| Shigella called as dysenteriae bacillary is one of the main pathogens of infectious diarrhea. The bacillary dysentery shigellosis, which is caused by Shigella, is a kind of acute intestines contagion. It is estimated that shigellosis causes around 600,000 deaths per year in the world, and two-thirds of the deceased are children under 10 years of age. In developing countries, the bacillary dysentery is difficult to prevent due to the complex of the bacterial type, the easy variance, without the crossover immunity in Shigella. Since antimicrobial agents are widely employed to treat shigellosis, antibacterial resistance in shigella spp. has been rapidly emerged, frequently appeared and is multiple-antibiotic-resistance, which bring great hard to the clinical management. Therefore, it is desirable to study the mechanism of antibacterial resistance of shigella spp.. Quinolones are an important kind of antibiotic to treat shigellosis in present, which were used in clinic from 1980' s. and shigella spp. were very suscept to them at first with the susceptable rate above 95%. So shigellosis had been under control for some time. But for resent years, the susceptable rate has fallen below 85%.Due to the importance of shigellosis and the common use of quinolones for treatmeat of bacterial infections, to study the antimicrobial susceptibility patterns of Shigella spp. with the particular reference of the quinolones and its possible mechanism is obviously necessary. Recent studies showed that the mechanisms of bacterial resistance to quinolones related with chromosome included target genes mutation, including gyrA and parC. membrane permeation decrease and active efflux pumps. It is believed that the target gene mutations play an important role in the mechanisms of bacterial quinolone resistance, and the other two are the main mechanisms of multiple-antibiotic-resistant including quinolones. Although the study on the antimicrobial resistance mechanism of quinolones in Shigella spp. includes the clinical isolates and the induced strains, and they deal with all the three mechanisms by now, but the relationships between them and each status in the form of Shigella spp. resistance need to be explored deeply.In order to know each quinolones resistance mechanism's status, the characteristics of their function, and the relationships between them in the form of Shigella spp., this study was planned to based on the theory of pressure of drug causing bacteria resistance, in the way of manual inducement, passage a susceptible strain of Shigella spp. onto norfloxacin -containing culture medium to obtain resistant mutants step by step in the laboratory. We measured the minimum inhibition concentration (MIC) of four kinds of antibiotics to each generation of induced strain to check the degree of quinolones resistance and multiple-antibiotic-resistance. And we amplified gyrA and parC gene of the stains of every generation by polymerase chain reaction (PCR) and applied single strand conformational polymorphism analysis (SSCP) to all PCR products to detect mutation strains. Then DNA sequencing analysis was adopted to them to define site and derection of the mutation of the target gene to probe into the relationship between antibiotic-resistance degree and the mutation of target gene. This study was amed to supervise clinicians to find resistant trains in forepart and apply quinolones reasonably and effectively in order to avoid the quinolones resistance increasing. And at the same time it would offer gist for the exploiture of new quinolones medicines. And we observed the MIC changes of Norfloxacin to the strains with CCCP choking back the active pump function in order to probe into the effect of active pump system in multiple-antibiotic-resistance of Shigella spp. and the relationship between it and the mutation of target genes in the form of Shigella spp. quinolones resistance to quest for theoretical argument and countermeasure for the prevention and treatment of bacillary dysentery shigellosis .Methods1. Resuscitating and identifying of the bacteria strains: There are 48 Shigella spp. strains saved in semi-solid agar in 4℃refrigeratory of our staff room. We inoculated them into LB solid culture medium and identified them with biochemical and serological methods over again to make sure the reliability of the strains.2. Filtrateing the wide type strains: Susceptibility tests (Kirby-Bauer) of 7 antimicrobial agents were