H

H. ESBLs in clinical isolates of spp. and and allowed detection of an ESBL even when potentially masked by a pAmpC. Since extended-spectrum -lactamases (ESBLs) were initially reported to be in in the first half of the 1980s in Germany, ESBLs have been spreading globally (2). The CLSI interpretive guidelines stated that strains of spp. and that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these brokers (4). Therefore, detection of ESBLs in spp. and isolates is crucial for optimal treatment of patients and to control the spread of resistance. The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the zone diameters of cefotaxime (CTX; 30 g) or ceftazidime (CAZ; 30 g) disks with or without clavulanic acid (CA; 10 g) in spp. and and isolates have been found to produce both plasmid-mediated AmpC -lactamases (pAmpCs) and ESBLs (6, 8). In Korea, 8.7% of pAmpC suppliers also produced ESBLs (8). AmpCs resist inhibition by CA, and hence, the presence of an ESBL can be masked by the expression of an AmpC. Thus, the coexistence of both pAmpCs and ESBLs in the same strain may result in false-negative assessments for the detection of ESBLs (5). There is a need, therefore, for an alternative method that can detect ESBLs in spp. and isolates with a high sensitivity, even though the isolates simultaneously harbor pAmpCs. Boronic acids (BAs) were reported as reversible inhibitors of AmpCs (1). There are a few reports of studies designed to detect pAmpC-producing isolates of spp. and by a method using BA as an inhibitor that closely resembles the phenotypic confirmatory assessments for ESBLs (3, 5). And Coudron (5) recently described that a BA disk also enhances detection of isolates that harbor both ESBLs and pAmpCs. The aim of this work was to modify the phenotypic confirmatory test for ESBL production by using BA disks to improve its sensitivity for the detection of ESBLs in spp. and isolates even though the isolates simultaneously harbor pAmpCs. MATERIALS AND METHODS Bacterial strains. A total of 182 nonrepeat ESBL- and/or pAmpC-producing clinical isolates of spp. (= 118) and (= 64) collected from 12 tertiary-care hospitals in Korea during 2004 and 2005 were included in this study (Table ?(Table1)1) : 62 harbored ESBLs but not pAmpCs, 80 harbored both ESBLs and pAmpCs, and 40 harbored pAmpCs but not ESBLs. The isolates were identified with API-20 E systems (bioMrieux, Marcy l’Etoile, France). Searches for genes coding for the class A ESBLs and pAmpCs were performed by PCR amplification as described previously (7, 8). The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730spp.isolate that produces SHV-2a, which showed a 2-mm increase in the zone diameter of a CAZ/BA disk in the presence of CA (Fig. 1E and F). The average increases in the zone diameters of the CTX/BA and CAZ/BA disks in the presence of CA were 11.8 mm and 11.1 mm, respectively. Open in a separate window Open in a separate window FIG. 1. Comparison of the results for the CLSI confirmatory test (A and B) and two new tests (C to F). The.Bae, E. zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs ( pAmpCs) and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The latter new interpretive guideline has enhanced detection of ESBLs in clinical isolates of spp. and and allowed detection of an ESBL even when potentially masked by a pAmpC. Since extended-spectrum -lactamases (ESBLs) were initially reported to be in in the first half of the 1980s in Germany, ESBLs have been spreading globally (2). The CLSI interpretive guidelines stated that strains of spp. and that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents (4). Therefore, detection of ESBLs in spp. and isolates is crucial for optimal treatment of patients and to control the spread of resistance. The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the zone diameters of cefotaxime (CTX; 30 g) or ceftazidime (CAZ; 30 g) disks with or without clavulanic acid (CA; 10 g) in spp. and and isolates have been found to produce both plasmid-mediated AmpC -lactamases (pAmpCs) and ESBLs (6, 8). In Korea, 8.7% of pAmpC producers also produced ESBLs (8). AmpCs resist inhibition by CA, and hence, the presence of an ESBL can be masked by the expression of an AmpC. Thus, the