RpoB Gene Mutation And Molecular Epidemiology Of Rifampicin-resistant Staphylococcus Aureus

Objective i)To explore the charcteristic of rpoB gene mutations inrifampicin-risistant Staphylococcus aureus and analyze the correlation between the level ofrifampicin resistance and the types of the mutations in rpoB gene. RAPD method and Spamethod was used to explore the molecular epidemiologic characters ofrifampicin-resistant Staphylococcus aureus in our area. ii)To study the influence ofCIP on frequencies of rifampin-resistant mutation and the rpoB gene mutation ofpartial mutational strains. Methords88non-duplicated Staphylococcus aureusstrains collected from Anhui province resistance monitoring center between Jan2009to May2010were analyzwed for antimicrobial susceptibility with16kinds of drugs. Asequence of432bp (nucleotides1216to1648) was amplified by PCR. This regionincludes the intact area. Analysis the most common resistance-associated mutationssite and the analysis the correlation between the level of rifampicin resistance and thetype and local of the mutations. Random amplified polymorphic DNA(RAPD) andStaphylococcus aureus protein A gene variable repeat region typing (Spa) were used togenotype them. Detecting the change of frequencies of rifampin-resistant mutantsin the presence of1/2,1/4or1/8MIC ciprofloxacin. Partical mutants wereanalyzed for the rifampicin MICs and its rpoB gene mutations. Results Thetotal88isolates were MRSA and resistant to Betalactam antibiotic, gentamicin,erythrocin, tetracycline, ciprofloxacin and levofloxacin, the resistant rate ofmoxifloxacin, clindamycin, SMZ/TMP, furantoin and quinupristin/dalfopristin were98.8%,88.6%,9.1%,3.4%and2.3%, respectively, the isolates were still sensitive tovancomycin, linezolid and Tigecycline in100%.83(94.3%) isolates were resistant torifampicin for high-level while the rest5(5.7%) were low-level resistant to rifampicin.Four nonsynonymous mutations were found in the total88Staphylococcus aureus,which were respectively466Leu/Ser（87.5%）,477Ala/Asp（6.8%）,481His/Asn （95.5%） and486Leu/Ser（4.5%）. All of the mutations present in the clusterⅠ.Thelow-level resistant one have only one nonsynonymous mutations on it while thehigh-level resistant one have two or more nonsynonymous mutations, the mostcommon pattern is481His/Asn in combination with466Leu/Ser(92.8%,77/83) and therest patterns were481His/Asn in combination with477Ala/Asp(6.0%，5/83) and481His/Asn in combination with466Leu/Ser＋477Ala/Asp（1.2%，1/83）. RAPDdivided the strains into four different types(A,B,Cand D),and the main types was D.And Spa type divided the strains into nine different types, among them t030is thesuperiority type which have37isolates while the last types has only one isolatesrespectively. Growth of rifampicin-susceptible strains in the presence of1/8,1/4or1/2MIC of ciprofloxacin led to higher frequencies of rifampin-resistant mutants onrifampin supplemented agar, compared to ciprofloxacin-free conditions. The rpoBgene mutations in rifampin-resistant mutants from ciprofloxacin-exposed cultures weredissimilar to the clinical isolates. Conclusion Spa t030clone featuring rifampicinhigh-level resistance is spreading in local hospital which associated wih rpoB genemutation in481in combination with466. Exposed in sub-MICs of ciprofloxacinespecially higher than1/4MIC of ciprofloxacin led to higher frequencies ofrifampin-resistant mutants on rifampin (1mg/L)-supplemented agar. But the types ofmutation are different from clinical isolates. The notable rising rate of rifampicinresistance could be attributable to the spread of minority resistance stain.