| Shigellosis is one of the most common diarrheal diseases in humans worldwide.Approximately 125 million cases of Shigella infections occur annually in Asia,of which 14,000 are fatal.In China,shigellosis is one of the top four notifiable infectious diseases,with 1.7 million episodes of bacillary dysentery,and 200,000 patients admitted to hospitals each year.Based on the structure of O antigen,Shigella includes 49 serotypes and subserotype,which can be divided into four species: Shigella boydii,Shigella.dysenteriae,Shigella flexneri,and Shigella sonni.Among them,S.sonnei is the most prevalent species in developed countries,whilst S.flexneri is most reported in developing countries.However,the distribution of Shigella spp.is changing in recent years.S.sonnei has become dominant in some Asian countries.Shigellosis is often treated with antibiotics to reduce excretion and to shorten the duration of illness.However,the progressive increase in antimicrobial resistance,especially multidrug resistance among Shigella due to the antimicrobial overuse,is becoming a critical public health problem.Because increased mobility of the population appreciates the propagation of Shigella spp.,distribution and drug resistance of Shigella spp.have changed largely.To control the prevalence of antimicrobial-resistant Shigella,it is crucial to further study the mechanism of multidrug resistance and to how to reduce the drug resistance.The objective of the research is to test biochemical characters,antibiotics sensitivity and serotype identification of 382 Shigella spp.isolated from clinic cases in 2010-2014.Through the study,we want to analyze the antibiotic resistant patterns and drug-resistance spectrum change of Shigella spp.A total of l39 strains of Shigella flexneri,meanwhile,was analysed on the resistance-related genes with decreased susceptibility to fluoroquinolones.These strains were further analyzed with the methods of virulence gene,genetic features,gene variety.The followings are the findings in detail:Firstly,serological and biochemical identification were adopted on the 382 strains isolated from clinical surveillances.The biochemical test was conducted with API 20 E biochemical assay kit(API Inc.,France),and serological identification was performed using slide agglutination with polyvalent somatic(O)antigen grouping sera,followed by testing with monovalent antisera(Denka Seiken,Japan)for specific serotype determination.The results showed that a total of 243 strains is shigella sonni and of 139 strains is shigella flexneri,containing 9 serotypes of F1a、F2a、F2b、F3a、F4a、F4c、F6、Fx and Fy.So the distribution of pathogen of Shigellosis is changing and the strains of S.sonnei gradually forms the preponderance in recently years.And the strains of F2 a still dominates its development.Bacterial susceptibility to antimicrobial agents was determined on 382 strains of shigella isolated in shanghai from 2010 to 2014 by modified Kirby-Bauer agarose method,and totally 14 kinds of antibiotics were tested,including ampicillin、ciprofloxacin、cefotaxime、cefepime、sulfamethoxazole/trimethoprim、levofloxacin、amoxicillin-clavulanic acid、chloramphenicol、cefoxitin、nalidixic acid、norfloxacin、streptomycin、tetracycline、compound sulphonamides.The result was that high levels of antimicrobial resistance among the Shigella isolates including nalidixic acid(97.4%),streptomycin(96.1%),ampicillin(84.4%),tetracycline(77.7%),sulfamethoxazole/trimethoprim(77.2%)and compound sulphonamides(67.8%).Among the 382 isolates tested,nearly all(92.7%)were resistant to ≥ three antimicrobial agents and 24 strains(6.3%)to 10~13 antimicrobials.In addition,strains resistant to quinolone and the third and fourth generation cephalosporins were also detected.Notably,The resistance of shigella flexneri was significant higher than shigella sonni.The resistance reaches as high as 40.3%,especialy,to quinolone ciprofloxacin.The emergence of co-resistance to fluoroquinolones,and 3rd-and 4th-generation cephalosporines in Shigella is particularly significant to the public health and poses a great challenge for the effective treatment of shigellosis.So the multidrug resistance of shigellosis was severe and complicated and we should enhance to detect and survey those isolates All these strains were subjected to the virulence genes testing with polymerase chain reaction(PCR)which included genes ipa H,ial,sen,set1 A and set1 B.The results showed that positive rates for ipa H,ial,sen,set1 A and set1 B genes were 100%,65.7%,64.5%,34% and 34%,respectively.Notably,the positive rates of setl were 92.8% and 0.4%for S.flexneri and S.sonnei,respectively.So the strains of S.flexneri carry more virulence genes than S.sonnei and we should pay more attention.Mutations within the quinolone resistance determining regions(QRDR)of gyr A and par C and the plasmid-mediated quinolone resistance(PMQR)genes,qnr A、qnr B、qnr S and acc(6’)-Ib-cr were also detected by PCR.All the products were then sequenced.It showed that a mutation in gyr A was present in 137(98.6%)of the isolates and a mutation in par C was present in 136(97.8%)of the isolates.Moreover,gyr A contains three point mutations(Ser83,Asp87 and His211)and par C contains only one point mutation(Ser80).In addition,qnr S and aac(6’)-Ib-cr were present in 9(6.5%)and 6(4.3%)of the isolates,respectively.There are High mutation rate of amino acids in the region QRDR in S.flexneri of shanghai and the point mutation in Asp87 plays the important role in the resistance ability of S.flexneri to quinolone.The genotypes were analyzed with the PFGE standard methods recommended by Pulse Net.The electrophoresis profiles were categorized by Bio Numerics software.l39 strains of Shigella flexneri could be divided into 97 band patterns and 7 clusters.A total of 128 strains belong to one cluster,accounting for 92.9% of the isolates and the similarity coefficient was more than 80.6%.243 strains of Shigella sonnei could be divided into 138 PFGE band patterns and 17 clusters.A total of 198 strains belong to one cluster,accounting for 81.5% of the isolates and the similarity coefficient was more than 89.3%.So there was a close genetic relationship between the Shigellosis of shanghai in recently years although they originates from multiple clones.Seven housekeeper genes provided by warwick database were amplified with PCR method,followed by sequence testing of the PCR product.After the results were uploaded to the warwick database and compared with the known sequences,the sequence types of each strain could be acquired.Based on this,we identified three STs,ST 245,ST 145 and ST 628.ST 245 was most abundant,accounting for 96.4% of the isolates.All of the four F6 strains belong to ST628 and one F3 a belongs to ST145.So the strains of S.flexneri in shanghai have close relationship and most of which is popular with ST245. |
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