Studies On The Resistant Mechanism And Genetic Diversity Of Multidrug Resistant Shigella Sonnei

Shigella is one of the common pathogenic bacteria responsible for gastrointestinal infection, transmitted mostly by the fecal-oral route and caused by the pollution of foods and water resources. The annual incidence globally approximates 164.7 million, about 163.2 of which occur in developing countries. Among the vulnerable population are infants, elders and the immunocompromised. 69% of the 1,1 million deaths are infants under five years old. Shigella spp is subdivided into shigella dysenteriae, shigella flexneri, shigella boydii and shigella sonnei. Shigella sonnei is a already prevalent in developed countries and is becoming more prevalent in developing countries that have been dominated by shigella flexneri. Rational use of antibiotics for treating bacillary dysentery can be reduce the duration of illness and severity of symptoms and may be used to prevent the spread of disease. At the same time, it also led to increased drug resistance of shigella sonnei whose drug resistance can be transmitted between different bacteria through plasmid horizontal transfer, resulting in the extensive drug resistance of enzymes produced by bacteria, including the drug resistance of beta-lactam antibiotics. Expect for horizontal transmission of drug resistance also existed vertical transmission of clones. The wide-spread drug resistance and complicated mechanisms pose a serious challenge to the clinical therapy of bacillary dysentery. Given this situation, a better understanding of the correlation between the prevalence, drug resistance, mechanism of drug resistance and genetic diversity of shigella sonnei within pathogenic bacteria of diarrhea is of great importance to surveillance of prevalent drug resistance, rational clinical use of drugs and control and prevention of the spread of epidemics. To serve this purpose, we have conducted studies listed as follows:1. Distributional characteristics, drug sensitive tests and detection of multidrug resistant shigella sonneiWe collected 1831 strains of shigella sonnei isolated from patients of diarrhea treated by our hospital between Jan 1990 and Dec 2007 before we tested the rate of drug resistance of 13 types of antibiotics using the Kirby-Bauer agar diffusion method recommended by CLSI. From 212 strains shigella sonnei collected between 2004 and 2007 were screened out 43 multidrug resistant strains (mainly AmR, SxtR and CtxR). The confirmatory test recommended by CLSI was used to detect ESBLs of multi-drug-resistant Shigella sonnei.The modified three-dimensional extract test was used to detect both ESBLs and AmpC.The ratio of males to females among victims of shigella sonnei was 1.25:1. It was also found that the peak season was from July to September and that 57.82% of the patients were minors under 14 and that 71.64% of the patients were under 20. Since 2005, the percentage of shigella sonnei in cases of infection with shigella spp has been remarkably increasing.Shigella sonnei was highly resistant to trimethoprim-sulfamethoxazole, ampicillin and ceftriaxone, the rate of drug resistance being as high as 87.7%, 23.0% and 9.5% respectively. However, it was much less resistant to fluoroquinolones, cefepime, cefmetazole, chloramphenicol, fosfomycin and gentamicin than to ceftriaxone and cefotaxime (P<0.05),The resistance rate was 1.2%,2.5%,0.3%,3.7%,1.5%,1.7% respectively.Between 2000 and 2007, the rate of drug resistance of ampicillin, ceftriaxone and cefotaxime was substantially increased. The 43 strains of multidrug resistant shigella sonnei accounted for 20.28%(43/212), all of which were ESBLs-positive strains without any AmpC-producing enzymmes being found. The conformity rate of the two methods of testing ESBLs was 100%, suggesting the dominance of ESBLs-producing shigella sonnei of multidrug resistance in Beijing.2. Studies on the resistance genes of ESBLs-producing shigella sonneiWe collected a total of 43 strains of multidrug resistant shigella sonnei isolated from diarrhea outpatients of our hospital between 2004 and 2007. The minimum inhibition concentration kit was used to test MIC of ampicillin, cefotaxime, ceftazidime, ciprofloxacin, trimethoprim-sulfamethoxazole