performed. Four quinolones were Nalidixic Acid(NAL), Ciprofloxacin(CIP), Ofloxacin(OFL), and Norfloxacin(NOR). Other three antimicrobial agents were Teracycline(TE), Chloramphenicol(C), Ampicillin(Am). The strains appearing susceptible to all above were called as wild type (WT). Selecting one strain to induce.3. The method of inducement: The concentration of Norfloxacin started from 1/4×MIC0, which were the MIC of original generation of induced strain, increasing by double, and the strain was induced in every concentration for two generations. When the concentration of Norfloxacin reached 1μg/ml, the concentration increased by 1μg/ml 256×MIC0. When the comcentration of the revulsant reached 4, 8, 16, 32μg/ml, after the strain had grown in this concentration for two days, they were not continued to induce momentarily. They were cultivated in culture medium without Norfloxacin for four times, then they were inoculated into LB solid culture medium with the same concentration's revulsant for one night. The next day we selected better clusters to continue inducing and cultivating, so that we could get the steady resistant strains.4. Mensurating the minimum inhibition concentration (MIC): we tested the MICs of NOR, TE, C and Am to every generation of induced strain by the agar doubling dilution method.5. PCR-SSCP of the target genes of quinolones, gyrA and parC: The two genes were amplified by PCR, the products of which were digested with restriction endonuclease Hinf I . Then SSCP analysis was performed. This can detect a single nucleotide change as well as the loss or insertion of oligonucleotide by electrophoresis.6. DNA sequence analysis of PCR products of several mutant strains were performed.7. The inhibiting detecting of CCCP, a kind of energy inhibitor, to the active efflux function of multiple-antibiotic-resistant Shigella spp. induced in laboratory: CCCP(10μg/ml) was added into M-H culture medium when antimicrobial susceptibilities of induced strains were tested by the agar doubling dilution method. We observed the changes of MICs of Norfloxacin to every generation of induced strain after the energy inhibitor was added.Results1. Resuscitating and identifying of the bacteria strains: A total of 48 clinical isolates were resuscitated. 11 strains of Shigella flexneri were isolated in Tonggu, Jiangxi. 13 strains of Shigella flexneri, 8 strains of Shigella sonnei, and 3 strains of Shigella dysenteriae were isolated in Zhengzhou, Henan. 12 strains of Shigella flexneri, 1 strain of Shigella sonnei were isolated in Sui county. Shangqiu.2. Filtrateing the wide type strains: 3 wide type strains. N8, Z10, Z11, were filtrated. It is 6.25% of the total. N8 was selected as the induced strain.3. The result of the inducement: The strain had been induced for 74 generations, and the ultima concentration of Norfloxacin was 32μg/ml. The MIC of Norfloxacin to the induced strain mounted up to 128μg/ml from 0.125μg/ml, 1024 times of the original. At the same time, the MIC of TE, C and Am went up to 128, 64 and 32 times of the original. The induced strain changed to multiple-antibiotic-resistant strain from susceptive one step by step. 4. PCR of gyrA and parC: Every generation of N8 induced by Norfloxacin had all been amplified two segments of 649bp and 469bp, which were the products of gyrA and parC.5. PCR-SSCP analysis detected gyrA and parC gene: The mutant of gyrA gene emergented from the 12th and 55th generation, and the mutant of parC gene emergented from the 21st and 52nd generation.6. DNA sequence analysis of PCR products of gyrA and parC gene: The sites of mutation in the. gyrA gene was the 87th and 221st codon, and the sites of mutation in parC gene was the 80th and 84th codon.7. The influence of CCCP on MICs: After the CCCP was added, the MICs of susceptive strains and resistant ones both reduced to different degrees.Conclusions1. Selectly pressure of drug causes bacteria resistance.2. GyrA and ParC might be primary and secondary target, respectively, of the quinolones. The more mutations in the target gene might lead to the higher level of quinolone-resistance. The mutation of target gene in quinolone-resistant strains may be polymorphic.3. The multi-drug resistance induced in vitro should be mediated by the active efflux system. AcrAB included. The active efflux mechanism together with the mutation of the target gene leads shigella spp. resistant to quinolones.
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