coexistence of both pAmpCs and ESBLs in the same strain may result in false-negative tests for the detection of ESBLs (5). There is a need, therefore, for an alternative method that can detect ESBLs in spp. and isolates with a high sensitivity, even though the isolates simultaneously harbor pAmpCs. Boronic acids (BAs) were reported as reversible inhibitors of AmpCs (1). There are a few reports of studies designed to detect pAmpC-producing isolates of spp. and by a method using BA as an inhibitor that closely resembles the phenotypic confirmatory tests for ESBLs (3, 5). And Coudron (5) recently described that a BA disk also enhances detection of isolates that harbor both ESBLs and pAmpCs. The aim of this work was to modify the phenotypic confirmatory test for Phenolphthalein ESBL production by using BA disks to improve its sensitivity for the detection of ESBLs in spp. and isolates even though the isolates simultaneously harbor pAmpCs. MATERIALS AND METHODS Bacterial strains. A total of 182 nonrepeat ESBL- and/or pAmpC-producing clinical isolates of spp. (= 118) and (= 64) collected from 12 tertiary-care hospitals in Korea during 2004 and 2005 were included in this study (Table ?(Table1)1) : 62 harbored ESBLs but not pAmpCs, 80 harbored both ESBLs and pAmpCs, and 40 harbored pAmpCs but not ESBLs. The isolates were identified with API-20 E systems (bioMrieux, Marcy l’Etoile, France). Searches for genes coding for the class A ESBLs and pAmpCs were performed by PCR amplification as described previously (7, 8). The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730spp.isolate that produces SHV-2a, which showed a 2-mm increase in the zone diameter of a CAZ/BA disk in the presence of CA (Fig. 1E and F). The average increases in the zone diameters of the CTX/BA and CAZ/BA disks in the presence of CA were 11.8 mm and 11.1 mm, respectively. Open in a separate window Open in a separate window FIG. 1. Comparison of the results for the CLSI confirmatory test (A and B) and.Chemother. showed frequent false-positive results (50%) for isolates that produce only pAmpCs. Meanwhile, when a 3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs ( pAmpCs) and showed less frequent false-positive results (5%) in isolates that create only pAmpCs. The second option new interpretive guideline has enhanced detection of ESBLs in medical isolates of spp. and and allowed detection of an ESBL even when potentially masked by a pAmpC. Since extended-spectrum -lactamases (ESBLs) were in the beginning reported to be in in the 1st half of the 1980s in Germany, ESBLs have been spreading globally (2). The CLSI interpretive recommendations stated that strains of spp. and that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these providers (4). Therefore, detection of ESBLs in spp. and isolates is vital for ideal treatment of individuals and to control the spread of resistance. The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the zone diameters of cefotaxime (CTX; 30 g) or ceftazidime (CAZ; 30 g) disks with or without clavulanic acid (CA; 10 g) in spp. and and isolates have been found to produce both plasmid-mediated AmpC -lactamases (pAmpCs) and ESBLs (6, 8). In Korea, 8.7% of pAmpC makers also produced ESBLs (8). AmpCs resist inhibition by CA, and hence, the presence of an ESBL can be masked from the expression of an AmpC. Therefore, the coexistence of both pAmpCs and ESBLs in the same strain may result in false-negative checks for the detection of ESBLs (5). There is a need, therefore, for an alternative method that can detect ESBLs in spp. and isolates with a high sensitivity, even though the isolates simultaneously harbor pAmpCs. Boronic acids (BAs) were reported as reversible inhibitors of AmpCs (1). There are a few reports of studies designed to detect pAmpC-producing isolates of spp. and by a method using BA mainly because an inhibitor that closely resembles the phenotypic confirmatory checks for ESBLs (3, 5). And Coudron (5) recently described that a BA disk also enhances detection of isolates that harbor both ESBLs and pAmpCs. The aim of this work was to modify the phenotypic confirmatory test for ESBL production by using BA disks to improve its level of sensitivity for the detection of ESBLs in spp. and isolates even though the isolates simultaneously harbor pAmpCs. MATERIALS AND METHODS Bacterial strains. A total of 182 nonrepeat ESBL- and/or pAmpC-producing medical isolates of spp. (= 118) and (= 64) collected from 12 tertiary-care private hospitals in Korea during 2004 and 2005 were included in this study (Table ?