Staphylococcus aureus has become a major source of hospital infections and the risk of colonisation and infection by community-acquired methicillin-resistant S. aureus (CA-MRSA) is increasingly higher. Because of the importance of S. aureus to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. In this study we evaluated the usefulness of multiplex PCR based Multi-Locus VNTR Fingerprinting (MLVF) as the first step method for rapid differentiation of Croatian and Polish S. aureus isolates in hospital and community settings. This is a first report of the usefulness of MLVF in typing of hospital-acquired methicillin-sensitive S. aureus (HA-MSSA) and four CA-MRSA isolates. A total of 47 isolates of S. aureus recovered in Croatia in 2004 and in Poland in 2006 and 2007 were tested. The MLVF results were compared to those produced by other typing methods, such as Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST) and spa typing. The MLVF analysis showed almost the same clonality results as the remaining typing methods although some differences were found. Epidemiological data about the relation among S. aureus isolates and the results produced by typing methods applied in the present study indicate that because of the advantages in ease and speed of Variable Number of Tandem Repeats (VNTR) procedure over PFGE, spa typing and MLST, MLVF can be used as a first screening method followed by additional typing.
One hundred seventy Staphylococcus aureus isolates, collected in 1996 to 2004, were reidentified by phenotypic and genotypic methods. One hundred ten of these (65%) were confirmed, as previously denoted, to be clumping factor (CF)-or free coagulase-deficient S. aureus, based on their phenotype. Based on the CF or coagulase production, three groups of phenotypically deficient S. aureus isolates were distinguished. Group 1 encompassed CF-positive and coagulase-deficient isolates, group 2 consisted of CF-deficient and coagulasepositive isolates, and group 3 included isolates that were CF positive, had delayed coagulase activity, and were deficient in other species-specific features. All investigated strains harbored the clfA, clfB, coa, spa, and nuc genes, but the presence of their products was not detected by the phenotypic methods. Glycopeptide susceptibility testing showed that 26 isolates (23.6%) were hetero-glycopeptide-intermediate S. aureus (hGISA) or hetero-teicoplanin-intermediate S. aureus (hTISA), based on the population analysis profile. The relatedness of the isolates was evaluated by multiple-locus variable number of tandem repeats analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. The phenotypically deficient S. aureus isolates were classified into PFGE types B (ST239-III) and D (ST246-IA) and were related to the common clones, Hungarian and Iberian, respectively, which have been widely disseminated in Poland and globally. The simultaneous occurrence of hGISA/hTISA and the CF-deficient phenotypes was found for 62.1% of isolates belonging to group 2. The majority of these isolates were assigned to the Iberian clone (PFGE type D; ST247-IA). An association between the defect in coagulase and that in thermonuclease production was observed, which concerned 59.2% of isolates of group 1. The majority of these isolates belonged to the Hungarian clone (PFGE type B; ST239-III).
The yet uncharacterized membrane protein SA2056 belongs to the ubiquitous RND (Resistance-Nodulation-cell Division) family of transmembrane efflux transporters. The sa2056 gene is located downstream of femX, the gene encoding the essential, non-ribosomal peptidyl-transferase adding the first glycine in the staphylococcal cell wall pentaglycine interpeptide. Due to its proximity to and weak co-transcription with femX, we assumed that sa2056 may somehow be involved in peptidoglycan synthesis. Specific antibodies against SA2056 showed that this protein is expressed during growth and present in the membrane fraction of cell preparations. Using a bacterial two hybrid system, SA2056 was shown to interact (i) with itself, (ii) with FemB, which adds glycines 4 and 5 to the peptidoglycan interpeptide and (iii) with the essential penicillin binding proteins, PBP1 and PBP2, required for cell division and incorporation of the peptidoglycan into the cell wall. Unexpectedly, deletion of sa2056 led to no phenotype regarding growth, antibiotic resistances or cell morphology; nor did sa2056 deletion in combination with femB inactivation alter β-lactam and lysostaphin sensitivity and resistance, respectively, pointing to possible redundancy in the cell wall synthesis pathway. These results suggest an accessory role of SA2056 in S. aureus peptidoglycan synthesis, broadening the range of biological functions of RND proteins.
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