| ObjectiveShigellosis was investigated among outpatients with diarrhea in Wuhan, serotype distribution and drug resistance of Shigellae was analyzed, the mechanism of fluroquinolones-resistant was studied and a genotyping method for Shigella spp. was set up. The aim of all above was to provide update data for antimicrobial resistant monitoring, clinical reasonable usage of antimicrobial agents, new drug or vaccine developing and epidemiology.MethodsStains were isolated from Stool samples of outpatients with diarrhea from four big hospitals in Wuhan from June to November in 2004 and the serotype was identificated. The minimum inhibition concentration to 12 kinds of antimicrobial agents was tested by agar dilution method and disc diffusion test was used to study the antimicrobial resistance of another 7 kinds of antimicrobial agents. WHONET 5.3 and SPSS 11.5 were applied to analysis the results and part of the results were compared with the data of 2003. Genes of gyrA and parC were amplified, sequenced and found mutation site. Repetitive extragenic palindromic-polymerase chain reaction was used for genotyping. Results29 strains of shigellae were isolated from 5079 samples from June to November in 2004 and the isolate rate was 0.57%. 12 stains were in group B (41%) and 17 stains in group D (59%). The prevalent serotypes were 1a(2/12, 16.7%),2a(4/12, 33.3%) and 4c (6/12, 50%) in group B .The results of resistance to 19 antimicrobial agents revealed that trimoxazole had the highest resistant rate which was 100%. 96.6%~72.4% of strains were resistant to nalidixic acid, tetracycline, spectinomycin and ampicillin, while 41.4% and 31% to chloramphenicol and amoxicillin-clavulanic acid. The resistant rates of gentamicin, ceftriaxone and cefetaxime were from 17.2% to 10.3%. The resistant rates of kanamycin, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin and nitrofurantoin were less than 10%. No trains were found to resistant to ceftazidime, cefepime and aztreonam.There are 20 kinds of resistance patterns in 29 isolates of Shigellae. No isolate was sensitive to all the antibiotics, but with co-resistance to 2 kinds at least and 12 at the most. Each serogroup has its superiority resistance pattern. The dominant pattern(50%,6/12) of B group was co-resistant to 6 antibiotics include ampicillin, spectinomycin, chloramphenicol, tetracycline, trimoxazole, nalidixic acid(ASHTRD). For group D(12.5%, 2/16), there was two main resistance patterns, one was 4 co-resistant to spectinomycin ,tetracycline,trimoxazole and nalidixic acid(STRD), the other was 9 co-resistant to ampicillin, ceftriaxone, spectinomycin, chloramphenicol , tetracycline, trimoxazole, nalidixic acid, amoxicillin-clavulanicacid and cefetaxime, both had 2 strains.Compared with the data of 2003, the isolate rate was higher (0.13%); the proportion of group D surpassed that of group B. The resistant rate to aztreonam decreased 16.7% and that to trimoxazole increased 20.8%, both of them had significant differences.The MIC of norfloxacin and ciprofloxacin for the two fluroquinolones -resistant strains numbering 3222 and 4194 were 16μg/ml and 4μg/ml.The MIC of levofloxacin and moxifloxacin for 3222 were 8μg/ml,while that for 4194 were 4μg/ml.Target gene mutation detection revealed that 3222 and 4194 had Asp(GAC)87→Asn(AAC) point mutation in quinolone resistance-determing region and Gly(GGT)177→Ser(AGT) point mutation out of quinolone resistance-determing region in gyrA gene. Out of QRDR, 4194 also have Met(ATG)120→Leu(CTG),Ile(ATC)203→Leu(CTC), Ser(AGC)204→Thr(ACC) and Ile(ATT)205→Leu(CTT) mutations,3222 also have the mutation of Ser(AGC)204→Ile(AAC). No point mutation had been found in parC gene.The results of Repetitive extragenic palindromic-polymerase chain reaction cued that 16 shigella sonnei strains had 7 fingerprinting patterns ,named A~G. Pattern A had 4 isoforms ,which were A1,A2,A3 and A4;All of the Isoforms had 1 strain except A1(3 stains) .Pattern B's isoforms were B1 and B2, each of them had 1 strains. Both C1 and C2 isoforms for Pattern C had 2 strains. Pattern D,E,F and G each had 1 stain.12 shigella flexneri had 2 patterns,named H and K. H1,H2 and H3 were 3 isoforms for pattern H,each had 1 stain.Pattern K had 4 isoforms ,which were K1(2 strains),K2(4 strains ),K3(2 strains),K4(1 strains)。Some pattens of isolated from sporadic cases were same or similar. Isolates with the same gene type had various drug resistance patterns. Various gene types could be within one kind of drug resistance pattern.ConclusionsThe infectious rate of Shigella spp. among outpatients with diarrhea in Wuhan in 2004 was a little higher than that of 2003.The count of group D strains was more than that of group B .The prevalent serotype in group B was f4c.Multiply antibiotic resistance is widespread and resistance patterns were complicated in Shigella spp. isolated in 2004. There was no significant change for resistance of Shigella spp. except aztreonam and trimoxazole. Among common used drug, trimoxazole.and ampicillin had the higher resistant rates, while fluroquinolones had the lower rate. Fluroquinolones could be chosed as empirical usage of antimicrobial agents for adult with emergency conditions, while for children the third or the fourth generation cephalosporins or aztreonam could be chosen. Fluroquinolones-resistance of the two strains has relationship with the 87 point mutation of gyrA gene.Sporadic isolates prevalent in Wuhan during summer and autumn in 2004 have same or similar clones.Shigella sonnei with single serotype presented gene diversity and serotype 2a shows more gene diversity than 4c among shigella flexneri.
|
PDF Full Text Request