(Table1)1) : 62 harbored ESBLs but not pAmpCs, 80 harbored both ESBLs and pAmpCs, and 40 harbored pAmpCs but not ESBLs. The isolates were recognized with API-20 E systems (bioMrieux, Marcy l’Etoile, France). Searches for genes coding for the class A ESBLs and pAmpCs were performed by PCR amplification as explained previously (7, 8). The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730spp.isolate that produces SHV-2a, which showed a 2-mm increase in the zone diameter of a CAZ/BA disk in the presence of CA (Fig. 1E and F). The average raises in the zone diameters of the CTX/BA and Phenolphthalein CAZ/BA disks in the presence of CA were 11.8 mm and 11.1 mm, respectively. Open in a separate window Open in a separate windowpane FIG. 1. Assessment of the results for the CLSI confirmatory test (A and B) and two fresh checks (C to F). The abbreviations (CTX/CA versus CTX, CAZ/CA versus CAZ, CTX/CA/BA versus CTX, CAZ/CA/BA versus CAZ,.S. and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The second option new interpretive guideline has enhanced detection of ESBLs in medical isolates of spp. and and allowed detection of an ESBL even when potentially masked by a pAmpC. Since extended-spectrum -lactamases (ESBLs) were in the beginning reported to be in in the 1st half of the 1980s in Germany, ESBLs have been spreading globally (2). The CLSI interpretive recommendations stated that strains of spp. and that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these providers (4). Therefore, detection of ESBLs in spp. and isolates is vital for ideal treatment of individuals and to control the spread of resistance. The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the zone diameters of cefotaxime (CTX; 30 g) or ceftazidime (CAZ; 30 g) disks with or without clavulanic acid (CA; 10 g) in spp. and and isolates have been found to produce both plasmid-mediated AmpC -lactamases (pAmpCs) and ESBLs (6, 8). In Korea, 8.7% of pAmpC makers also produced ESBLs (8). AmpCs resist inhibition by CA, and hence, the presence of an ESBL can be masked from the expression of an AmpC. Therefore, the coexistence of both pAmpCs and ESBLs in the same strain may result in false-negative checks for the detection of ESBLs (5). There is a need, therefore, for an alternative method that can detect ESBLs in spp. and isolates with a high sensitivity, even though the isolates simultaneously harbor pAmpCs. Boronic acids (BAs) were reported as reversible inhibitors of AmpCs (1). There are a few reports of studies designed to detect pAmpC-producing isolates of spp. and by a method using BA mainly because Phenolphthalein an inhibitor that closely resembles the phenotypic confirmatory checks for ESBLs (3, 5). And Coudron (5) recently described that a BA disk also enhances detection of isolates that harbor both ESBLs and pAmpCs. The aim of this work was to modify the phenotypic confirmatory test for ESBL production by using BA disks to improve its sensitivity for the detection of ESBLs in spp. and isolates even though the isolates simultaneously harbor pAmpCs. MATERIALS AND METHODS Bacterial strains. A total of 182 nonrepeat ESBL- and/or pAmpC-producing clinical isolates of spp. (= 118) and (= 64) collected from 12 tertiary-care hospitals in Korea during 2004 and 2005 were included in this study (Table ?(Table1)1) : 62 harbored ESBLs but not pAmpCs, 80 harbored both ESBLs and pAmpCs, and 40 harbored pAmpCs but not ESBLs. The isolates were recognized with API-20 E systems (bioMrieux, Marcy l’Etoile, France). Searches for genes coding for the class A ESBLs and pAmpCs were performed by Sele PCR amplification as explained previously (7, 8). The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730spp.isolate that produces SHV-2a, which showed a 2-mm increase in the zone diameter of a CAZ/BA disk in the presence of CA (Fig. 1E and F). The average increases in the zone diameters of the CTX/BA and CAZ/BA disks in the presence of CA were 11.8 mm and 11.1 mm, respectively. Open in a separate window Open in a separate windows FIG. 1. Comparison of the results for the CLSI confirmatory test (A and B) and two new assessments (C to F). The abbreviations (CTX/CA versus CTX, CAZ/CA versus CAZ, CTX/CA/BA versus CTX, CAZ/CA/BA versus CAZ, CTX/CA/BA versus CTX/BA, and CAZ/CA/BA versus CAZ/BA) are explained in footnote to Table ?Table2.2. Black circle, spp.; white circle, spp. (= 118) and (= 64) spp. (= 31)31 (100)31 (100)31 (100)????(= 31)31 (100)31 (100)31 (100)????Subtotal (= 62)62 (100)62 (100)62 (100)Producing both ESBLs and pAmpCs????spp. (= 64)55 (86)64 (100)64.Meanwhile, when a 3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result for ESBL production, the test also detected all clinical isolates that harbor ESBLs ( pAmpCs) and the test showed less frequent false-positive results (2/40, 5%) for clinical isolates that produce pAmpCs but not ESBLs. ESBL even when potentially masked by a pAmpC. Since extended-spectrum -lactamases (ESBLs) were in the beginning reported to be in in the first half of the 1980s in Germany, ESBLs have been spreading globally (2). The CLSI interpretive guidelines stated that strains of spp. and that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these brokers (4). Therefore, detection of ESBLs in spp. and isolates is crucial for optimal treatment of patients and to control the spread of resistance. The CLSI recommends a phenotypic confirmatory test for ESBL production that consists of measuring the zone diameters of cefotaxime (CTX; 30 g) or ceftazidime (CAZ; 30 g) disks with or without clavulanic acid (CA; 10 g) in spp. and and isolates have been found to produce both plasmid-mediated AmpC -lactamases (pAmpCs) and ESBLs (6, 8). In Korea, 8.7% of pAmpC suppliers also produced ESBLs (8). AmpCs resist inhibition by CA, and hence, the presence of an ESBL can be masked by the expression of an AmpC. Thus, the coexistence of both pAmpCs and ESBLs in the same strain may result in false-negative assessments for the detection of ESBLs (5). There is a need, therefore, for an alternative method that can detect ESBLs in spp. and isolates with a high sensitivity, even though the isolates simultaneously harbor pAmpCs. Boronic acids (BAs) were reported as reversible inhibitors of AmpCs (1). There are a few reports of studies designed to detect pAmpC-producing isolates of spp. and by a method using BA as an inhibitor that closely resembles the phenotypic confirmatory assessments for ESBLs (3, 5). And Coudron (5) recently described that a BA disk also enhances detection of isolates that harbor both ESBLs and pAmpCs. The aim of this work was to modify the phenotypic confirmatory test for ESBL production by using BA disks to improve its sensitivity for the detection of ESBLs in spp. and isolates even though the isolates simultaneously harbor pAmpCs. MATERIALS AND METHODS Bacterial strains. A total of 182 nonrepeat ESBL- and/or pAmpC-producing clinical isolates of spp. (= 118) and (= 64) collected from 12 tertiary-care hospitals in Korea during 2004 and 2005 were included in this study (Table ?(Table1)1) : 62 harbored ESBLs but not pAmpCs, 80 harbored both ESBLs and pAmpCs, and 40 harbored pAmpCs but not ESBLs. The isolates were recognized with API-20 E systems (bioMrieux, Marcy l’Etoile, France). Searches for genes coding for the class A ESBLs and pAmpCs were performed by PCR amplification as explained previously (7, 8). The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730spp.isolate that produces SHV-2a, which showed a 2-mm increase in the zone diameter of a CAZ/BA disk in the presence of CA (Fig. 1E and F). The average increases in the zone diameters of the CTX/BA and CAZ/BA disks in the presence of CA were 11.8 mm and 11.1 mm, respectively. Open in a separate window Open in a separate windows FIG. 1. Comparison of the results for the CLSI confirmatory test (A and B) and two new assessments (C to F). The abbreviations (CTX/CA versus CTX, CAZ/CA versus CAZ, CTX/CA/BA versus CTX, CAZ/CA/BA versus CAZ, CTX/CA/BA versus CTX/BA, and CAZ/CA/BA versus CAZ/BA) are explained in footnote to Table ?Table2.2. Black circle, spp.; white circle, spp. (=.