Research On The Resistance Of Intestinal Flora In Mice And Klebsiella Pneumoniae To Fluoroquinolones

Background and purposesIn resent years,Because of fluoroquinolones widly using.Enterobacteriaceaes, represented by Escherichia coli and Klebsiella pneumoniae,have resistant to quinolone severe gradually.therefore,the study on the resistance mechanism of Escherichia coli and Klebsiella pneumoniae to fluoroquinolones are of importance meaning for decreasing and arresting the emergence and development of resistance and for administrating antibacterial agents reasonably.For this purpose,the vitro experiment on Escherichia coli and Klebsiella pneumoniae for the induced resistance to antibacterial agents is carried out.The genes of gyrA and parC in Klebsiella pneumoniae induced to success were amplified by PCR and their part DNA sequences were compared.Experimentation on animal imitate medication methods of clinical to observe the influnce of intestinal flora in mice for regular medication and irregular medication.Methods24 KM mice in this animal experiment were randomly divided into 3 groups, each of 8 mice.The mice of control group received saline and the mice in other 2 experimental groups received gatifloxacin regularly and irregularly respectively for 7 days.Feces samples of the mice were taken by aseptic manipulation and cultured for assessment of amount of enterobacteriaceae,enterococcus,bacteroides, bifidobacteriums and lactobacillus growing in the intestine.At the same time samples of feces of the mice were directed smear to Gram straining methods for checking bacterias.Escherichia coli and Klebsiella pneumoniae were identified to species level correctly according to the method of phenotype ascertainment,119 strains Escherichia coli and 73 strains Klebsiella pneumoniae were detecteded by ESBLs phenotype affirmative test in accordance with drug sensitive tests were detected by Kirby-Bauer test.The susceptibility of ciprofloxacin,levofloxacin and gatifloxacin was measured by determining the MIC using agar dilution method for Escherichia coli and Klebsiella pneumoniae.Then 15 of clinical isolated Escherichia coli and 10 of Klebsiella pneumoniae were choosed as the test strains according to the results of strain identification and MIC determination.These test strains were divided into six groups for inducing the resistance to ciprofloxacin,levofloxacin and gatifloxacin, respectively.Escherichia coli ATCC 25922 and ATCC 700603 was used as control strains.In vitro experiments on some test strains were carried out by multiple-step method.The procedure was as follows.The test strains were inoculated to Macconkey plate,Emergence of new bacterial colony was observed and reported at 35℃after 18～24 hour incubation.Taking 2 or 3 pure colony to make the bacterial suspension of 0.5 McBurney unit,then diluted to 1×10~7 CFU /ml.Taking 10ul bacterial suspension to 2ml MH broth containing 1/4 MIC antibacterial drug.Its ultimate inoculation was 5×10~4CFU/ml,At 35℃incubation 24 hours.if the bacterial can grow,using pure bacterial cultures of 24h to make the bacterial suspension of 1×10~7CFU/ml.Taking 10 ul bacterial suspension to 2ml MH broth containing 1/2MIC antibacterial drug at 35℃incubation 24 hours.As the above procedure,the test strains were cultured in MH broth with the same level of drug up to 128 ul/ml.The induced strains resistant to the antibacterial agents were incubated drug-free MH broth for 5 generations at 35℃,then the resistance to three antibacterial agents were determined by the agar dilution method.The resistant genes including gyrA and parC of Klebsiella pneumoniae were amplified by using PCR,then the nucleotide sequence of these genes was analysed. ResultsGatifloxacin by intragastrical irrigation for 7 day to cause intestinal flora imbalance in mice.Compared to previous medicines,the result of antibiotic susceptibility to Escherichia coil and Enterococcus has no obvious changes. Escherichia coil keeps sensitive to CIP,LEV,GAT,AM,CRO,AMK and NOR. Enterococcus keeps sensitive to AM,LEV,VA and GAT,different sensitivity to NOR,CIP and CRO.Two medications pattern lead to the amount of Escherichia coli decrease notability(P＜0.05) and the amount of enterococcus increase somewhat (P＞0.05).while the amount of bacteroides,bifidobacteriums and lactobacillus have no obvious changes.119 strains Escherichia coli were checked 54 strains ESBLs.Its Positive rate is 45.4 percent.73 strains Klebsiella pneumoniae were checked 36 strains ESBLs.Its Positive rate is 49.3 percent.ESBLs Producers were resistant to multiple antibiotics and their resistance rate was significantly highter than non-ESBLs Producers.There were 10 sensitive strains(MIC≤1μg/ml),2 intermediary resistant strains (MIC=2μg/ml) and 3 resistant strains(MIC≥4μg/ml) of E.coli in ciprofloxaein inducement group.15 strains E.coli were induced 13 strains high drug-resistant to ciprofloxaein(MIC≥128μg/ml).There were 10 sensitive strains(MIC≤2μg/ml),3 intermediary resistant strains(MIC=4μg/ml) and 2 resistant strains (MIC≥8μg/ml) of E.coli in levofloxacin inducement group and gatifloxacin inducement group.15 strains E.coli were induced 8 strains and 5 strains high level drug-resistant(MIC≥128μg/ml) by levofloxacin and gatifloxacin respectively.10 strains Klebsiella pneumoniae in each group were induced 8 high level drug-resistant strains to ciprofloxaein,7 high level drug-resistant strains to levofloxacin and 4 high level drug-resistant strains to gatifloxacin.The MICs of Escherichia coli and Klebsiella pneumoniae induced by quinolone in vitro vary notability(with 16 to 16410-fold )increase than the previous inducement.MIC value increase with 8 to 8205-fold for the other two drugs via to one drug induced successfully.The results verify furtherly the fact that Escherichia coli strains emerged resistant is a step by step resistant process.The mutation of gyrA and parC gene are the primary mechanisms responsing for resistance of Klebsiela pneumortiae to FQS.This research DNA sequence show that 8 strains had mutated in gyrA gene.The 83 amino acid occurs mutation (TCC→ATC/TTC/TAC).Giving rise to corresponding amino acid change(Ser-83→Ile/Phe/Tyr).There were 4 strains appearing the 87 amino acid change at the same time(GAC→GAA/GCC).Giving rise to amino acid from Asp to Glu and Ala.5 strains high level drug-resistant all variant on Ser80 in parC.Simultaneously the 5 strains have mutated in gyrA.The alterations in gyrA exist commonly in the strains of Klebsiella pneumoniae resistant to FQS.The alteration at Ser83(TCC) is common. This explanation that When alteration occurs in gyrA companied by that in parC,the FQS resistance of bacteria tends to intensify.This research only observed that Ser80 (AGC) mutate Ile(ATC),no other mutation was found.Conclusions1.Using of gatifloxacin for 7 day has no deep influnce of intestinal flora with two medications pattern in this animal experiment.gatifloxacin is not easy to come into being drug resistance for short-term.2.Echerichia coli and Klebsiella pneumoniae were performed by multiple-step method,show that acquired resistance could be occurred when exposuring them to low level of some antibacterial agents for long term.3.The mutation of gyrA and parC gene are the primary mechanisms responsing for resistance of Klebsiela pneumortiae to FQS.Alterations in gyrA existed generally in resistant strains,The alteration at Ser83(TCC) is common.When alteration occurs in gyrA companied by that in parC,the FQS resistance of bacteria tends to intensify.