Can We Use Imipenem and Meropenem Vitek 2 MICs for Detection of Suspected KPC and Other-Carbapenemase Producers among Species of Enterobacteriaceae?

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Lucero, Celeste. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Soloaga, Rolando. Hospital Naval Buenos Aires Cirujano Mayor Pedro Mallo. Servicio de Microbiología; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Imipenem and meropenem Vitek 2 MICs were evaluated for a panel of 104 Enterobacteriaceae for identification of carbapenemase producers. The sensitivity and specificity values for the new CLSI interpretative criteria (CLSI document M100-S20-U, 2010) were 98% and 83% for imipenem and 76% and 83% for meropenem, respectively. We propose an algorithm that is highly sensitive (98%) and specific (94%) for carbapenemase screening based on the combined use of imipenem and meropenem MICs

Can We Use Imipenem and Meropenem Vitek 2 MICs for Detection of Suspected KPC and Other-Carbapenemase Producers among Species of Enterobacteriaceae?

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Lucero, Celeste. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Soloaga, Rolando. Hospital Naval Buenos Aires Cirujano Mayor Pedro Mallo. Servicio de Microbiología; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Imipenem and meropenem Vitek 2 MICs were evaluated for a panel of 104 Enterobacteriaceae for identification of carbapenemase producers. The sensitivity and specificity values for the new CLSI interpretative criteria (CLSI document M100-S20-U, 2010) were 98% and 83% for imipenem and 76% and 83% for meropenem, respectively. We propose an algorithm that is highly sensitive (98%) and specific (94%) for carbapenemase screening based on the combined use of imipenem and meropenem MICs

Sensitive and specific modified Hodge test for KPC and metallo-beta- lactamase detection in Pseudomonas aeruginosa by use of a novel indicator strain, Klebsiella pneumoniae ATCC 700603

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Veliz, Omar. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.We evaluated the ability of the modified Hodge test to discriminate between KPC- and metallo-betalactamase (MBL)-producing Pseudomonas aeruginosa isolates and carbapenemase nonproducers. With Escherichia coli ATCC 25922 as the indicator strain, the MHT resulted in low sensitivity, specificity, and repeatability. Replacing the indicator strain with Klebsiella pneumoniae ATCC 700603 led to an improved performance (100%, 97%, 0%, and 100% sensitivity, specificity, indeterminate results and repeatability, respectively)

Sensitive and specific modified Hodge test for KPC and metallo-beta- lactamase detection in Pseudomonas aeruginosa by use of a novel indicator strain, Klebsiella pneumoniae ATCC 700603

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Veliz, Omar. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.We evaluated the ability of the modified Hodge test to discriminate between KPC- and metallo-betalactamase (MBL)-producing Pseudomonas aeruginosa isolates and carbapenemase nonproducers. With Escherichia coli ATCC 25922 as the indicator strain, the MHT resulted in low sensitivity, specificity, and repeatability. Replacing the indicator strain with Klebsiella pneumoniae ATCC 700603 led to an improved performance (100%, 97%, 0%, and 100% sensitivity, specificity, indeterminate results and repeatability, respectively)

Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Mendez, Tania. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.The detection of class A serine-carbapenemases among species of Enterobacteriaceae remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated Enterobacteriaceae strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of Enterobacteriaceae was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of -lactamases (extended-spectrum -lactamases, AmpCs, and metallo- -lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control

Controlling false-positive results obtained with the Hodge and Masuda assays for detection of class a carbapenemase in species of enterobacteriaceae by incorporating boronic Acid

The modified Hodge method (MHT) has been recommended by the CLSI for confirmation of suspected class A carbapenemase production in species of Enterobacteriaceae. This test and the Masuda method (MAS) have advantages over traditional phenotypic methods in that they directly analyze carbapenemase activity. In order to identify the potential interferences of these tests, we designed a panel composed of diverse bacterial genera with distinct carbapenem susceptibility patterns (42 carbapenemase producers and 48 nonproducers). About 25% of results among carbapenemase nonproducers, mainly strains harboring CTX-M and AmpC hyperproducers, were observed to be false positive. Subsequently, we developed an optimized approach for moreaccurate detection of suspicious isolates of carbapenemase by addition of boronic acid (BA) derivatives (reversible inhibitor of class A carbapenemases and AmpC cephalosporinases) and oxacillin (inhibitor of AmpCs enzymes). The use of the modified BA- and oxacillin-based MHT and MAS resulted in high sensitivity (>90%) and specificity (100%) for class A carbapenemase detection. By use of these methodologies, isolates producing KPCs and GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of ß-lactamases (extended-spectrum -lactamases [ESBLs], AmpC -lactamases, metallo- -lactamases [MBLs], etc.). These methods will provide the fast and useful information needed for targeting of antimicrobial therapy and appropriate infection control.Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Mendez, Tania. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina

Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Mendez, Tania. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio Antimicrobianos; Argentina.The detection of class A serine-carbapenemases among species of Enterobacteriaceae remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated Enterobacteriaceae strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of Enterobacteriaceae was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of -lactamases (extended-spectrum -lactamases, AmpCs, and metallo- -lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control

Novel variant (bla(VIM-11)) of the metallo-{beta}-lactamase bla(VIM) family in a GES-1 extended-spectrum-{beta}-lactamase-producing Pseudomonas aeruginosa clinical isolate in Argentina

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Faccone, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Petroni, Alejandro. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Galas, Marcelo F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Vázquez, Miryam. Hospital de Niños Dr. Ricardo Gutiérrez. Sección Microbiología; Argentina.Fil: Procopio, Adriana. Hospital de Niños Dr. Ricardo Gutiérrez. Sección Microbiología; Argentina.Two major groups of acquired β-lactamases have emerged in Pseudomonas aeruginosa: Ambler class A extended-spectrum β-lactamases (ESBLs) and class B metallo-β-lactamases (MBLs) (7, 9). MBLs are an expanding group of carbapenemases that includes the VIM family. To date, multiple allelic variants, namely, VIM-1 to VIM-10 (http://www.lahey.org/studies ), have been described in Europe, Asia, and the Americas, VIM-2 being the most ubiquitous enzyme by far (7, 8; R. E. Mendes, M. Castanheira, P. Garcia, M. Guzman, M. A. Toleman, T. R. Walsh, and R. N. Jones, Letter, Antimicrob. Agents Chemother. 48:1433-1434, 2004). Additionally, five types of ESBLs have been detected in P. aeruginosa: TEM, SHV, PER, VEB, and IBC/GES (9). Association between VIM and ESBLs still appears to be a rare event in P. aeruginosa and was reported only for VIM-2 with either PER-1 or IBC-2 (3; J. D. Docquier, J. D., F. Luzzaro, G. Amicosante, A. Toniolo, and G. M. Rossolini, Letter, Emerg. Infect. Dis. 7:910-911, 2001). MBL- or ESBL-producing P. aeruginosa isolates have not yet been reported in Argentina, while the coexistence of VIM with GES-1 in a single clinical isolate has not been documented anywhere

CMY-2-type plasmid-mediated AmpC beta-lactamase finally emerging in Argentina

Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio Antimicrobianos; Argentina.Fil: Monzani, V. Hospital Interzonal Especializado Materno Infantil. Servicio Laboratorio, Bacteriología; Mar del Plata, Argentina.Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio Antimicrobianos; Argentina.Fil: Morvay, L. Hospital Interzonal Especializado Materno Infantil. Servicio Laboratorio, Bacteriología; Mar del Plata, Argentina.Fil: Faccone, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio Antimicrobianos; Argentina.Fil: Petroni, Alejandro. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio Antimicrobianos; Argentina.Fil: Galas, Marcelo F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio Antimicrobianos; Argentina

Tigecycline and intravenous fosfomycin zone breakpoints equivalent to the EUCAST MIC criteria for Enterobacteriaceae

Fil: Pasteran, Fernando. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Lucero, Celeste. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Rapoport, Melina J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Guerriero, Leonor. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Barreiro, Irene. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Albornoz, Ezequiel. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Veliz, Omar. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Fil: Corso, Alejandra. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología. Servicio de Antimicrobianos; Argentina.Introduction: Tigecycline and intravenous (i.v.) fosfomycin could be alternative therapeutic options for the treatment of carbapenemase-possessing Enterobacteriaceae bacterial infections. However, routine laboratories are forced to test these drugs using minimum inhibitory concentration (MIC) methods as zone breakpoints are not available for the disc diffusion technique. Methodology: Clinical and Laboratory Standards Institute methods for agar dilution and disc diffusion were compared to determine tentative zone breakpoints that best correlate to tigecycline and i.v. fosfomycin MIC breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing. A total of 195 Enterobacteriaceae with defined mechanisms of resistance were tested in duplicate assays. Half of the strains were characterized as carbapenemase producers (KPC-2, OXA-48, OXA-163, VIM-1, VIM-2, IMP-8, NDM-1). Results: Corresponding zone diameters of susceptible ≥15mm, resistant ≤12mm and susceptible ≥17mm, resistant ≤15mm for the 50μg fosfomycin plus 50μg glucose-6-phosphate and 200μg fosfomycin plus 50μg glucose-6-phosphate discs, respectively, allowed categorization of the strains with an acceptable level of error ( 90%). For the 15μg tigecycline disc, the best performance was achieved with the corresponding zone diameters of susceptible ≥21mm and resistant ≤16mm, which eliminated the very major and major errors but not the minor errors (34.4%). Conclusions: Based on these results, tigecycline and fosfomycin can be included in the routine panel of antibiotics for susceptibility testing by disc diffusion to provide fast and reliable information for the selection of treatment alternatives, especially for strains with extreme resistance, as carbapenemase producers