The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant Staphylococcus aureus. Five staphylococcal VNTR loci (sdr, clfA, clfB, ssp, and spa) were subjected to analysis, and it was shown that the method allows typing of S. aureus strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.During the past few years, the most remarkable advances in molecular typing were achieved by analysis of variable numbers of tandem repeat (VNTR) loci identified in the genomes of eukaryotic and prokaryotic species during genome sequencing projects. The number of repeat units at the same locus often varies from strain to strain and can be detected by PCR with flanking primers, a technique also commonly used for DNA fingerprinting of eukaryotic and prokaryotic species (1,5,7,9,11). However, to the best of our knowledge, this method has never been used to type Staphylococcus aureus strains, although several genes with repetitive sequences were analyzed for the purpose of S. aureus typing (16).The sequencing of the S. aureus genome indicated the presence of several VNTR loci, including sdr, clfA, clfB, ssp, coa, and spa. The sdr locus (10) comprises two or three closely linked and tandemly arrayed open reading frames containing sdrC, sdrD, and sdrE, which encode Sdr proteins. The Sdr proteins, together with clumping factor A (ClfA) (12) and clumping factor B (ClfB) (13), are members of a family of surface proteins which are characterized by the presence of an R region containing various numbers of the repeated Ser-Asp dipeptides encoded by an 18-nucleotide DNA repeat at the 3Ј region of the sdr genes. The ssp locus contains a gene (sspA) encoding a serine protease (V8 protease) (14), the C-terminal fragment of which is built of multiple, variable numbers of tripeptide repeats encoded by 9-nucleotide repeating units (2). Finally, coa and spa genes coding for collagen binding protein and protein A, respectively, have various numbers of degenerated repeats of 81 and 24 bp, respectively (15). This polymorphism has been commonly used for differentiating S. aureus isolates (6,8). In this paper, we describe a multiple-locus VNTR analysis (MLVA) system to discriminate among different S. aureus clinical isolates based on the analysis of five (sdr, clfA, clfB, ssp, and spa) tandem repeat loci composed of seven individual genes. This method of exploring the VNTR polymorphism enables the typing of clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, determination of their diversity and evolutionary relationships with discriminatory power, and a reproducibility matching the commonly used pulsed-field gel electrophoresis (PFGE) technique.The strain collection encompa...

Multiple-locus variable-number tandem-repeat analysis (MLVA), a new PCR-based method of typing Staphylococcus aureus, was compared to pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) on a group of 59 S. aureus (mostly methicillin-resistant) clinical isolates. The aim of the study was to establish possible criteria of clustering MLVA patterns and to check concordance levels between the results produced by MLVA and the three other typing methods. As in our earlier study, MLVA turned out to have discriminatory power similar to that of PFGE. Comparison of data obtained by the two approaches allowed us to propose a 70% or ca. 80% cutoff value of the similarity between two MLVA patterns, depending on a cutoff level applied to interpret the PFGE results, 75% or ca. 90%, respectively. The cutoff values corresponded to the difference of up to six or four bands, respectively, among maximum 14 bands in total produced by two isolates in the analysis. The MLVA clusters matched well those obtained by PFGE, and they were also consistent in general with clusters generated by spa typing and MLST, these latter methods characterized lower resolution. Our results suggest that MLVA may be reliable in shorter-term S. aureus epidemiological studies, including analyses of outbreaks and hospital-to-hospital strain transmission events. Well-known advantages of typing methods based on PCR (low cost, short time, and easiness of performance) make MLVA a method that may be useful in a variety of laboratories, including those performing routine microbiological analyses within medical centers.

In this paper we have identified homologous proteins encoded in the genome of S. aureus and other coagulase-negative Staphylococci . Collectively we refer to these proteins as staphostatins as they specifically inhibit cysteine proteinases (staphopains) from Staphylococcus spp. The primary structure of staphostatins seems to be unique, although they resemble cystatins in size (105-108 residues). Recombinant staphostatin A, a product of the scpB gene and staphostatin B (SspC) from S. aureus have been characterized in details. Similar to the cystatins, the staphostatins interact specifically with their target proteinases forming tight and stable non-covalent complexes, staphostatin A with staphopain A and staphostatin B with staphopain B. However, in contrast to the cystatins, each of which inhibits broad range of cathepsins, complex formation between staphostatin and staphopain appears to be exclusive, with no cross interaction observed. In addition, the activities of several tested cysteine proteinases of prokaryotic-and eukaryoticorigin were not affected by staphostatins. Such narrow specificity limited to staphopains is presumed to be required to protect staphylococcal cytoplasmic proteins from being degraded by prematurely activated/folded prostaphopains. This function is guaranteed through the unique co-expression of the secreted proteinase and the intracellular inhibitor from the same operon, and represents a unique mechanism of regulation of proteolytic activity in Grampositive bacteria.

The aim of this study was to develop an easy-to-use culture-free diagnostic method based on next generation sequencing (NGS) of PCR amplification products encompassing whole 16S-23S rRNA region to improve the resolution of bacterial species identification. To determine the resolution of the new method 67 isolates were subjected to four identification methods: Sanger sequencing of the 16S rRNA gene; NGS of the 16S-23S rRNA region using MiSeq (Illumina) sequencer; Microflex MS (Bruker) and VITEK MS (bioMérieux). To evaluate the performance of this new method when applied directly on clinical samples, we conducted a proof of principle study with 60 urine samples from patients suspected of urinary tract infections (UTIs), 23 BacT/ALERT (bioMérieux) positive blood culture bottles and 21 clinical orthopedic samples. The resolution power of NGS of the 16S-23S rRNA region was superior to other tested identification methods. Furthermore, the new method correctly identified pathogens established as the cause of UTIs and blood stream infections with conventional culture. NGS of the 16S-23S rRNA region also showed increased detection of bacterial microorganisms in clinical samples from orthopedic patients. Therefore, we conclude that our method has the potential to increase diagnostic yield for detection of bacterial pathogenic species compared to current methods.