High-level Methicillin-resistant Staphylococcus Aureus Research Articles

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to β-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

The role of femA regulating gene on methicillin-resistant Staphylococcus aureus clinical isolates

ObjectiveThe authors investigated the role of femA regulating gene on methicillin-resistant Staphylococcus aureus (MRSA). MethodsHigh-level MRSA, low-level MRSA, and methicillin-susceptible Staphylococcus aureus (MSSA) were determined by agar diffusion methods. β-lactamase was then detected by nitrocefin and the presence of mecA was determined by PCR. Only β-lactamase-negative but mecA-positive isolates were included in further studies. The femA gene and its 250bp upstream sequence were amplified by PCR. Expression levels of femA were determined by real-time fluorescent quantitative PCR. The 250bp upstream sequence of femA was labeled by BrightStar Psoralen-Biotin and was detected by electrophoretic mobility shift assay (EMSA). ResultsThe expression levels of femA in the three different groups (MSSA, low-level MRSA, and high-level MRSA) ranged from 3.53×10–3% to 29.91%, 5.54×10–3% to 3.1×102%, and 13.88% to 5.50×104%, respectively. EMSA could detect the signal shift in 55 high-level MRSA isolates but not in four low-level MRSA and four MSSA strains. ConclusionThe expression levels of femA in high-level MRSA (non-β-lactamase-producing) were higher than in low-level MRSA and MSSA. The femA regulating gene probably lies in the 250bp upstream sequence in MRSA. High-level expression of femA seems to be essential for high-level MRSA.

Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC </=2 mg/L) are increasingly reported. To gain molecular and functional insights on this observation, we focused on additional factors possibly contributing to phenotypic susceptibility. Methods The nucleotide content of mecA, femA, femB and femX genes, which are considered essential for methicillin resistance, was determined in four OS-MRSA clinical isolates and a genetically similar low-level MRSA control (oxacillin MIC 6 mg/L). Gene expression was quantified compared with the low- and a high-level MRSA (MIC 256 mg/L) control. The tertiary structure of Fem proteins was predicted based on protein structure homology modelling, using web-based automated comparative protein modelling. Growth kinetics were tested for the study and control isolates, to determine whether FemXAB mutations lead to reduced fitness. Results Genes mecA, femA, femB and femX were expressed similarly in the study and the control isolates. Mutations in the gene mecA were not present in any isolate. However, several mutations leading to amino acid substitutions in positions possibly affecting Fem enzyme activity were detected in all fem genes. Two OS-MRSA that had no oxacillin heteroresistance had more mutations in the Fem proteins compared with the remaining isolates that were heteroresistant. The low-level MRSA control had considerably fewer mutations. No differences between growth rates of the OS-MRSA and the MRSA controls were observed. Conclusions Accumulation of amino acid changes in Fem proteins might affect intact cell wall synthesis, even though not causing reduced viability, thus contributing to atypical oxacillin responsiveness.

A Study of the Regulating Gene of femA from Methicillin-resistant Staphylococcus aureus Clinical Isolates

The regulating gene of femA was studied in methicillin-resistant Staphylococcus aureus (MRSA). High-level MRSA, low-level MRSA and methicillin-sensitive S. aureus (MSSA) were identified by agar diffusion. Beta-lactamases were detected by nitrocephin and the presence of the mecA gene was determined by polymerase chain reaction (PCR). Only isolates that were both beta-lactamase-negative and mecA-positive were used. The femA gene and its 250 base pair (bp) upstream sequence were amplified by PCR and expression was determined by real-time fluorescent quantitative PCR. The 250 bp upstream sequence was labelled by BrightStar Psoralen-Biotin and detected by electrophoretic mobility shift assay (EMSA). Expression levels of femA in MSSA, low-level MRSA and high-level MRSA were 3.53 x 10(-3)% - 29.91%, 5.54 x 10(-3)% - 3.1 x 10(2)% and 13.88 - 5.50 x 10(4)%, respectively. EMSA detected a signal shift in 57 high-level MRSA isolates but not in four low-level MRSA and four MSSA strains. Expression of femA in high-level MRSA (non-beta-lactamase-producing) was higher than in low-level MRSA and MSSA. The femA regulating gene probably lies in the 250 bp upstream sequence in MRSA and high-level expression is essential for high-level methicillin resistance.

Efficacy of an alcohol/chlorhexidine hand hygiene program in a hospital with high rates of nosocomial methicillin‐resistant Staphylococcus aureus (MRSA) infection

To assess the effect of a multifaceted hand hygiene culture-change program on health care worker behaviour, and to reduce the burden of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Timetabled introduction of interventions (alcohol/chlorhexidine hand hygiene solution [ACHRS], improved cleaning of shared ward equipment, targeted patient decolonisation, comprehensive "culture change" package) to five clinical areas of a large university teaching hospital that had high levels of MRSA. Health care worker hand hygiene compliance; volume of ACHRS used; prevalence of patient and health care worker MRSA colonisation; environmental MRSA contamination; rates of clinical MRSA infection; and rates of laboratory detection of ESBL-producing Escherichia coli and Klebsiella spp. In study wards, health care worker hand hygiene compliance improved from a pre-intervention mean of 21% (95% CI, 20.3%-22.9%) to 42% (95% CI, 40.2%-43.8%) 12 months post-intervention (P < 0.001). ACHRS use increased from 5.7 to 28.6 L/1000 bed-days. No change was observed in patient MRSA colonisation or environmental colonisation/contamination, and, except in the intensive care unit, colonisation of health care workers was unchanged. Thirty-six months post-intervention, there had been significant reductions in hospital-wide rates of total clinical MRSA isolates (40% reduction; P < 0.001), patient-episodes of MRSA bacteraemia (57% reduction; P = 0.01), and clinical isolates of ESBL-producing E. coli and Klebsiella spp (90% reduction; P < 0.001). Introduction of ACHRS and a detailed culture-change program was effective in improving hand hygiene compliance and reducing nosocomial MRSA infections, despite high-level MRSA endemicity.