Analysis Of Drug Resistance Of Shigella Flexneri And Study On Ralation Between Mutant In GyrA,parC And Quinolone Resistance

Analysis of drug resistance of Shigella flexneri and study on relationbetween mutant in gyrA~ parC and quinolone resistanceAbstractShigella is one of most important pathogens to infectious diarrhea ,In pathogen pattern to infectious diarrhea, Shigella ranks the third ,next to rotavirus and ETEC is the first.ln 1950s resistant Shigelia was found and was believed to be related to resistance plasmid.At present, Shigella is resistance not only to a kind of antibiotic,but also to many kinds of antibiotic. Shigella resistance forming and spreadding lead more difficulties to cure severity bacillary dysentery.Analysising of resistance of 112 Shigella flexneri,we knew the rate of resistance is 44.6% to 21 kinds of antibacterial.The majority of them is multi-antibiotic-resistance.Even some is resistance to 14 antibiotics. 33.9% Shigella flexneri are resistance to norfloxacin.Quinolones is main drug to treat bacillary dysentery. Quinolone affect topoisomerase II and IV,interfere with replication,transcription and repair.In the last 30 years,because quinolones was widely used,quinolone resistance already formed step by step,and it was reported by all countries in world.Recent studies showed that the mechanisms of bacterial resistance to quinoiones include chromosomal mutants ,reduce membrane permeation and action efflux pumps.In early years when Escherichia coil quinoione resistance was studied,and it was found quinolone resistance mutants in gyrA of Escherichia coil were clustered within a small region of the 5?end between nucieotides 1 99(Ala-67) and 318(Gln-106) which is known as the quinolone resistance-determining region(QRDR).In this study,two pairs primer were designed,and QRDR of gyrA(294bp) and parC(365bp) in 112 Shigella flexneri were ampliified respectively by polymerase chain reaction(PCR).Single-Strand Conformation Polymorphism-3.(SSCP) analysis was applied to all PCR products,and showed mutants in gyrAoflh J3~. J13~ Hl6~ H2O~ H25~ SH1~ N3andinparCofJl3.Then DNA sequence revealed the serine at positive 83 was changed to leucine in gyrA of 8 mutant strains,in addition of J1 3 the aspcine at positive 87 was changed to gly in gyrA and the serine at 58 positive was changed to isoleucine in parC.We found tolerence level of mutant strains to quinolone was higher than that of normal strains,and toierence level of J 13 to quinolone was the highest in 112 Shigella flexneri.Jt showed there was dose-reaction relation between point mutant and tolerence level.In addition, mutant of Ser-83 to Leu existed in gyrA of all mutant strains,showed mutant in gyrA was main reason for Shigella flexneri resistance to quinolone.To fixed point mutant, mismatch primer was designed and Shield flexneri were detected by mismatch PCR.In this study,by inducing Shigella flexneri to quinolone resistance step by step,and DNA sequence,revealed a C-to-T trasition at nucleotide position 248 leading to a Ser-83-to 桳eu in gyrA of frail strain when Sbigclia flexneri of experiment group MIC is 16 ii g/nil ,and control strain was no any change;There were a C-to-T trasition at nucleotide position 248 leading to a Ser-83-to 桳eu and GCG (Ala) -84梩o-TTT (Phe) arid a A-to-C trasition at nucleotide position 260 leading to a Asp-87-to桮ly in gyrA ,a silent mutant at nucleotide position 207 and a C-to-C trasition at nucleotide position 173 leading to a Ser-5 8-to條ie in parC of trail strain when Shigelia flexneri of experiment group MIC is 32 LL g/ml ,and control strain was no any change.Therefore,it proved that seiectly pressure of drug caused bacteria resistance;on the other hand ,it suggested the mutant of gyrA was main reason of Shigella flexneri quinolone resistance.