reaction and sequence analysis confirmed the presence of the plasmidassociated h-lactamase gene bla CMY-2 in the 2 available MicroScan-screened E. coli isolates and in 5 of 6 of the cefoxitin-resistant group. These data suggest that in the presence of AmpC, ESBLs in E. coli may not be detected by the currently recommended confirmatory test. Published by Elsevier Inc. Keywords: Extended-spectrum h-lactamase; ESBL; AmpC; Syn2190
1. Introduction The detection of organisms that produce extendedspectrum h-lactamases (ESBLs) is an important challenge for clinical microbiology laboratories. Failure to detect an ESBL may result in treatment failure if the appropriate antimicrobial drugs are not given (Pai et al., 2004; Paterson et al., 2001; Paterson et al., 2004). Therefore, the Clinical and Laboratory Standards Institute (CLSI) recommends confirmation using both ceftazidime and cefotaxime in the presence and absence of clavulanic acid. In this test, increase in the zone of inhibition by z 5 mm in the presence of clavulanic acid is considered a positive test for the presence of an ESBL (CLSI, 2005). Many automated microbiology analyzers, B
This study was supported in part by the Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, and Shands at the University of Florida Hospital, Gainesville, FL. 4 Corresponding author. Tel.: +1-352-392-5621; fax: +1-352-392-4693. E-mail address: [email protected] (K.H. Rand). 0732-8893/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2007.01.017
including the Dade MicroScanR, include screening tests for ESBL-producing isolates in their Gram-negative identification and sensitivity panels. However, the CLSI confirmatory method is still required. Confounding the detection of ESBLproducing organisms is the coproduction of an AmpC hlactamase. This enzyme is resistant to clavulanic acid, and the presence of an AmpC h-lactamase in Gram-negative pathogens could potentially cause false-negative reporting of ESBL-producing organisms. Syn2190 (1,5-dihydroxy-4pyridone monobactam) is an AmpC h-lactamase inhibitor that does not inhibit ESBL activity (Nishida et al., 1999; Babini and Livermore, 2000). Syn2190 inhibited both class A and D h-lactamases in Pseudomonas aeruginosa and many Enterobacteriaceae, with the exception of Klebsiella sp. Its activity was significantly increased when tested in the presence of the iron chelator conalbumin, although the precise mechanism of action of Syn2190 has not been identified. The addition of this compound to media when performing the CLSI confirmatory test for ESBLs could
346
T.C. Netzel et al. / Diagnostic Microbiology and Infectious Disease 58 (2007) 345 – 348
theoretically permit detection of an ESBL in the presence of an AmpC h-lactamase. In this study, we tested 193 isolates of E. coli and 80 isolates of Klebsiella pneumoniae for the presence of ESBLs using the CLSI confirmatory disk diffusion test in the presence and absence of Syn2190. 2. Materials and methods 2.1. Isolates Escherichia coli and K. pneumoniae isolates were collected from specimens processed in the clinical microbiology laboratory at Shands Hospital at the University of Florida (UF), Gainesville, FL, between June 2002 and July 2005. These isolates were identified by the Dade MicroScanR automated microbiology analyzer. A possible ESBLpositive E. coli or K. pneumoniae isolate is flagged by the MicroScan Gram-negative breakpoint combo 30 panels with one or more of the following: cefpodoxime z 4 Ag/mL, aztreonam z 16 Ag/mL, cefotaxime z 32 Ag/mL, ceftazidime z 8 Ag/mL, or ceftriaxone z 32 Ag/mL. The criteria are the same for the Gram-negative breakpoint Combo 11 panels, with the exception that the cefpodoxime MIC must be N1 Ag/mL to be considered a positive screen for ESBLs. One hundred seventy-three isolates of E. coli and 80 isolates of K. pneumoniae were tested with either the MicroScan Gram-negative breakpoint combo 30 panel or Gram-negative breakpoint combo 11 panel and subsequently tested for the presence of ESBLs by the CLSI confirmatory method (UF group). In addition, 20 strains of cefoxitin-
nonsusceptible E. coli were provided by Creighton University Medical Center (CUMC group), Omaha, NE. 2.2. Syn2190 Syn2190 was generously provided by Naeja Pharmaceuticals, Edmonton, Alberta, Canada. As of January 2007, Naeja Pharmaceuticals has sold this compound to an undisclosed company and no longer supplies it. 2.3. CLSI confirmatory test in the presence and absence of Syn2190 The diameters of the zones of inhibition for ceftazidime and cefotaxime without clavulanic acid were compared with those for ceftazidime and cefotaxime with clavulanic acid. A positive result was obtained when the zone with clavulanic acid was at least 5 mm larger than the corresponding zone without clavulanic acid for either ceftazidime or cefotaxime (CLSI, 2005). Mueller–Hinton agar was prepared in 90-mm Petri dishes (30 mL each) according to the instructions of the manufacturer (Difco, Sparks, MD). Syn2190 was added to the agar to give a final concentration of 4 Ag/mL. The coproduction of both an ESBL and AmpC h-lactamase was predicted when the zone with clavulanic acid was at least 5 mm larger than the corresponding zone without clavulanic acid for either ceftazidime or cefotaxime in the presence of Syn2190, but not in the absence of Syn2190 (Fig. 1). 2.4. Molecular analysis Multiplex ampC polymerase chain reaction (PCR) was performed on DNA template obtained from the 2 available
Fig. 1. A positive test for ESBL detection in the presence of AmpC interference. An E. coli isolate known to produce the CMY-2 AmpC h-lactamase was tested for the presence of an ESBL in the presence and absence of the AmpC inhibitor Syn2190. Without Syn2190, the plate on the left shows zones of inhibition for ceftazidime and ceftazidime + clavulanic acid (top left and top right) of 7 and 10 mm, respectively, whereas the zones of inhibition for cefotaxime and cefotaxime + clavulanic acid (bottom left and bottom right) are 11 and 12 mm, respectively. The plate on the right contains 4 Ag/mL of Syn2190. The zones of inhibition for ceftazidime and ceftazidime + clavulanic acid (top left and top right) are 16 and 24 mm, respectively, whereas those of cefotaxime and cefotaxime + clavulanic acid (bottom left and bottom right) are 17 and 24 mm. The appearance of a 5-mm zone size increase in the presence of Syn2190, without a corresponding increase in its absence, indicates the presence of the AmpC h-lactamase, CMY-2, which would otherwise inhibit detection of the ESBL.
