The Role Of IR In The Mtr System On Drug Resistance Of Neisseria Gonorrhoeae

PartⅠSurveillance on susceptibility of six antimicrobial agents of Neisseria gonorrhoeae in WuhanObjective To study the susceptibility of six antimicrobial agents of Neisseria gonorrhoeae(N.G.) in Wuhan, and to collect samples for study in the role of IR in Mtr system on drug resistance of N.G..Methods The 76 clinical isolates from pations with gonorrheas were collected. Agrose-dilution-method was used to test minimal inhibitory concentration(MIC) of antimicrobial agents including penicillin, tetracycline, ciprofloxacin, azithromycin, ceftriaxone and spectinomycin. Drug resistance of N.G. was decided according standards of NCCLS.Results The 76 strains were collected from April to December in 2004. Positive rates ofβ-lactamase test was 7.89%(6). Positive rates of resistance to penicillin, tetracycline, azithromycin, ciprofloxacin were 60.00%(42), 71.42%(50), 37.14%(26) and 54.28% (38) respectively, but clinical isolates of resistance to ceftriaxone or spectinomycin were not tested. Positive rates of across or multiple resistance within penicillin, tetracycline, azithromycin and ciprofloxacin were 80%(56).Conclusions Positive rates of across or multiple resistance increased rapidly, and the resistance to ciprofloxacin also increased rapidly. Clinical isolates of resistance to ceftriaxone or spectinomycin were not tested and not changed compared with that of 1996～1998. Ceftriaxone and spectinomycin should be the first choice for the treatment of gonorrhes at present in Wuhan area. PartⅡThe relationship between mutation of IR in the mtr system of Neisseria gonorrhoeae and multiple antibiotic resistanceObjective To study the relationship between mutation of the 13 inverted repeat sequence(IR) in the cross or multiple transferable resistant system(mtr) of N.G. and its antibiotic resistance. To study the difference of mutation of IR between single and cross or multiple antibiotic resistance groups. To study the difference of mutation of IR between clinical isolates and laboratory induces.Methods The susceptibility of the clinical isolates was tested by agar plate dilution method for minimum inhibitory concentrations(MICs), at the same time the different antibiotic resistant strains were induced with subordinate minimal inhibitory concentration(sMIC) from clinic isolated sensitive strains. The mtr system's IR gene of strains obtained differently was sequenced after amplification by polymerase chain reaction (PCR), and the sequence was compared.Results In clinical strains: all susceptive and penicillin-resistant strains had no base mutation in IR gene, while cross or multiple antibiotic resistant groups had a single-base A/T deletion except one with a single-base A/T insert. In induced strains: single antibiotic resistant groups and only one cross or multiple antibiotic resistant strain had no base deletion in IR gene while cross or multiple antibiotic resistant groups(7) had a single base A/T deletion. There was a single-base A/T deletion or insert in IR gene among induced or clinical cross or multiple antibiotic resistant groups.Conclusion There are no mutation of IR gene in susceptive and single antibiotic resistant strains but there are in cross or multiple antibiotic resistant strains. There was a single-base A/T deletion or insert in IR gene among induced or clinical cross or multiple antibiotic resistant groups. The single-base mutation of the IR gene in mtr system of N.G. might result in cross or multiple antibiotic resistance. PartⅢThe relationship between IR gene mutation and mtrC in the mtr system of Neisseria gonorrhoeaeObjective To study the variability of transcriptional and expressive level in mtrC among different antibiotic resistant groups obtained differently. To study the relationship between mutation in IR and the transcriptional and expressive level in mtrC of mtr system. To explain the mtrR-independent regulation of mtr.Methods The susceptibility of the isolated strains was tested by agar plate dilution method for minimum inhibitory concentrations(MICs), at the same time the different antibiotic resistant strains were induced with subordinate minimal inhibitory concentration(sMIC) from clinic isolated sensitive strains. Transcriptionof mtrC was tested by reverse transcriptional polymerase chain reaction(RT-PCR). Expression of mtrC was tested by western blot. The levels among different antibiotic resistant goups obtained differently were compared according mutation of IR.Results In clinical strains: there was significant variance in transcriptional and expressive level in mtrC between All susceptive and penicillin-resistant strains(P<0.05), which all had no IR gene mutation, and there was no difference among cross or multiple antibiotic resistant groups and the level of cross or multiple antibiotic resistant group (IR-gene-mutation group) was significant higher than that of single group (no IR-gene-mutation group). In induced strains: transcriptional and expressive levels in mtrC of single antibiotic resistant groups was significant higher than that of susceptive groups, but there were no significant variance among single antibiotic resistant groups. Among antibiotic resistant strains, the levels of cross or multiple antibiotic resistant strains (IR-geng-mutation group, 8) was significant higher than that of single groups(no IR-geng-mutation group, 24). There are no difference in levels of cross or multiple antibiotic resistant strains between induces and clinics.Conclusions There is the mtrR-independent regulation of mtr, mtrC gene of N.G. participates the drug resistant mechanism, mutation in IR domain together with other mechanism may enhance transcription and expression of mtrC and further elevate the drug resistance of N.G., and during the drug resistant formation of N.G., firstly mtrR-dependent regulation of mtr may result in low-resistance,and secondarily IR-dependent regulation result in high-resistance.