Genetic Bases of Methicillin Susceptible And Methicillin Resistant Staphylococci

Abstract

One thousand specimens (wound swab, urine, vaginal swab and prostatic fluid) were collected form patients attending Omdurman military hospital during the period February 2004 to October 2004. The code of each specimen, age and the gender of the patient were recorded. All the specimens were cultured on appropriate media and incubated at 37oC overnight. Plates which showed bacterial growth were examined by Gram-stain. All isolates which showed Gram-positive spherical cells arranged in clusters or chains were subjected to subsequent bacteriological tests. The primary selected isolates were examined for catalase, bound coagulase and tube coagulase test. Organisms showing positive results to the above tests were tested for Mannitol fermentation and sub cultured on nutrient agar for further investigations. Production of β .lactamase enzyme in the above isolates was studied. A total of 270 strains were recovered and confirmed to be S.aureus. Using eight antibiotics, Kirby-Bauer technique was adopted as the ideal method to study the antimicrobial activity of the isolates. Depending on the susceptibility testing result, forty MRSA isolates were selected in addition to forty MSSA β - lactamase producer and twenty MSSA non β .lactamase producer were randomly selected for genotyping. Wizard R genomic DNA purification kit was used to isolate the DNA. To ensure that all the strains were S.aureus. PCR was assayed for the amplification of S.aureus specific sequence gene with 30 nucleotide forward and reverse primers and 107 amplicon size. Another PCR program was adopted to isolate mecA gene with 22 nucleotide forward and reverse primers and 532 amplicon size. All of the 270 Saureus isolates showed high degree of sensitivity to vancomycin (100%) and high resistance to penicillin (98.5%). For the other antibiotics the S.aureus isolates showed different degree of sensitivity. Methicillin 85.2%, Meropenem 80%. Amoxicillin /clavulanic acid 74% ciprofloxacin 57% gentamicin 62% and cephalexin 62%. The PCR product electrophoresis revealed a clear band of 107 amplicon size which confirmed that all the isolates were S.aureus. Another PCR electrophoresis product revealed a gene of 532 base pair (mecA) in 36 strains. When the result was compared with the traditional sensitivity method, it confirmed that all the 36 strains were MRSA. They expressed their resistance with both resistance to methicillin and presence of mecA gene. The other four strains of S.aureus showed resistance to methicillin by the classical method but did not express mecA gene on their PCR product and those are classified as border line methicillin resistant S.aureus (BORSA). Five strains from the non β .lactamase producer expressed mecA gene and resistance to methicillin.