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UF E. coli isolates that had positive CLSI confirmatory tests only in the presence of Syn2190 and on the entire CUMC group (Perez-Perez and Hanson 2002). Sequencing was carried out by automated-cycle sequencing using an ABI Prism 3100-Avant Genetic analyzer. Sequence analysis of ampC was performed by computer-generated amino acid analysis using the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST/). 3. Results One hundred seventy-three E. coli isolates were tested using the Dade MicroScanR panels NBP 11 or NBP 30. Fifty-four of these isolates tested positive for a possible ESBL. Eight of these isolates were shown to produce an ESBL using the CLSI confirmatory method in the absence of Syn2190; 9 additional isolates met CLSI criteria for ESBL production when tested in the presence of Syn2190 (Fig. 1). Multiplex ampC PCR confirmed the presence of an imported ampC of Citrobacter origin in the 2 available isolates from this group. From one strain, the ampC gene was sequenced and found to be bla CMY-2. The other isolates were not available for molecular characterization. None of the 119 screen-negative E. coli isolates tested were shown to produce ESBLs using the CLSI confirmatory disk test with or without the addition of Syn2190. Twenty cefoxitin-nonsusceptible E. coli isolates from CUMC were also screened using the CLSI confirmatory test with and without 4 Ag/mL of Syn2190. All tested negative for the presence of an ESBL without Syn2190 in the agar, but 6 isolates tested positive by the CLSI confirmatory method in the presence of 4 Ag/mL Syn2190. Fourteen of these isolates were confirmed to carry an imported ampC gene of Citrobacter origin by ampC multiplex PCR. In this group, 5 of the 6 that tested positive for an ESBL only in the presence of Syn2190 were positive for a bla CMY-2–like gene by multiplex PCR. Among the CUMC isolates, we observed a marked increase in the mean F SD for ceftazidime and cefotaxime zone sizes without Syn2190 in the agar from 14.3 F 6.1 and 16 F 6.1 mm, respectively, to 27.7 F 4.0 and 29.3 F 4.4 mm with 4 Ag/mL Syn2190 in the agar. Only one strain showed no such increase in zone size. In contrast, only 14% of the MicroScan screen-positive isolates from the UF group showed a comparable increase in zone size, although 40% of the isolates did exhibit a z 5- to V 10-mm increase. Nine of 80 isolates of K. pneumoniae tested positive in the Dade MicroScanR for a possible ESBL; 2 tested positive for ESBLs without Syn2190, but no additional isolates were found to produce ESBLs in the presence of Syn2190. 4. Discussion Eight of 173 strains of E. coli collected from Shands Hospital at the UF and no strains tested from CUMC were positive in this study for ESBLs using the
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CLSI-recommended confirmatory method. However, when Syn2190 was added to the test agar, an additional of 9 strains collected in Shands Hospital at the UF and 6 strains collected from CUMC met the CLSI criteria, indicating the presence of an ESBL. These data indicate that a significant proportion of the UF and CUMC isolates (617% and 30%, respectively) were false negative for ESBL production by the CLSI-recommended methodology. These findings suggest that ESBL production was masked by the presence of an AmpC h-lactamase. MIC results were recorded for 4 of the 9 UF strains. Three of the 4 were reported susceptible (V 8 Ag/mL) to ceftazidime or cefotaxime or both using the MicroScan panel. Several disk-based methods have been described to detect ESBLs in the presence of AmpC h-lactamases. Thomson and Sanders (1992) originally described the 3dimensional test that detected ESBLs by distortion of individual antibiotic disk zones using a direct inoculum of the test organism. Subsequent studies by this group have used disks impregnated with EDTA to release bound hlactamases from an inoculum of the test organism also applied to the disk (Black et al., 2005). Coudron (2005) has reported using boronic acid to inhibit AmpC permitting detection of ESBLs using CLSI-recommended disks with and without clavulanic acid. Others have used agar containing cloxacillin to inhibit AmpC, but this was only useful for isolates lacking chromosomal AmpC (Poirel et al., 2003). With the exception of the boronic acid method, these procedures are time consuming and require subjective interpretation of zone distortion. Both the boronic acid and Syn2190 methods make use of the more objective measurement of a 5-mm increase in zone diameter to identify the presence of an ESBL. A recent study by Tenover et al. (2003) identified 41 (31.3%) of 131 E. coli isolates that