Pseudomonas aeruginosa and Staphylococcus aureus virulence factors as biomarkers of infection.

The gold standard for the diagnosis of bacterial infections in clinical samples is based on culture tests that are time-consuming and labor-intense. For these reasons, an extraordinary effort has been made to identify biomarkers as the tools for sensitive, rapid and accurate identification of pathogenic microorganisms. Moreover, biomarkers have been tested to distinguish colonization from infection, monitor disease progression, determine the clinical status of patients or predict clinical outcomes. This mini-review describes Pseudomonas aeruginosa and Staphylococcus aureus biomarkers, which contribute to pathogenesis and have been used in culture-independent bacterial identification directly from patient samples.

Staphylococcal cassette chromosome mec containing a novel mec gene complex, B4.

OBJECTIVES: To describe a new subclass of mec class B complex identified in Staphylococcus epidermidis. METHODS: Four S. epidermidis isolates obtained from bloodstream infections in patients at University Medical Center Groningen (UMCG) were analysed by phenotypic antibiotic susceptibility testing and WGS. RESULTS: Sequence analysis revealed a new staphylococcal cassette chromosome mec (SCCmec) structure in isolate UMCG335. In this structure, plasmid pUB110 was found to be integrated into SCCmec IVc, creating a new SCCmec subtype, IVUMCG335. SCCmec IVc and a copy of plasmid pUB110 were found in other isolates, UMCG364 and UMCG341, respectively, indicating a probability that SCCmec IVUMCG335 could have evolved at the UMCG. SCCmec of UMCG337 contained a new genetic organization of the mec complex (IS431-ΔmecR1-mecA-IS431-pUB110-IS431-ψIS1272) that we have named B4. This new subclass of mec class B complex originated by IS431-mediated inversion of the DNA segment encompassing the plasmid and most of the genes of the mec complex with the exception of IS1272. As the SCCmec organization in UMCG337 differed by the inversion of an ∼10 kb sequence compared with SCCmec IVUMCG335, we have named it SCCmec subtype IVUMCG337. Isolates UMCG335 and UMCG337 carrying SCCmec IVUMCG335 and IVUMCG337, respectively, were associated with a restriction-modification system and a CRISPR-Cas system, creating a composite island of almost 70 kb. CONCLUSIONS: Our findings highlight the importance of IS431 in the evolution of the SCCmec region. The increasing genetic diversity identified in the SCCmec elements imposes a great challenge for SCCmec typing methods and highlights possible difficulties with the SCCmec nomenclature.

Misidentification of meticillin-resistant Staphylococcus aureus by the Cepheid Xpert MRSA NxG assay, the Netherlands, February to March 2021.

We describe two false-negative results in the detection of meticillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 and spa type t011 using the Cepheid Xpert MRSA NxG assay. The isolates were recovered in late February and early March 2021 from two patients in different hospitals in the northern Netherlands. Variations between the two isolate genomes indicate that this MRSA strain might have been spreading for some time and could have disseminated to other regions of the Netherlands and other European countries.

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Whole-genome analysis of an oxacillin-susceptible CC80 mecA-positive Staphylococcus aureus clinical isolate: insights into the mechanisms of cryptic methicillin resistance.

OBJECTIVES: The mec and bla systems, among other genetic factors, are critical in regulating the expression of methicillin resistance in Staphylococcus aureus. We examined by WGS a naturally occurring oxacillin-susceptible mecA-positive S. aureus isolate to identify the mechanism conferring oxacillin susceptibility. METHODS: The mecA-positive oxacillin-susceptible S. aureus isolate GR2 (penicillin and oxacillin MICs 0.094 and 1 mg/L, respectively), belonging to clonal complex 80, was characterized. DNA fragment libraries were sequenced on Roche 454 and Illumina MiSeq sequencers and de novo assembly of the genome was generated using SeqMan NGen software. Plasmid curing was conducted by SDS treatment. Expression of mecA was quantified without/with ß-lactam pressure. RESULTS: The genome of GR2 consisted of a 2 792 802 bp chromosome and plasmids pGR2A (28 895 bp) and pGR2B (2473 bp). GR2 carried SCCmec type IV, with a truncated/non-functional mecR1 gene and no mecI. A single copy of the bla system, with an organization unique for S. aureus, was found, harboured by plasmid pGR2A. Particularly, the blaZ gene was orientated like its regulatory genes, blaI and blaR1, and a gene encoding transposase IS66 was integrated between blaZ and the regulatory genes deleting the 5'-end of blaR1; blaI, encoding blaZ/mecA repressor, was intact. After plasmid loss, GR2 became penicillin and oxacillin resistant (MICs 0.5 and 6 mg/L, respectively). CONCLUSIONS: We can conclude that after exposure to ß-lactams, the non-functional BlaR1 does not cleave the mecA repressor BlaI, derepression does not occur and mecA is not efficiently expressed. Removal of the bla system after curing of pGR2A allows constitutive expression of mecA, resulting in oxacillin and penicillin resistance.

