Study On Epidemic Characteristics, Antimicrobial Resistance And Variation Of Shigella Sonnei In China

Shigella spp., the causative agent of bacillary dysentery, is a gram-negative, facultative anaerobic bacillus of the family Enterobacteriaceae. According to the World Health Organization, the annual number of Shigella episodes throughout the world was estimated to be 165 million, of which 163 million were in developing countries with poor sanitation. Approximately 1.1 million episodes resulted in deaths and 61% of all deaths attributable to shigellosis involved children under 5 years of age. Despite a significant decrease in Shigella-related mortality in recent years, shigellosis was still a leading infectious cause of mortality among children younger than five years old.Based on the O-antigen structures of the membrane-associated lipopolysaccharide, Shigella species are classified into four serogroups: S. dysenteriae, S. flexneri, S. boydii and S. sonnei. S. flexneri is the most frequently isolated species in underdeveloped countries, while S. sonnei is the most commonly isolated species in industrialized areas. Recently, this trend is gradually being changed that S. sonnei has become increasingly prevalent in some Asian countries. S. flexneri was the most prevalent species of Shigella in China and the predominant serotype was S. flexneri 2a over a long period of time. However, in recent years, S. sonnei is increasingly exceeding S. flexneri to become the dominant pathogen of bacillary dysentery, especially in some developed regions of China. Given the epidemic change, we explore the surveillance of Shigella spp..In this study, 3758 strains of Shigella spp. were collected from 14 areas of China, among which 2084 strains were identified as S. flexneri and 1630 were S. sonnei. According to the serotype, the incidence of S. sonnei was increasing yearly, especially in the last decade. And S. sonnei is replacing S. flexneri to become the dominant pathogen of bacillary dysentery. This trend was more obvious in well-developed regions, such as Southeast, North and Central of China. In addition, we identified a prevalence of biochemically atypical variants of S. sonnei, which could not ferment lactose and thus were negative for the phenotype of O-nitrophenyl-β-D-galactopyranoside(ONPG). And the ONPG-negative variants had exceeded ONPG-positive isolates as the main pathogens in some regions of our country. A negative reaction in ONPG hydrolysis reduces the likelihood of correct species identification as S. sonnei from 99% to 50% and 47% by VITEK 2 and API 20 E methods, respectively. Only this feature may complicate microbiologic identification of S. sonnei with these commercial systems, to some extent, which will lend to misdiagnosis and result in delaying treatment and prolonged illness. Therefore, the epidemic and changing trends of S. sonnei have posed serious threats to public health.Among the 1630 S. sonnei isolates, 451(451/1630, 27.7%) were ONPG-negative variants. The first ONPG-negative variant was found in 2005. Then the ONPG-negative variants sharply increased in 2009. The prevalence of the ONPG-negative variants obviously increased over time. In 2011, the ONPG-negative variants firstly surpassed ONPG-positive isolates as the dominant pathogens.In order to get much more materials and have a deep understanding of the genetic polymorphism and genetic relationship of S.sonnei in our country, we analyzed the S. sonnei isolates from different regions by pulsed field gel electrophoresis(PFGE), especially ONPG-negative variants. The result showed that ONPG-negative variants and ONPG-positive isolates had apparent difference in banding patterns and were divided into different clusters. And ONPG-negative variants found in Shanghai, Beijing, Gansu and Henan had same banding pattern. Genetic relationships analysis based on PFGE showed that the ONPG-negative variants formed three major clone groups that mainly occupied by isolates from Shanghai, Gansu and Beijing. These results suggested that ONPG-negative variants had widely disseminated in China.In order to understand the antimicrobial resistance characteristics of S. flexneri and S. sonnei in our country, antibiotic susceptibility test was performed. We found that S. flexneri and S. sonnei had high resistance rate to some conventional antimicrobials including ampicillin(88.1%), ticarcillin(87.8%), tetracycline(87.5%), trimethoprim/sulfamethoxazole(73.2%) and chloramphenicol(49.2%). It was noteworthy that S. flexneri and S. sonnei had also developed high resistance rate to