Structure-function studies of the staphylococcal methicillin resistance antirepressor MecR2.

Methicillin resistance in Staphylococcus aureus is elicited by the MecI-MecR1-MecA axis encoded by the mec locus. Recently, MecR2 was also identified as a regulator of mec through binding of the methicillin repressor, MecI. Here we show that plasmid-encoded full-length MecR2 restores resistance in a sensitive S.

Associations between dru Types and SCCmec cassettes.

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists.

Redefining the role of the β-lactamase locus in methicillin-resistant Staphylococcus aureus: β-lactamase regulators disrupt the MecI-mediated strong repression on mecA and optimize the phenotypic expression of resistance in strains with constitutive mecA expression.

In response to β-lactam chemotherapy, Staphylococcus aureus has acquired two resistance determinants: blaZ, coding for β-lactamase, which confers resistance to penicillins only, and mecA, coding for an extra cell wall cross-linking enzyme with reduced affinity for virtually all other β-lactams. The transcriptional control of both resistance determinants is regulated by homologous repressors (BlaI and MecI, respectively) and sensor inducers (BlaR1 and MecR1, respectively). There is a cross-talk between the two regulatory systems, and it has been demonstrated that bla regulators stabilize the mecA acquisition.

Proteolysis of mecA repressor is essential for expression of methicillin resistance by Staphylococcus aureus.

Recently, we have demonstrated that the cognate regulatory locus of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) is in fact a three-component system containing the novel mecR2 gene coding for an antirepressor. MecR2 interacts with the repressor MecI, disturbing its binding to the mecA promoter and fostering its proteolysis. Here, we engineered a point mutation in the putative cleavage site of MecI and demonstrated that MecI proteolysis is strictly required for the optimal expression of β-lactam resistance.

The anti-repressor MecR2 promotes the proteolysis of the mecA repressor and enables optimal expression of β-lactam resistance in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, which is cross-resistant to virtually all β-lactam antibiotics. MRSA strains are defined by the presence of mecA gene. The transcription of mecA can be regulated by a sensor-inducer (MecR1) and a repressor (MecI), involving a unique series of proteolytic steps.

Methicillin-resistance in Staphylococcus aureus is not affected by the overexpression in trans of the mecA gene repressor: a surprising observation.

Methicillin-resistant Staphylococcus aureus (MRSA) is intrinsically cross-resistant to virtually all β-lactam antibiotics. The central determinant for the MRSA phenotype is the mecA gene, whose transcriptional control may be mediated by a repressor (mecI) and a sensor/inducer (mecR1). The mecI-mecR1-mediated induction of mecA takes several hours rendering the strains phenotypically susceptible in spite of the presence of the resistance gene.

Structural and functional analyses reveal that Staphylococcus aureus antibiotic resistance factor HmrA is a zinc-dependent endopeptidase.

HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or β-lactamase but a nonspecific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface.

Evidence for a purifying selection acting on the β-lactamase locus in epidemic clones of methicillin-resistant Staphylococcus aureus.

Background: The β-lactamase (bla) locus, which confers resistance to penicillins only, may control the transcription of mecA, the central element of methicillin resistance, which is embedded in a polymorphic heterelogous chromosomal cassette (the SCCmec element). In order to assess the eventual correlation between bla allotypes and genetic lineages, SCCmec types and/or β-lactam resistance phenotypes, the allelic variation on the bla locus was evaluated in a representative collection of 54 international epidemic methicillin-resistant Staphylococcus aureus (MRSA) clinical strains and, for comparative purposes, also in 24 diverse methicillin-susceptible S. aureus (MSSA) strains.

Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing.

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype.

Identification of a novel variant of staphylococcal cassette chromosome mec, type II.5, and Its truncated form by insertion of putative conjugative transposon Tn6012.

We identified two novel staphylococcal cassette chromosome mec (SCCmec) elements in sequence type 8 methicillin-resistant Staphylococcus aureus strains isolated in Japan: type II.5 SCCmec, whose J1 region was highly homologous to that of type I.2 SCCmec of strain PL72 (previously isolated in Poland), and its J1 region variant caused by the deletion/insertion of putative conjugative transposon Tn6012, identified in four S.

Epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among patients and healthcare workers in a Portuguese hospital: a pre-intervention study toward the control of MRSA.

This two-year study investigated the epidemiology of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) among patients and healthcare workers (HCWs) in two wards with a high frequency of MRSA isolation, at Hospital Geral de Santo António (HGSA), Portugal. Three point-prevalence surveys per year were carried out. A case-control approach was used to identify potential risk factors associated with MRSA carriage among patients.

Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S.

Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus.

Staphylococcal cassette chromosome mec (SCCmec) typing is important for the identification and definition of methicillin-resistant Staphylococcus aureus clones, and for routine purposes, multiplex PCR assays are the most adequate for SCCmec typing. Here, we describe an update to the multiplex PCR strategy for SCCmec typing that we described in 2002 so that SCCmec types IV and V may be properly identified.

Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: 'SCCmec IV multiplex'.

Objectives: To develop and validate a new multiplex PCR strategy for subtyping SCCmec type IV methicillin-resistant Staphylococcus aureus (MRSA) strains-SCCmec IV multiplex PCR.

Methods: Seven primer pairs were designed to detect the ccrB allotype 2 (internal positive control), the five polymorphic J1 regions described so far for SCCmec type IV and the new J1 region specific for EMRSA-15. Primer sets were tested for specificity and robustness with prototype strains for each subtype of SCCmec type IV.

Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI.

Previously we identified a SCCmec variant similar in size to type IV but with a new ccrAB allotype, allotype 4. We addressed the epidemiological relevance of this variant and found it among several strains belonging to the same clone. We propose to rename this structural variant SCCmec type VI.

Assessment of allelic variation in the ccrAB locus in methicillin-resistant Staphylococcus aureus clones.

Objectives: To evaluate the allelic variability of ccrA and ccrB among methicillin-resistant Staphylococcus aureus (MRSA) strains belonging to different genetic lineages and carrying different SCCmec types.

Methods: Internal fragments from both genes were obtained by PCR amplification with pairs of degenerate primers and the nucleotide sequence was determined for both strands. Nucleotide sequences were aligned and the phylogenetic trees were reconstructed.

Partial excision of the chromosomal cassette containing the methicillin resistance determinant results in methicillin-susceptible Staphylococcus aureus.

We report a detailed characterization of methicillin-susceptible Staphylococcus aureus isolates from five French hospitals negative for both the mecA and the ccrAB loci but positive for the IS431::pUB110::IS431::dcs structure, present in some Staphylococcus cassette chromosome mec (SCCmec) types. The presence of SCCmec-associated elements suggests that this unusual resistant phenotype is due to a partial excision of SCCmec from epidemic methicillin-resistant S. aureus.

Epidemiology of emerging methicillin-resistant Staphylococcus aureus (MRSA) in Denmark: a nationwide study in a country with low prevalence of MRSA infection.

Strict infection control measures introduced during the 1970s have kept the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections extremely low in Denmark. Nevertheless, similarly to other countries, MRSA infections began to appear in the community in the late 1990s. A nationwide surveillance program has collected and stored all MRSA isolates since 1988 and, since 1999, clinical information has been also recorded.

Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus.

Full characterization of methicillin-resistant Staphylococcus aureus (MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous mec element these bacteria carry, which is associated with drug resistance determinant mecA. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the mec element types based on the structural features that were shown to be typical of mec elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated mec elements was previously determined by hybridization and PCR screenings and also by DNA sequencing.

Secrets of success of a human pathogen: molecular evolution of pandemic clones of meticillin-resistant Staphylococcus aureus.

The first European isolate of meticillin-resistant Staphylococcus aureus (MRSA) was detected in 1960. Since then MRSA has become a leading cause of nosocomial infections worldwide. Using molecular typing techniques--primarily pulsed-field gel electrophoresis (PFGE)--we identified five major MRSA clones that accounted for almost 70% of the over 3000 MRSA isolates recovered in hospitals mainly in southern and eastern Europe, South America, and the USA.