were screen positive for ESBLs but did not demonstrate a clavulanic acid (CA) effect. These isolates produced either a TEM extendedspectrum cephalosporinase (n = 36) or an OXA extendedspectrum cephalosporinase (n = 5) in addition to enhanced production of AmpC chromosomal h-lactamase. The relatively high proportion of isolates containing ESBLs despite a negative CA effect may have been due to selection of these strains as part of project ICARE, rather than from isolates from routine clinical care. Although the ESBL screening methods such as those in the MicroScanR negative breakpoint combo panels may overestimate the number of ESBL-containing strains, the current confirmatory CLSI method appears to underestimate ESBL-producing E. coli in the presence of AmpC production. These data suggest that a reservoir of ESBL-producing strains could go undetected, resulting in potential nosocomial dissemination of these resistant organisms. ESBL false-negative strains may be reported as susceptible to 3rd-generation cephalosporins and semisynthetic penicillins. These types of errors have the potential to result in a poor therapeutic outcome or even treatment failure for the
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patient if a cephalosporin is used for therapy (Pai et al., 2004; Paterson et al., 2001; Paterson et al., 2004). The data from this report indicate that further laboratory studies are needed to optimize and simplify detection of AmpC and ESBLcoproducing strains. Once such methods are developed, the clinical significance of these strains can be determined. Acknowledgment The authors gratefully acknowledge the generous support of the staff of the Clinical Microbiology laboratory of the Shands at the University of Florida Hospital, Gainesville, FL. They would also like to acknowledge Drs. Ashfaque Hossain and Mark Reisbig for molecular analysis. Appendix We have identified another 8 (6.2%) of 129 strains of E. coli that were ESBL screen positive by MicroScan, CLSI confirmatory ESBL test negative, but were CLSI ESBL confirmatory test positive in the presence of Syn2190. These 8 strains were tested for the presence of AmpC using the EDTA disk test (Black et al., 2005), and 6 of 8 were positive. When cloxacillin 200 Ag/mL was incorporated into Mueller–Hinton agar (Poirel et al., 2003), zone diameters of cefotaxime and ceftazidime increased by 14.8 F 5.2 mm ( P b 0.001, paired differences t test; range, 6.5–20 mm) and 16.1 F 6.5 mm ( P b .001, paired differences t test; range, 3–23 mm), respectively, for the 8 strains, consistent with inhibition of AmpC. References Babini GS, Livermore DM (2000) Effect of conalbumin on the activity of Syn 2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC beta-lactamases. J Antimicrob Chemother 45:105 – 109.
Black JA, Moland ES, Thomson KS (2005) AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 43:3110 – 3113. Clinical and Laboratory Standards Institute (CLSI) (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement. 25[1]. Coudron PE (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. Nishida K, Kunugita C, Uji T, Higashitani F, Hyodo A, Unemi N, Maiti SN, Phillips OA, Spevak P, Atchison KP, Salama SM, Atwal H, Micetich RG (1999) In vitro and in vivo activities of Syn2190, a novel betalactamase inhibitor. Antimicrob Agents Chemother 43:1895 – 1900. Pai H, Kang CI, Byeon JH, Lee KD, Park WB, Kim HB, Kim EC, Oh MD, Choe KW (2004) Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamase–producing Klebsiella pneumoniae. Antimicrob Agents Chemother 48:3720 – 3728. Paterson DL, Ko WC, Von GA, Casellas JM, Mulazimoglu L, Klugman KP, Bonomo RA, Rice LB, McCormack JG, Yu VL (2001) Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory. J Clin Microbiol 39:2206 – 2212. Paterson DL, Ko WC, Von GA, Mohapatra S, Casellas JM, Goossens H, Mulazimoglu L, Trenholme G, Klugman KP, Bonomo RA, Rice LB, Wagener MM, McCormack JG, Yu VL (2004) Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum beta-lactamases. Clin Infect Dis 39:31 – 37. Perez-Perez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153 – 2162. Poirel L, Menuteau O, Agoli N, Cattoen C, Nordmann P (2003) Outbreak of extended-spectrum beta-lactamase VEB-1–producing isolates of Acinetobacter baumannii in a French hospital. J Clin Microbiol 41:3542 – 3547. Tenover FC, Raney PM, Williams PP, Rasheed JK, Biddle JW, Oliver A, Fridkin SK, Jevitt L, McGowan Jr JE (2003) Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE. J Clin Microbiol 41:3142 – 3146. Thomson KS, Sanders CC (1992) Detection of extended-spectrum betalactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests. Antimicrob Agents Chemother, 36:1877 – 1882.