The Analysis Of Phenotypic And Molecular Epidemiology Of Shigella Flexneri Serotype4c

Shigella flexneri subserotype4c (S.F4c) was first isolated and predicted wouldbe the popular strains by the former Soviet Union scientists in1988. Since October2003, outbreak and prevalence of F4c happened in several provinces and cities ofChina and showed a trend of rising year by year and become dominate serotype ofShigella flexneri in some provinces and cities. But the difference among F4c indifferent areas was poorly understood. In order to understand the biologicalcharacteristics, pattern of transmission and variation, about204strains of F4c werecollected from5provinces and2municipalities during the period of2003to2013.Biochemical and serological characteristics, virulence genes and drug-resistantspectrum, the plasmid profiles, pulsed field gel electrophoresis, multiple loci sequenceclassification analysis were performed for understand the biological characteristicsand the variation pattern. This study will be important for further understand thespread of bacterial dysentery and control strategy.In this study, a total of204Shigella F4c strains were collected from more than30sentinel hospitals which belong to Beijing, Shanghai, Shenyang, Ganshu, Henan,Guangxi and Xinjiang from2003to2013. All strains were performed onbiochemical identification, serum agglutination test, antibiotics sensitivity test anddetection of virulence genes and O antigen genes of11strains which withoutepidemiological relation and background of genetic were detected. Pulsed field gelelectrophoresis(PFGE) was used to analyze the different among204strains andplasmids of60strains with the different virulence genes which collected fromdifferent areas were isolated and analyzed.37strains of Shigella F4c were detected byMultilocus sequence typing(MLST) and one strain which isolated from Beijing wasperformed on whole genome sequencing analysis.The results of serum agglutination test used Denka Seiken serum showed thatShigella F4c can agglutinate with multivalent antiserum of Shigella flexneri andShigella flexneri IV and group factors antiserum7(8). These results had highsimilarity with Shigella F4c.. And reactive formula of the monoclonal antibodies forShigella flexneri (MASF) was7(8)+, Ⅳ-1+, and negative reaction with other monoclonal antiserum and the same as Shigella FXv which reported by China CDC.Only the wzx1-5and gtrX gene were detected in11strains.The biochemical identification was done by API20E identification system andthe results showed most strains can ferment glucose and decompose mannitol, butsome differences present in four reaction including indole test, gelatin liquefactiontest, melibiose experiment and arabinose test. The positive strains of indole testgelatin liquefaction test, melibiose experiment and arabinose test were28,2,26and201.The positive rate of indole test was higher in strains isolated from Henan andShenyang than other area and most strains isolated in2007. The results of melibioseexperiment showed the positive strains which collected from Henan, Ganshu andXinjiang were higher and most of them isolated from2003,2006and2011.All strainswith the positive results of gelatin liquefaction test isolated from Guangxi without thespecific years. The negative result of arabinose test was showed on3strains whichisolated from Beijing and Xinjiang in2004and Shanghai in2007. Only2strains ofGuangxi with positive result of gelatin liquefaction test were first detected among theF4c strains.The antibiotic sensitive test according the CLSI was used for test the sensitivityrate for all204strains. The sensitive rate of gentamycin, tobramycin,second and thirdgeneration of cephalosporin antibiotics was higher, the penicillin class antibioticsampicillin sensitive rate was only3.92%, piperacillin sensitive rate was75.49%, thebeta lactamase inhibitor compound drugs ampicillin/shubajotham sensitive rate wasonly4.41%, and piperacillin/heazole sensitive rate was100%. Cephalosporinantibiotics sensitivity test of sensitive rate ranged from76.96%to99.51%. Threekinds of amino glucoside of antibiotics sensitive rate above84.8%.Two kinds offluoroquinolone antibiotics drug sensitive rate were86.27%and88.24%respectively.Sulfa antibiotics (folicacid metabolism pathway inhibitor) sensitive rate was29.41%and the nitrofurantoin, aztreonam sensitive rate was above96.08%. The prevalence ofmultiple resistance strains resistant to more than3kinds of antibiotics accounted for77.45%in Shanghai, Gansu, Henan. Only some strains isolated from Beijing andShanghai can resistant more than10kinds of antibiotics which the proportion ofBeijing was3.84%and17.89%for Shanghai.To understand virulence genes of Shigella F4c,12virulence genes associatedwith the pathogenic were selected to test.158strains (77.45%) carried all12virulence genes and all204strains harbored the ipaH1.4and shiD-Full gene, the ial gene