Strain characterisation, antibiotic resistance and Meca Gene analysis of Staphylococci

[摘要] S. aureus, is undoubtedly the most pathogenic of the Staphylococcus species,having the ability to produce invasive and toxigenic infections. Historically, theless virulent coagulase-negative staphylococci (ENS) were regarded asclinically insignificant contaminants but they have become increasinglyimplicated as opportunistic nosocomial pathogens. The increasing frequency ofmethicillin and multiple-antibiotic resistance in staphylococci over the last fourdecades has seriously compromised therapeutic options.The study was designed to (a) identify and type staphylococcal species, (b)undertake standardized antimicrobial susceptibility testing, and (c) determine theprevalence of methicillin resistance in staphylococcal isolates.Presumptive staphylococcal strains were isolated from the diagnosticmicrobiology laboratories of Pelonomi (147 strains) and Universitas (144 strains)hospitals. Subsequently, these strains were identified using conventionalbiochemical methods. Species-specific PCR identification assays wereperformed on selected ENS strains. Antimicrobial susceptibilities weredetermined for 13 clinically available antibiotics on 144 staphylococcal isolates and on selected strains for 5 developmental agents. RAPD and plasmid profileswere generated to assess possible epidemiological strain relatedness. For thedetection of methicillin resistance in staphylococci the following methods wereused: (a) oxacillin MICs detecting phenotypic methicillin resistance levels (b) amultiplex-PCR detecting the mecA gene, and (c) a slide agglutination test(MASTALEX-MRSA) detecting PBP2' production.The inclusion of bile-aesculin agar plates and a bacitracin susceptibility test intothe diagnostic laboratory protocol for the identification of staphylococci wouldreduce misidentification of non-staphylococcal isolates by 12.8%. Colonymorphology in combination with the coagulase test could be instrumental in theimproved differentiation of S. aureus from CNS. Although expensive, when arapid and fairly comprehensive identification of CNS species is required, theSTAPH ID 32 API system was found to be satisfactory. Due to the apparentinaccuracy of the PCR identification assay based on API, its use in the clinicalmicrobiology laboratory would be argued against; although if standardized andexpanded it could be considered for future incorporation in routine practice.The presence of unique RAPD profiles for each specific Staphylococcus speciessuggests RAPD profiling could offer a molecular identification technique for themajority of commonly isolated CNS in the clinical microbiology laboratory. Goodtypeability was observed for Primer I and III in CNS strains, however, forS. aureus strains, poor typeability and discrimination was observed. It has beenfound by other researchers that longer oligonucleotide primers (>10 bp in length)are more efficient for S. aureus strain typing, but to the contrary in the presentstudy primers ERIC 1 and 2 were totally unsatisfactory. Combined susceptibilitydata and plasmid profile analysis revealed strain relatedness in S. haemolyticusisolates but RAPD Primers I and III indicated otherwise.All staphylococcal strains isolated were vancomycin-susceptible. Of thestaphylococci isolated in the Universitas hospital, 34.3% were oxacillin-resistant.Similarly, 30.1% staphylococci isolated in Pelinomi hospital were oxacillinresistant.Resistance to ciprofloxacin, erythromycin, gentamicin andclindamycin was found in 49% of oxacillin-resistant staphylococci. Incomparison to the other quinolones tested, moxifloxacin showed superioractivity against oxacillin-resistant CNS. The glycylcyclines, LY333328 and Q/Dmay well be considered excellent alternatives to vancomycin for the treatment ofMRSA. Of the developmental agents investigated, linezolid showed consistentin vitro activity against all staphylococci.The inadequacy of a single diagnostic method for the detection of methicillinresistance in staphylococci was evident when comparing (a) susceptibility data,(b) multiplex-PCR for mecA gene detection, and (c) PBP2' detection. None ofthese methods was seen to correlate with each other at the 100%-level. Thedetection of PBP2' was rapid although, in comparison to mecA gene detectionand antimicrobial susceptibility tests, inaccurate for the identification of methicillin resistance in staphylococci. DNA sequencing of a fragment of themecA gene in selected staphylococcal strains revealed minimal sequencevariation. This was an indication that variable levels of methicillin resistance instaphylococci can be attributed to different mechanisms of methicillin resistanceor variations in the expression of the mecA gene, rather than mutations withinthe gene itself. The low sequence variation observed in the mecA gene isprimarily responsible for initial assumptions of a clonal origin for methicillinresistancein staphylococci. As of yet, pharmaceutical companies have failed toproduce an analogous antimicrobial agent to β-Iactam agents that would be ableto specifically target PBP2'. The development of such an agent would beinstrumental in the reduction of glycopeptide selection pressure. It is imperativethat correct identification, strain typing, susceptibility testing and methicillinresistance detection is performed to direct therapy and epidemiologicallymonitor methicillin-resistant strain types.

[发布机构] University of the Free State