cephalosporins, including cefazolin(32.4%), ceftriaxone(31.1%) and cefoperazone(27.5%). Compared to S. flexneri, S. sonnei not only had high resistance rate to some conventional antimicrobials, but also higher to cephalosporins, including cefazolin, ceftriaxone and cefoperazone. The resistance rate of S. sonnei to the three cephalosporins was greater or equal to 35%, whereas the resistance to fluoroquinolones was maintained at low level. The resistance rate of S. sonnei that isolated from different regions and time was divergent. The isolates from the Southeast, Northwest, Central and North regions of China were more resistant to cephalosporins. Also S. sonnei showed increasing resistance to ceftriaxone, cefoperazone and cefazolin from 2004 and the peaked resistance rate appeared during 2007-2008 with 62.2%, 59.6% and 61.7% to the three cephalosporins respectively. And then the rates held steady at approximately 30%-40%. Compared to the resistance characteristics of ONPG-negative variants, the ONPG-positive isolates were more resistant to the antibiotics. Notably, the ONPG-negative variants showed slowly increasing resistance to cefazolin, ceftriaxone and cefoperazone with time. Particularly in 2012, they had a sharp increase in resistance to those cephalosporins, to which should attach great importance. These findings had important value for the accurate diagnose of S. sonnei and the rational use of antibiotics.To further explore the molecular mechanism of antimicrobial resistance, we chose the isolates with resistance to cephalosporins to detect the resistance genes. The result showed that S. sonnei with resistance to cephalosporins harbored β-lactamase genes extensively. Among the S. sonnei isolates with resistance to cephalosporins, 82.8% and 70.5% contained the blaCTX-M and blaTEM genes respectively. The blaCTX-M genes were sequenced and the result showed that blaCTX-M-14 gene was the most common type, followed by blaCTX-M-79 and blaCTX-M-15. Additionally, the coexistence of two different blaCTX-M genes was found in S. sonnei. Integron genes testing result showed that 93.1% of S. sonnei had integrase II and 86.0% had integron II. The research findings showed that S. sonnei with resistance to cephalosporins commonly contained resistance genes, which could lead to severe antibiotic resistance by horizontal gene transfer(HGT)Based on the research findings above, 67 S. sonnei were chose for the whole genome sequence, including 58 ONPG-negative variants. Significant difference was found in lac operon locus between ONPG-positive S. sonnei and ONPG-negative variants. Further analysis by PCR found that ONPG-negative variants lacked lac operon(lacZ, lacY and lacI genes), mhp operon(mhpA, mhpB and mhpR genes), gene of hypothetical protein and two insertion sequences(IS1 and IS600). Based on genome data and methods published by Holt in 2012, we detected the single nucleotide polymorphisms(SNPs) and constructed a phylogenetic tree. The results showed that the 67 S. sonnei isolated from our country located in a same big branch. The finding implied that S. sonnei isolated in our country may descend from abroad.To explore the influence of lacking lac operon on S. sonnei, λ-Red recombination method was used to construct S. sonnei mutant lacking lac operon. Biochemical examination results showed that ONPG phenotype of S. sonnei mutant became negative and 6 other phenotypes also changed. The metabolism of gentiobiose, turanose, D-melibiose, D-mannose and D-serine were enhanced, while the metabolism of formic acid was weakened. Compared with the wild-type strains of S. sonnei, the growth curve of mutant strains had no difference in different incubation temperature. However, the mice injected with mutant strains didn’t showed higher mortality rate. These results demonstrated that the lack of lac operon is the reason for the appearance of ONPG-negative variants. However, what advantage leading to the variants prevalance is still unknow. Much more efforts and researches will be needed to address this issue.In summary, this study explored the the epidemic change and variation of S. sonnei, especially the ONPG-negative strains and determined their antimicrobial resistance characteristics from different places of China. By means of the whole genome sequence of typical strains, we clarified the genome characteristics and the molecular mechanism of the appearance of ONPG-negative variants. These finding can provide the scientific basis for the S. sonnei surveillance, source-tracking, transmission survey, the formulation of the control and prevention strategy.