with lowest rates(86.27%). Strains with ial gene which isolated from Shanghaiand Guangxi were69.57%and27.27%, respectively, and the number of strainswhich with ipaH genes was lower than in other regions. According the patterns of12virulence genes, all204strains were classified21genotypes and the pattern of1which found in six areas (77.45%)was the dominated genotype except Guangxivirulence gene patterns of4and14. Strains with positive indole test negativearabinose reaction almost harbored all12virulence genes. The results suggested thestrains with positive indole test, positive of dense disaccharide fermentation and thenegative sugar fermentation had the stronger ability of pathogenic and is easy to causeoutbreaks. In addition, the virulence and antibiotic resistance of strains isolated fromBeijing and Shanghai were analyzed. The result showed that strain harbored differentvirulence genes had obvious correlation with antibiotic sensitive rate.60strains with different virulence genes and obvious different in antibioticresistance pattern were selected to analyzed the pattern of plasmid. The resultsshowed21strains harbored the plasmid with the size of220kb and23strainsharbored the plasmid with the size of140kb;3different small plasmid with the sizefrom1.8kb to4.1kb:60strains with the size of4.1kb,56strains with the size of2.2kb,55strains with the size of1.8kb and without the plasmid with the size of10.5kband4.8kb.60strains carried the virulence genes shiD-Full, ipaH1.4, could extractthe plasmid with the size of4.1kb and7strains with3to5different plasmid. Inaddition, about25strains without220kb plasmid which did not harbored the ialvirulence gene.All of204strains were analyzed by Pulsed field Gel electrophoresis (PFGE).The14fragments obtained which digested by NotI restriction enzyme were between20kbto135kb. Dice coefficient of204strains of Shigella F4c were between80%to100%, the dominated PFGE band patterns was FN11.004in all81PFGE bandpatterns. All PFGE band patterns were classified about28cluster which named from1to28, the cluster1(FN11.001-FN11.023), cluster2(FN11.024-FN11.029), cluster4(FN11.032-FN11.036), cluster10(FN11.046-FN11.050),cluster18(FN11.064-FN11.067) including about108strains (52.94%),17strains,8strains,7strains and9strains. The rest of the cluster number of strains are under5.The analysis resultsshowed the strains which isolated before2009with higher similarity. The dominatedPFGE band pattern was FN11.004among Beijing, Shenyang and Xinjiang, and11strains isolated from Guangxi had11PFGE band pattern can be divided7cluster. Multilocus sequence typing (MLST) including7housekeeper genes were uploadBlast database which showed all Shigella F4c had the same sequence type named ST245. ST245and ST651, ST633, ST627, ST255and ST240belongs to ST245Cplxand only one locus variation among them and2to3locus differences with ST629,ST631, ST264and ST248, ST630which also belong to ST245Cplx. All resultsshowed the ST245probably evolved from ST270EIEC.Whole genome sequencing result analysis of one Shigella F4c strains isolatedfrom Beijing showed almost sequence including virulence genes and O antigen genehad high similarity with Shigella FXv. But F4c with some of genes and plasmidderived between F2a and E coli had influenced the difference the monoclonalantiserum and the F4c isolated from Beijing and other areas were different with theFXv which isolated from Henan. Different genotypes of the F4c and FXv belong tothe same serotype at this stage.Based on the analyzed method mentioned above, the Shigella F4c serotype wascomplex and O antigen specific genes gtrX as same as the Fx and Fxv and themonoclonal serum agglutination test was same with Fx and Fxv, and may be moreappropriate named F4not4a and4b type or F4v type. Shigella F4c strains withpositive test of glucose fermentation, mannitol and gelatin liquefaction positive strainswere first isolated from Gansu at first time. In addition the strains with Arabian sugarfermentation test negative harbored more virulence genes. Shigella F4c were restantto ampicillin, ampicillin/sulbactam, and resistant to piperacillin, andTrimethoprim/sulfamethoxazole, gentamycin, tobramycin, and second and thirdgeneration of cephalosporin antibiotics but Quinolones was more sensitive. Theantibiotic resistance rate was lower in Beijing than Shanghai, and the virulence genescarrying rate was more high than Shanghai. Virulence genes carrying rate of ShigellaF4c isolated from Shanghai and Guangxi is lower than other areas. Shigella F4cmainly carrying plasmid with the size of220kb,140kb plasmid and4.1kb,2.2kb and1.8kb.204strains of bacteria are divided into81PFGE band patterns and thedominated pattern was FN11.004type. MLST classification is given priority to withST245. Whole genome sequence of Shigella F4c with almost same as referencestrains of Shigella FXv including O antigen sequenceand, are the same serotype.