The increasing incidence of coagulase-negative staphylococci (CoNS) in hospital-acquired infections underlines the need for an accurate and simple identification of Staphylococcus isolates at the species level. Sequencing of the tuf gene has been shown to be the most accurate for the species identification of CoNS. We determined the species of 62 consecutive clinical and 31 reference CoNS isolates by tuf gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Species assignment by MALDI-TOF-MS and tuf sequencing was congruent in all cases. We conclude that MALDI-TOF-MS is accurate for identifying CoNS in routine clinical practice. The study also identified an unexpectedly high number of cases of Staphylococcus capitis infections among 62 consecutive CoNS isolates in 2009 at the University Medical Center Utrecht, the Netherlands.

The high rate of identical ccrB sequences in a geographically, temporally and genotypically diverse set of S. aureus and CoNS isolates indicates frequent horizontal transfer of SCCmec between CoNS and S. aureus, which may have contributed to the emergence of MRSA.

These data combined with the country of origin of the patient suggest that ST80 S0924 was probably relatively recently introduced into the USA from Syria.

IntroductionCommunity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are increasingly isolated, with USA300-0114 being the predominant clone in the USA. Comparative whole genome sequencing of USA300 isolates collected in 2002, 2003 and 2005 showed a limited number of single nucleotide polymorphisms and regions of difference. This suggests that USA300 has undergone rapid clonal expansion without great genomic diversification. However, whole genome comparison of CA-MRSA has been limited to isolates belonging to USA300. The aim of this study was to compare the genetic repertoire of different CA-MRSA clones with that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH) to identify genetic clues that may explain the successful and rapid emergence of CA-MRSA.Materials and MethodsHierarchical clustering based on CGH of 48 MRSA isolates from the community and nosocomial infections from Europe and the USA revealed dispersed clustering of the 19 CA-MRSA isolates. This means that these 19 CA-MRSA isolates do not share a unique genetic make-up. Only the PVL genes were commonly present in all CA-MRSA isolates. However, 10 genes were variably present among 14 USA300 isolates. Most of these genes were present on mobile elements.ConclusionThe genetic variation present among the 14 USA300 isolates is remarkable considering the fact that the isolates were recovered within one month and originated from a confined geographic area, suggesting continuous evolution of this clone.

BackgroundMicroarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliably determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes.When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes.ResultsIn this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively.ConclusionA definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal.