Rapid discrimination of Staphylococcus epidermidis genotypes in a routine clinical microbiological laboratory using single nucleotide polymorphisms in housekeeping genes.

PURPOSE: Staphylococcus epidermidis colonies often display several morphologies and antimicrobial susceptibility patterns when cultured from device-related infections, and may represent one or multiple genotypes. Genotyping may be helpful in the clinical interpretation, but is time consuming and expensive. We wanted to establish a method for rapid discrimination of S. epidermidis genotypes for use in a routine microbiology laboratory. METHODOLOGY: A real-time PCR targeting eight discriminatory class I or II single-nucleotide polymorphisms (SNPs) in six of the seven housekeeping genes was constructed. Post PCR, high-resolution melt (HRM) analysis using EvaGreen as fluorophore discriminated amplicons based on their percentage GC content. RESULTS: In silico, 42 representative sequence types (STs), including all major MLST group and subgroup founders, were separated into 23 different cluster profiles with a Simpson's index of diversity of 0.97. By HRM-PCR, 11 commonly encountered hospital and outbreak STs were separated into eight HRM patterns. CONCLUSION: This method can rapidly establish whether S. epidermidis strains belong to different genotypes. It can be used in patients with S. epidermidis infections, as an aid in outbreak investigations and to select strains for investigation with more discriminatory methods, saving workload and costs. Results may be obtained the same day as culture results. Its strength lies mainly in indicating differences, as some STs may have the same melt profile. Changes in S. epidermidis epidemiology may warrant alterations in the inclusion of SNPs. We believe this method can reduce the threshold for performing genotyping analysis on an increasingly important nosocomial pathogen.

Legionella pneumophila in Norwegian naval vessels.

BACKGROUND: Little is known about the occurrence of Legionella pneumophila in water supply systems on board ships. Our aim was to study the occurrence of L. pneumophila in the water supply system on board Norwegian naval vessels as the basis for framing preventive strategies against Legionella infection. MATERIAL AND METHOD: Water samples were collected from technical installations and from the water distribution network on board 41 vessels and from ten water filling (bunkering) stations, the sampling taking place in two rounds separated by a one-year interval. The samples were subjected to analysis, including serotyping and genotyping, with a view to identifying the presence of L. pneumophila and of free-living amoebae. RESULTS: L. pneumophila was found in 20 out of a total of 41 vessels in the first round of sampling, and live L. pneumophila serogroup 1 was isolated in seven of the 20 vessels. Free-living amoebae were found in the water supply system in most of the vessels, including all the vessels with L. pneumophila. The same genotype of L. pneumophila was identified in the water in bunkering stations and in the water on board the vessels. INTERPRETATION: L. pneumophila was not present in all the vessels, but all the vessels where the bacterium was found were also contaminated with free-living amoebae. We have demonstrated the probability of the fresh water from bunkering stations being the source of the contamination. In framing preventive strategies, importance should therefore be attached to identifying the source of contamination and the presence of free-living amoebae, as a premise for the establishment and growth of L. pneumophila in onboard water supply systems.

A comprehensive microbiological evaluation of fifty-four patients undergoing revision surgery due to prosthetic joint loosening.

The diagnosis of a chronic prosthetic joint infection (PJI) is challenging, and no consensus exists regarding how best to define the criteria required for microbiological identification. A general view is that culture of periprosthetic biopsies suffers from inadequate sensitivity. Recently, molecular analyses have been employed in some studies but the specificity of molecular analyses has been questioned, mainly due to contamination issues. In a prospective study of 54 patients undergoing revision surgery due to prosthetic joint loosening, we focused on two aspects of microbiological diagnosis of chronic PJI. First, by collecting diagnostic specimens in a highly standardized manner, we aimed at investigating the adequacy of various specimens by performing quantitative bacteriological culture. Second, we designed and performed real-time 16S rRNA gene PCR analysis with particular emphasis on minimizing the risk of false-positive PCR results. The specimens analysed included synovial fluid, periprosthetic biopsies from the joint capsule and the interface membrane, and specimens from the surface of the explanted prosthesis rendered accessible by scraping and sonication. No antibiotics were given prior to specimen collection. Based on five diagnostic criteria recently suggested, we identified 18 PJIs, all of which fulfilled the criterion of ≥2 positive cultures of periprosthetic specimens. The rate of culture-positive biopsies from the interface membrane was higher compared to specimens from the joint capsule and synovial fluid, and the interface membrane contained a higher bacterial load. Interpretational criteria were applied to differentiate a true-positive PCR from potential bacterial DNA contamination derived from the reagents used for DNA extraction and amplification. The strategy to minimize the risk of false-positive PCR results was successful as only two PCR results were false-positive out of 216 negative periprosthetic specimens. Although the PCR assays themselves were very sensitive, three patients with low bacterial numbers in periprosthetic specimens tested negative by real-time PCR. This overall lowered sensitivity is most likely due to the reduced specimen volume used for PCR analysis compared to culture and may also be due to interference from human DNA present in tissue specimens. According to the protocol in the present study, 16S rRNA gene real-time PCR did not identify more cases of septic prosthetic loosening than did culture of adequate periprosthetic biopsies.