| Quinolones and disinfectants play an important role in clinical, but resistant of various kinds of bacteria to quinolones and disinfectants has become increasingly serious, Resistance rates showed an increasing trend of different bacteria in different regions for quinolones and disinfectants. Resistance mechanisms of Escherichia coli to quinolones and quaternary disinfectants has been reported, and there were many reports show cross-resistance mechanisms of bacteria between disinfectants and quinolones, however, the cross-resistance to changes of the molecular mechanisms have not been reported. This study was fond on the different resistant phenotype artificial induction of E. coli different frequency induced by different concentrations Enrofloxacin or Benzalkonium bromide, in order to explore the molecular mechanism of cross-resistance changes and provide the theory basis for rational use of antimicrobial agents and disinfectants the study was carried out.1. The different resistant phenotype strains were obtained by inducting E. coli control stain ATCC25922 by Enrofloxacin or Benzalkonium bromide in vitro Method: The MIC(minimal inhibitory concentration) values for Enrofloxacin or Benzalkonium bromide for E. coli control strain ATCC25922 determined using standard broth micro dilution method. And then E. coli control strain ATCC25922 was induced on some selections steps with different concentrations(1/2×MIC, 1×MIC……128×MIC) Enrofloxacin or Benzalkonium bromide. The MIC values for Enrofloxacin or Benzalkonium bromide for the strains were determined after every concentration every inducting. Result: E. coli control strain ATCC25922 induced by different concentrations 1/2 × MIC, 1 × MIC...... 128 × MIC of enrofloxacin for inducing of 20 generations, has been obtained 5 resistant strains and these 5 resistant strains MIC to enrofloxacin has increased 8, 16, 32, 64, 128 times higher than control strain ATCC25922, mark as: E8, E16, E32, E64, E128. E. coli control strain ATCC25922 induced by different concentrations 1/2 × MIC, 1 × MIC...... 128 × MIC of Benzalkonium bromide for inducing of 50 generations, has been obtained 4 resistant strains and these 4 resistant strains MIC to Benzalkonium bromide has increased 2, 4, 8, 16 times higher than control strain ATCC25922, mark as: B2, B4, B8, B16. Conclusion: Different concentrations of enrofloxacin and benzalkonium bromide induced E. coli strain can obtain different resistance phenotypes strain, resistant strains of enrofloxacin more readily available than resistant strains of benzalkonium bromide, E. coli control strain ATCC25922 induced by benzalkonium bromide cannot obtain high-power resistance strains.2. Cross-resistance analysis of different resistant phenotype strains to enrofloxacin and benzalkonium bromide Method: The MIC values for Enrofloxacin for E. coli B2, B4, B8, B16 and the MIC values for benzalkonium for E8, E16, E32, E64, E128 determined using standard broth micro dilution method. Result: The MIC values for Enrofloxacin for E. coli B2, B4, B8, B16 has increased 4, 8, 16, 32 times higher than control strain ATCC25922, and The MIC values for benzalkonium bromide for E8, E16, E32, E64, E128 has increased 4, 8, 16, 16, 64 times higher than control strain ATCC25922. Conclusion: Resistance phenotype different strains were induced by one drug also showed varying degrees resistance to another drug, and the resistance to another drug is an increasing trend.3. Analysis of resistance change mechanism of E. coli to Enrofloxacin or benzalkonium bromide Method: The mutation of gyr A, gyr B, par C, par E, acr A, acr B, tol C, acr Z of E8, E16, E32, E64, E128 were determined, the detection rate of plasmid-mediated resistance gene qnr A, qnr B, qnr C, qnr D, qnr S, aac-6’-Ib-cr, qep A/B were determined, the m RNA expression quantity of acr A, acr B, tol C, acr Z, emr A/B, mdf A of E8, E16, E32, E64, E128 were determined by using real-time quantitative reverse transcription-PCR method. The mutation of teh A/B, ydg E/F, acr A, acr B, tol C, acr Z of B2, B4, B8, B16 were determined, the m RNA expression quantity of sug E, teh A/B, ydg E/F, acr A, acr B, tol C, acr Z, emr A/B, mdf A were determined by using real-time quantitative reverse transcription-PCR method.Result: The detection rate of plasmid-mediated resistance gene of resistance strains were induced by Enrofloxacin was 0; All the genes gyr A, gyr B, par C, par E, teh A/B, ydg E/F, acr A, acr B, tol C, acr Z except teh A/B, ydg E/F and acr Z occurred mutation, and the amino acid that transcribed by these genes also occurred mutation, and the mutation sites has increased by resistance increase. The expression quantity of sug E, teh A/B, ydg E/F, acr A, acr B, tol C, emr A/B, mdf A were significantly higher than the control strain ATCC25922, and the expression quantity of acr Z significantly reduced than the control strain ATCC25922. Conclusion: The resistance phenotype of resistance strains is closely related to genes mutation and expression quantity.4. Analysis of cross-resistance change molecular mechanism of E. coli to Enrofloxacin and benzalkonium bromide Method: The mutation of acr A, acr B, tol C, acr Z of E16, E32, E64, E128, B2, B4, B8, B16 were determined, and the m RNA expression quantity of acr A, acr B, tol C, acr Z, emr A/B, mdf A of E8, E16, E32, E64, E128, B2, B4, B8, B16 were determined by using real-time quantitative reverse transcription-PCR method. Result: All the genes acr A, acr B, tol C, except acr Z occurred mutation, and the amino acid that transcribed by these genes also occurred mutation, and the mutation sites has increased by resistance increase. The expression quantity of acr A, acr B, tol C, emr A/B, mdf A were significantly higher than the control strain ATCC25922, and the expression quantity of acr Z significantly reduced than the control strain ATCC25922. And in all resistance strains, the expression quantity of acr A was significantly higher than the control strain ATCC25922 between the continuous resistant phenotype strains, but others genes not. Conclusion: The cross-resistance change mechanism of E. coli to Enrofloxacin and benzalkonium bromide is caused by all genes mutation and expression quantity change, but in the determined genes, gene acr A plays a more important role, it prompt that acr A gene may be the main factors by causing cross-resistance Changes mechanism of Escherichia coli to enrofloxacin and benzalkonium bromide. |
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