Characterization of bacteria causing outbreaks by using molecular methods in a clinical microbiology laboratory

Outbreaks of bacteria causing hospital and community acquired infections have dramatically increased in number over the recent years. For example, travelers become colonized by resistant bacteria and may transmit the strains to other people and to medical care settings when they return home. The challenge is new and problematic for laboratories performing bacterial diagnostics. Fast and highly specific tools are needed to identify and characterize resistant bacterial strains in order to prevent outbreaks in local hospitals. This thesis studies the fast identification of virulence and resistance genes of the most important hospital acquired (HA) bacteria. In addition, the applicability of rapid outbreak analysis for HA-bacteria using molecular methods outside of the national reference laboratory was studied. The aim of the study was to establish a sensitive, reliable multiplex-PCR method suitable for daily use in a microbiological diagnostic laboratory and to speed up the reporting of bacteria causing outbreaks. Another aim was to study the usefulness and functionality of a commercial repetitive PCR (DiversiLab) and compare it with reference molecular typing methods. The thesis also gives an overall view of the occurrence of HA-bacteria witnessed in the district of Helsinki and Uusimaa over the recent years. The thesis consists of six studies on four different bacterial species/types causing outbreaks: MRSA (methicillin-resistant Staphylococcus aureus), ESBL (Extended Spectrum Beta-Lactamase)-producing Enterobacteriaceae, Acinetobacter baumannii and Clostridium difficile. Consecutive and retrospective bacterial isolates of the each bacteria were collected and examined. The methods used were conventional multiplex PCR and real-time multiplex PCR. The typing methods were repetitive PCR (DiversiLab), PFGE (pulsed field gel electrophoresis), PCR ribotyping, spa typing, and sequencing-based methods. During this thesis three usable in-house multiplex PCRs were established for the detection of MRSA, carbapenemase genes, and virulence genes in C. difficile. The repetitive PCR method, DiversiLab, was found to be a fast and proprietary typing method, very beneficial in the first-line identification of outbreaks. In addition, the Diversilab system may be used in the comparison of resistant bacterial isolates. The method produced better results with Gram-negatives (ESBL-producing Enterobacteriaceae and A. baumannii). However, the reference methods still serve their purpose in global isolate comparison. In the future, whole genome sequencing will, however, most likely replace contemporary typing methods.