Detection and characterization of ESBL positive Klebsiella pneumoniae clinical isolates

Abstract

Klebsiella pneumoniae is an important nosocomial pathogen that has the potential to cause severe morbidity and mortality, particularly in intensive care units, as well as in medical and surgical wards. In recent years, following extensive use of the expanded-spectrum cephalosporins, outbreaks of infections caused by extended-spectrum beta-lactamase (ESBL)- producing K. pneumoniae isolates have been reported throughout the world. Laboratory detection of ESBL-producing bacteria is important, if left undetected it can result in treatment failure, leading to serious consequences, such as an increased mortality rate. It is essential for diagnostic laboratories to use rapid and reliable methods for the detection of ESBL-producing bacteria. In this study, 150 K. pneumoniae clinical isolates were collected from the diagnostic laboratory of the Department of Medical Microbiology, National Health Laboratory Service (NHLS). The isolates were collected within a four month period (September 2009 to December 2009). The isolates were analyzed and characterized using phenotypic and genotypic methods. The aim of the study was to investigate the prevalence of ESBLproducing K. pneumoniae isolates. Secondly, to compare the sensitivity and specificity of the Vitek2 advanced expert system (AES) (bioMérieux, France) and Multiplex PCR assay (targeting blaSHV, blaTEM and blaCTX-M genes) against the combination disc method (Gold standard) in detecting ESBL-production. Lastly, the study investigated the clonal relatedness of the collected isolates using ERIC, REP and BOX PCR fingerprinting assays. The combination disc method was performed and interpreted according to the Clinical and Laboratory Standards Institute 2009 guidelines. The prevalence of ESBL-positive K. pneumoniae isolates according to the combination disc method was 57.4% (85/148). The sensitivity and specificity of the Vitek2 AES in detecting ESBL production was 99% (84/85) and 98% (62/63) respectively when compared to the combination disc method. The sensitivity and specificity of the multiplex PCR assay (using blaCTX-M gene as a true marker for ESBL-production) were 96% (82/85) and 98% (62/63) respectively. A good correlation was obtained between the three assays (combination disc method, Vitek2 AES and multiplex PCR assay) evaluated in this study. There is a high prevalence of ESBLproducing isolates in the Tshwane area. The high prevalence of ESBL-producing isolates is of great concern; proper infection control measures to reduce the spread of ESBL-producing K. pneumoniae isolates is recommended. Genotyping revealed a high level of similarity among ESBL and non ESBL-producing K. pneumoniae isolates. However, there was no sign of an outbreak among the ESBLproducing K. pneumoniae isolates because ESBL and non-ESBL producing isolates were uniformly distributed in the groups obtained with the ERIC and BOX PCR dendrogrammes.