Genome-wide analysis reveals two novel mosaic regions containing an ACME with an identical DNA sequence in the MRSA ST398-t011 and MSSA ST8-t008 isolates.

OBJECTIVES: The presence of the arginine catabolic mobile element (ACME) in Staphylococcus aureus has been reported to enhance the colonization of the human host. The aim of this study was to determine the genetic organization of composite islands harbouring ACME. METHODS: Two ACME-positive S. aureus isolates obtained during two different surveys conducted in the Netherlands and Poland were characterized in this study. The isolates were analysed by spa typing, DNA microarrays and whole-genome sequencing. RESULTS: The two isolates harboured a truncated yet fully functional ACME type II with an identical nucleotide sequence, but differed in their adjacent mobile genetic elements. The first strain was a livestock-associated ST398-t011 MRSA, which had a staphylococcal cassette chromosome mec (SCCmec) composite island composed of SCCpls adjacent to orfX followed by ACME type II and SCCmec type IVa. The second ACME-positive isolate was an ST8-t008 MSSA. Its composite island showed an SCC-like element carrying the ccrC gene followed by ACME II. CONCLUSIONS: This is the first report of an ACME in a livestock-associated MRSA ST398. It is also the first presentation of an ACME composite island structure in an MSSA isolate. Our findings indicate an extensive mosaicism of composite islands in S. aureus, which has implications for the transmissibility among humans and thus for public health.

The complete genome sequence of unculturable Mycoplasma faucium obtained through clinical metagenomic next-generation sequencing.

Introduction: Diagnosing Mycoplasma faucium poses challenges, and it's unclear if its rare isolation is due to infrequent occurrence or its fastidious nutritional requirements. Methods: This study analyzes the complete genome sequence of M. faucium, obtained directly from the pus of a sternum infection in a lung transplant patient using metagenomic sequencing. Results: Genome analysis revealed limited therapeutic options for the M. faucium infection, primarily susceptibility to tetracyclines. Three classes of mobile genetic elements were identified: two new insertion sequences, a new prophage (phiUMCG-1), and a species-specific variant of a mycoplasma integrative and conjugative element (MICE). Additionally, a Type I Restriction-Modification system was identified, featuring 5'-terminally truncated hsdS pseudogenes with overlapping repeats, indicating the potential for forming alternative hsdS variants through recombination. Conclusion: This study represents the first-ever acquisition of a complete circularized bacterial genome directly from a patient sample obtained from invasive infection of a primary sterile site using culture-independent, PCR-free clinical metagenomics.

Novel organization of the arginine catabolic mobile element and staphylococcal cassette chromosome mec composite island and its horizontal transfer between distinct Staphylococcus aureus genotypes.

In this study, 425 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in the Dutch-German Euregio were investigated for the presence of the arginine catabolic mobile element (ACME). Sequence analysis by whole-genome sequencing revealed an entirely new organization of the ACME-staphylococcal cassette chromosome mec composite island (SCCmec-CI), with truncated ACME type II located downstream of SCCmec. An identical nucleotide sequence of ACME-SCCmec-CI was found in two distinct MRSA lineages (t064-ST8 and t002-ST5), which has not been reported previously in S. aureus.

Evaluation of a modular multiplex-PCR methicillin-resistant Staphylococcus aureus detection assay adapted for mecC detection.

A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection.

Rapid and high-resolution distinction of community-acquired and nosocomial Staphylococcus aureus isolates with identical pulsed-field gel electrophoresis patterns and spa types.

Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the 'classical' community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control.

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398.

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.

High-resolution typing by MLVF unveils extensive heterogeneity of European livestock-associated methicillin-resistant Staphylococcus aureus isolates with the sequence type 398.

Methicillin-resistant Staphylococcus aureus sequence type 398 (MRSA ST398) has emerged in livestock worldwide. In particular, areas in Europe with high densities of livestock farming are affected. Consequently, the incidence of human colonization and infection with ST398 is rapidly increasing. Distinguishing different ST398 isolates with standard typing tools is problematic. The objective of this study was to examine the discriminatory power of Multiple-Locus Variable number tandem repeat Fingerprinting (MLVF) on a highly diverse ST398 collection. Our data show that MLVF combined with spa-typing is an attractive approach for high-resolution typing of ST398 isolates and unveiling their relatedness.

Detection of new methicillin-resistant Staphylococcus aureus strains that carry a novel genetic homologue and important virulence determinants.

In this study, 18 methicillin-resistant Staphylococcus aureus (MRSA) isolates harboring staphylococcal cassette chromosome mec (SCCmec) type XI, recovered in the Dutch-German Euregio, were characterized by DNA microarrays. In contrast to previous data, we found two MRSA strains of different clonal lineages possessing SCCmec XI that carried important virulence determinants. The worrisome emergence of such toxigenic MRSA strains raises concerns that MRSA strains with enhanced virulence potential and impaired detectability by standard molecular assays may spread in Europe.

Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus.

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Case Report: Necrotizing fasciitis caused by Staphylococcus aureus positive for a new sequence variant of exfoliative toxin E.

Objectives: Necrotizing fasciitis (NF) caused by S. aureus is a rare, aggressive and rapidly progressing superficial fascia infection with a high mortality rate. The aim of this study was to identify virulence-related genes from a complete genome sequence of a methicillin-susceptible S. aureus (MSSA) isolate recovered from a monomicrobial case of NF. Materials and methods: The MSSA isolate UMCG579 was cultured from a pus collection from the subcutis of a patient with NF. The genome of isolate UMCG579 was sequenced using MinION (Oxford Nanopore) and MiSeq (illumina) platforms. Results: The genome of the UMCG579 isolate was composed of a 2,741,379 bp chromosome and did not harbor any plasmids. Virulence factor profiling identified multiple pore-forming toxin genes in the UMCG579 chromosome, including the Panton-Valentine leukocidin (PVL) genes, and none of the superantigen genes. The UMCG579 isolate harbored a new sequence variant of the recently described ete gene encoding exfoliative toxin (type E). A search in the GenBank database revealed that the new sequence variant (ete2) was exclusively found among isolates (n = 115) belonging to MLST CC152. While the majority of S. aureus ete-positive isolates were recovered from animal sources, S. aureus ete2-positive isolates originated from human carriers and human infections. Comparative genome analysis revealed that the ete2 gene was located on a 8777 bp genomic island. Conclusion: The combination of two heterogeneously distributed potent toxins, ETE2 and PVL, is likely to enhance the pathogenic ability of S. aureus isolates. Since anti-virulence therapies for the treatment of S. aureus infections continue to be explored, the understanding of specific pathogenetic mechanisms may have an important prophylactic and therapeutic value. Nevertheless, the exact contribution of ETE sequence variants to S. aureus virulence in NF infections must be determined.

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration.

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847-treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region.

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region.

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.

Polymorphism, genetic exchange and intragenic recombination of the aureolysin gene among Staphylococcus aureus strains.

BACKGROUND: Staphylococcus aureus expresses several proteases, which are thought to contribute to the virulence of this bacterium. Here we focus on aureolysin, the major thermolysin-like metalloprotease. Despite the importance of aureolysin in the physiology and pathogenesis of S. aureus, relatively little information was so far available concerning the aur gene diversity and mobility within and between the major subdivisions of the S. aureus population. Therefore, an epidemiologically and genetically diverse collection of S. aureus strains was used to determine the range of aureolysin (aur) gene polymorphism. RESULTS: Sequence analyses support the conclusion that the aur gene occurs in two distinct types of related sequences. The aur gene was much more polymorphic but, at the same time, showed higher purifying selection than genes utilized for multilocus sequence typing (MLST). Gene trees constructed from aur and concatenated MLST genes revealed several putative assortative recombination events (i.e. entire aur gene exchanges) between divergent lineages of S. aureus. Evidence for intragenic recombination events (i.e. exchanges of internal aur segments) across aur genes was also found. The biochemical properties and substrate specificity of the two types of aureolysin purified to homogeneity were studied, revealing minor differences in their affinity to low molecular weight synthetic substrates. CONCLUSION: Although numerous nucleotide differences were identified between the aur alleles studied, our findings showed that a strong purifying selection is acting on the aur gene. Moreover, our study distinguishes between homologous exchanges of the entire aur gene (assortative recombination) between divergent S. aureus lineages and recombination events within aur genes.

Molecular surveillance of methicillin-resistant Staphylococcus aureus by multiple-locus variable number tandem repeat fingerprinting (formerly multiple-locus variable number tandem repeat analysis) and spa typing in a hierarchic approach.

In this study, clonal relatedness of 202 Staphylococcus aureus (mostly methicillin-resistant) isolates recovered in 29 Polish hospitals was investigated by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa typing. Our analysis yielded 69 MLVF patterns and 34 spa types. Almost all isolates (97.4%) identical by MLVF were also indistinguishable by spa typing. Therefore, the MLVF method can be a cheap and fast screen before spa typing. Moreover, results obtained by MLVF suggest a set of simple criteria for grouping of spa types. The proposed algorithm groups isolates into the same cluster when spa sequences differ by a single mutation event: i) a single deletion or insertion of repeat unit(s) at the X region of the protein A gene or ii) a single nucleotide polymorphism within a repeat sequence. The combined use of these 2 methods, MLVF in local laboratories and spa typing of selected isolates in reference centers, can improve the monitoring of hospital-to-hospital strain transmission events and public health interventions on a huge scale.