Low-level Methicillin-resistant Staphylococcus Aureus Research Articles

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.

Détection simple des staphylocoques résistants à la méticilline grâce à un disque de céfoxitine ou de latamoxef

Very-low level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We aimed at detecting the low-level MRSA by the disk diffusion method on a NaCl-free Mueller-Hinton agar plate flooded with 10 6 UFC/ml bacterial inoculum using the cephamycin antibiotics cefoxitin and latamoxef for 18 hours' incubation at 37°C. Detection of the mecA gene by PCR was the ⪡gold standard⪢. The cefoxitin and latamoxef disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm and latamoxef inhibition zone diameters <24 mm. Consequently, we evaluated the cefoxitin and latamoxef disk diffusion method to detect very low-level coagulase-negative staphylococci (MRCNS) or class 1 MRCNS. The cefoxitin and latamoxef disk diffusion detected 94% and 97% of all MRCNS respectively. Cefoxitin inhibition diameters were <27 mm and latamoxef diameters <22 mm. We studied the Slidex MRSA detection® (bioMérieux) to test the PLP2a in all S. aureus and coagulase negative staphylococci included in our trials with cephamycins. This test detected 98% MRSA, and 98% MRCNS so long as the CNS sample was grown on the edge of an oxacillin or a cephamycin disk. Therefore, we recommend the use of latamoxef disk diffusion method for routine detection of all classes of MRSA and MRCNS, and the subsequent PLP2a detection in cases of inhibition zone diameters in the range of 22–24 mm. Taken together, both tests allow one to detect all class 1 methicillin-resistant staphylococci within 24 hours.