and ESBLs-producing strains were given amplification using the universal primers and sequencing primers of coding genes of TEM, SHV, CTX-M1, CTX-M2, CTX-M8, CTX-M9, OXA, PER,5,CS,3,CS and hep74,hep51 of class 1 and 2 integrons .These strains received plasmid conjugation experiments. The results of tests indicated that 27 of these strains carried CTX-M, 16 of which carried CTX-M1 and 11 carried CTX-M9, making up 62.79%(27/43)of the positive isolated strains. Besides, 18 of the tested strains carried TEM genes, accounting for 41.86 %(18/43), but no SHV, OXA or PER genes were identified. Ten strains co-carried CTX and TEM (23.26 %) while four strains co-carried CTX-M1 and TEM genes9.30%(4/43). Six strains co-carried CTX-M9 and TEM 13.95% (6/43). 95.35%(41/43)of the shigella sonnei carried Class 2 integrons. the amplification fragments were within about 2000bp;one stain carried Class 1 integrons, the amplification fragments were within about 1600bp. The blast comparison via GenBank after sequencing showed that CTX-M1 group was mostly CTX-M-15like; one strain was CTX-M-64; one was CTX-M-15 while CTX-M9 group was mostly CTX-M-14: ten were CTX-M-14 and one was CTX-M-27. TEM was all TEM-1 .During MIC experiments,CTX-M-15like,CTX-M-15,CTX-M-27,CTX-M-64 resistant to ceftazidime were different extents, which was associated with the substitution of amino acid Asp240 for Gly.Class 2 integrons generally carried gene cassettes dfrA1, sat1 and aadA1; Class 1 integrons carried gene cassettes dfrA17 and aadA5. The success rate of plasmid conjugation experiments was 92.7%. ESBLs-producing shigella sonnei in Beijing carried mostly CTX-M genes, The main groups were CTX-M1 groups and CTX-M9 groups,followed by TEM. Drug-resistant genes were mainly transmitted through plasmid horizontal transfer. The prevalent subtype was CTX-M-15like, CTX-M-14 and TEM-1. Two strains Shigella sonnei carrying CTX-M-27 or CTX-M-64 have not been reported in China .3. Analyses of genetic correlation of multidrug resistant shigella sonneiPFGE was used in molecular typing of strains were digested with restriction endonucleases XbaI to determine the affinity of the 43 multidrug resistant shigella sonnei and to identify prevalent strains and the pattern of prevalence. The detection rate of multidrug resistant shigella sonnei rose from 4.44% in 2004 to 34.09% in 2007. The 43 multi-drug resistant shigella sonnei was all Phase 1 shigella sonnei. PFGE typing showed 97.6 % of these strains (40/41) were prevalent. There were at least six A– A5 clone strains prevalent in Beijing, the percentage being 39.0%(16/41), 24.4%(10/41), 17.1%(7/41), 7.3%(3/41), 4.9%(2/41), 4.9%(2/41)respectively. The band difference between A1, A2, A3, A4, A5 and prevalent strain A was 1,2,2,3,4 bands respectively. The year 2006 was dominated by A and A1 while 2007 by A alone. Type A1 carried mostly CTX-M-15like while Type A3 carried mostly TEM-1. The clone transmission strains existed in multidrug resistant shigella sonnei in BeiJing.From the above-mentioned studies the primary results can be concluded :Infection of shigella sonnei happens largely to children and teenagers. The percentage of shigella sonnei in cases of infection with shigella spp was mounting.Shigella sonnei are quite resistant to ceftriaxone, cefotaxime, trimethoprim-sulfamethoxazole and ampicillin and the resistance is rising. Multi-drug resistant shigella sonnei is becoming more prevalent each year, the drug resistance Mechanism of which is the production of ESBLs without any AmpC involved.Further typing of ESBLs shows the dominance of ESBLs carryiny the CTX-M type. The most commonplace is CTX-M-15like, CTX-M-14 and TEM-1. Some strains carry two types of drug-resistant genes . Drug-resistant genes were mainly transmitted through plasmid horizontal transfer. Most of strains carry Class 2 integrons. Shigella sonnei carrying CTX-M-27 or CTX-M-64 have not been reported in China. 97.6% of the multi-drug resistant shigella sonnei strains are prevalent by PFGE .There is both horizontal transmission of drug-resistant genes among the drug resistant shigella sonnei and vertical transmission among cloned strains. Thus, it is necessary to enhance surveillance while ensuring proper clinical treatment.