Characterization of Clinical Isolates ofKlebsiella pneumoniaefrom 19 Laboratories Using the National Committee for Clinical Laboratory Standards Extended-Spectrum β-Lactamase Detection Methods

Abstract

Extended-spectrum ␤-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were >2 g/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of >5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by >3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were >32 g/ml, suggesting either the presence of an AmpC-type ␤-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a ␤-lactamase with a pI of >8.3 suggestive of an AmpC-type ␤-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla OXA genes. The IEF profiles of the remaining 16 isolates showed a variety of ␤-lactamase bands, all of which had pIs of <7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla OXA , and bla CTX-M genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained ␤-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained ␤-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.

Prevalência das famílias TEM, SHV e CTX-M de β-lactamases de espectro estendido em Escherichia coli e Klebsiella spp no Hospital Universitário de Santa Maria, Estado do Rio Grande do Sul

“…Com base no método do disco combinado, alguns estudos confirmam o baixo desempenho da ceftazidima principalmente na presença de ESBLs do tipo CTX-M 22 30 . Contrariamente, Steward e cols 28 relataram melhor desempenho da ceftazidima em relação à cefotaxima entre isolados de Klebsiella pneumoniae provenientes dos Estados Unidos. No Brasil, Nogueira e cols 16 comparando substratos para a detecção de ESBLs relataram melhor desempenho da cefotaxima e menor eficiência da cefpodoxima e ceftazidima.…”

“…Tal achado é importante porque a resistência a cefoxitina foi correlacionada com a possível presença de outros mecanismos de resistência que podem prejudicar a detecção de ESBLs pelos métodos fenotípicos 28 .…”

“…Contudo, ao analisarmos esses resultados devemos levar em conta a presença das β-lactamases TEM-1, TEM-2 e SHV-1, também detectadas pelos primers utilizados neste estudo. Além disso, a presença de mecanismos adicionais de resistência, como enzimas do tipo AmpC 19 30 , mudanças nos poros e β-lactamases do tipo TEM e SHV com reduzida afinidade aos inibidores de β-lactamases, pode mascarar a inibição do ácido clavulânico resultando em resultados falsos negativos 28 .…”

“…Os microrganismos que evidenciaram ausência de ESBLs em qualquer um dos métodos fenotípicos foram avaliados frente a cefoxitina (30μg) (Oxoid; Basingstoke, Ing). A resistência à cefoxitina está associada a presença de outros mecanismos de resistência que podem interferir na detecção de ESBLs pelos métodos fenotípicos 28 .…”

Assessment the Effects of Low Power Laser Light on Some Virulent Factors of Klebsiella pneumoniae Isolated from Respiratory Tract Infections

“…This bacteria has many virulence factors as a whole make it pathogenicity, included Capsule (Steward et al, 2001). In addition to the different types of adhesions or colonization factors like Pili (fimbriae) such as Type Ι pili or mannose-sensitive Klebsiella hemagglutination (MS/K-HA), Type ΙΙΙ Pili or mannose-resistant Klebsiella hemagglutination (MR/K-HA) (Podschun et al, 1991), both can contribute to biofilm formation (Podschun et al, 1991;.…”