Resistant Analysis And Fluoroquinolone-Resistant Mutations Of Staphylococcus Aureus From Animal

Drug sensitive test is done about S.aureus from six region of JinLin to 20 antibiotic to use K-B way contrast at ATCC 25923 norm S.aureus.201 S.aureus have different resistance to antibiotic.The resistant rate is highest to Penicillin, Ampicillin,Norfloxacin,Cidoniycin,Aficentin and Kyonamycin.It's 61.1%, 55.7%, 82.6%,93.5%,55.7% and 93.5%.It's morderate to Abomacetin, Econmycin, Ciprofloxacin,Enoxacin,Chemitrim,Chamamyn and resistant rate is 22.3%-38.6%. It's more sensitive to Ofloxacin, Furazoliclone, Amika cin,Clindamycin,Rifadin and resistance rate is 0.5%-7.5%.It's most sensitive to Brizolina,Cefetaxime.The resistance of S.aureus have mutiplex character.The study on fluoroquinolone-resistant mutations of the gyrA and grlA gene of staphylococcus.aurcus was carried out.Genomic chromosome DNA were abstracted from five isolated and three manual-induced S.aureus that were high- or low-level resistant or susceptible to fluoroquinolone.Fragment of gyrA and grlA including the quinolone-resistant determined region(QRDR) were amplified by PCR and cloned,sequenced.The sequencing results were analysted with software WDNASIS.The minimum inhibitory concentration(MIC) of S.aureus to fluoroquin olone wre determined with tuber twice dilution for all isolates.The range of MIC to ciprofloxcin,norflonxcin,enrofloxacin and ofloxcinum is 4-128 ug/ml.This result showed that five isolated S.aureus were resistant to fluoroquinolone.At the same time,stable,high- and low-level enrofloxcin(EN) resistant of S.aureus (28001c, 28001e,28001g) were obtained with serial passage on agar containing subinhibitory concentration of indicated EN.The MIC of three strains to EN is 4 ug/ml,16 ug/ml and 128ug/ml.The range of MIC of three strains to ciprofloxacin, norflonxcin and ofloxcinum is 4-> 128ug/ml,64- > 128ug/ml and 2-128ug/ml respectively.The result showed that the manual-induced S.aureus exist cross-resistance to ciprofloxacin, norflonxcin,enrofloxacin and ofloxcinum.Chromosomal DNA was prepared according to molecular cloning:A laboratory Manual. Amplification of gyrA and grlA were achieved by PCR with different primers designed from sequence in the flanking region of theQRDR of S.aureus.The resulted DNA fragment with expected size was cloned into vector pMD 18-T and subsequently subjected to restriction enzyme analysis and PCR reaction and sequencing.The results showed that all sequence have over 90% homogenous to published data,the fragment amplified of grlA gene of 28001 was 618bp comprising QRDR and 99.4%homgenous with replacement of 4 nucleotides not resulting in amino acid changed,and the majority of replacement were common mutation from C to T.The fragment of gyrA was 618bp comprising QRDR and 100% homogenous too.All resistant strains contained a single mutation in grlA at position 80.Two isolated and two manual-induced strains have a single Ser80 to Tyr(Phe),not any mutation in gyrA.Other three isolated and one manual-induced strains contained a mutation not only in grlA Ser80 to Tyr(Phe),but also in gyrA Ser84 to Leu(Ala) or Glu88 to Lys.All these results showed that maybe the mutation in grlA ser80 and in gyrA at ser84 or Glu88 were the mechanisms of resistance to fluoroquinnolone of S.awrews.When strains of isolated and manual-induced S.aureus were low-level resistance.there was a single mutation in gr/A,but high-level fluoroquinolone-resistant S.aureus required amutation not only in grlA but also in gyrA. All resistant mutation have grlA Ser80 - Tyr(Phe) point mutation. This suggest that the primary target enzyase to Staphylococcus aureus of Fluoroquinolone (ciprofloxa- cin,norfloxcin, enrofloxacin and ofloxcinum) is topoisomerase IV.