Rapid detection of GES-type extended-spectrum β-lactamases in Pseudomonas aeruginosa with a peptide nucleic acid-based realtime PCR assay

Abstract

Extended-spectrum β-lactamases (ESBLs) constitute a major problem given their broad substrate specificity and ability to hydrolyse many of the extended-spectrum third-generation cephalosporins currently in use in hospital settings. Guiana extended-spectrum-type (GES-1 – GES-9) ESBL enzymes have mainly been found in Pseudomonas aeruginosa (P. aeruginosa) and only at a limited number of geographical sites, mainly France, Greece and South Africa. Detection of GES-type ESBL-producing P. aeruginosa isolates in the clinical microbiology laboratory using conventional methods is problematic with molecular methods yielding better results. The aim of this study was to utilise various molecular techniques to determine the prevalence of GES-type ESBLs, characterise their genetic determinants and determine their clonal relatedness. The study further aimed to apply a sequence-selective, competitive PNA-based multiplex PCR in real-time for the identification and differentiation of GES-type enzymes. The prevalence of GES-type ESBLs was determined successfully through DNA sequencing. An increase in GES-2 prevalence since 2000 was noted which emphasised the importance of constant surveillance to monitor antibiotic determinants, their spread and overall prevalence. The knowledge on prevalence could be used in turn to monitor the efficacy of infection control measures and antibiotic regimens. Repeated sequencing confirmed the presence of blaGES-5 in P. aeruginosa isolates. As far as could be established, this study reported the first occurrence of GES-5 in South Africa and was the second description of GES-5 in P. aeruginosa. Application of a sequence-specific, competitive PNA-based multiplex PCR in real-time utilising SYBR Green was not suitable for the identification and differentiation of the blaGES genes. Although the method achieved different melting temperatures for the bla<GES genes tested, these temperatures were not suitable for accurate differentiation. Melting temperatures obtained for the same blaGES gene varied and those for different genes overlapped. An approach exploiting the high temperature shift caused by the PNA-probe rather than its competitive nature might be more successful. Random amplified polymorphic DNA typing has been described as a fast and simple method with high discriminatory power for the typing of P. aeruginosa and was thus used to determine the clonal relatedness of the bla<GES positive P. aeruginosa isolates. The occurrence of identical or similar P. aeruginosa isolates producing ESBLs in a single hospital setting emphasised the importance of constant surveillance. The study further identified identical P. aeruginosa clones that occurred in different hospitals indicating spread from a common external reservoir into these hospitals. The occurrence of highly drug-resistant P. aeruginosa in the environment has serious implications in a country with an ever increasing immune-compromised population. These finding were of concern since they demonstrated that acquired GES ESBLs can rapidly emerge and become a major cause of broad-spectrum β-lactam resistance among nosocomial pathogens. The information obtained in this study should be used to create awareness of the potential ESBL problem threatening current antimicrobial regimens in South Africa.