Optimization Of Pulsed-Field Gel Electrophoresis And MLVA Subtyping For Salmonella Enterica Paratyphi A

Salmonella enterica serovar Paratyphi A is the aetiologic agent of paratyphoid fever.During the past decade,especially after 1990s',outbreaks of paratyphoid fever due to S.Paratyphi A were continually and increasingly reported in some countries,especially in east and south Asia.In those countries,the incidence of paratyphoid fever due to S.Paratyphi exceeded that of due to S.Typhi.Pulsed-field gel electrophoresis(PFGE),which is generally considered as one of the most reproducible and highly discriminatory genome based subtyping techniques,has successfully used for epidemiology investigation and the characterization of multiplebacterial pathogens including Salmonella.However,with PulseNet 1-day standardized PFGE protocol used by PulseNet laboratories for the subtyping of Salmonella serotypes,limited patterns were obtained when the strains from multiple years and multiple provinces during our surveillance of S.Paratyphi A.Then whether this protocol is not suitable for the S.Paratuphi A isolates or it is really a national wide outbreak which spanned so many years and provinces should be clarified.In order to explore possible more suitable PFGE protocol for the subtyping of S.Paratyphi A,in this study,based on PulseNet International 1-day standardized PFGE protocol used by PulseNet laboratories for the subtyping of Salmonella serotypes,we selected 33 test strains from different years and provinces,compared different restriction enzymes and electrophoresis parameters,and make a preliminary comparison and optimization of PFGE protocols for the subtyping of S.Paratyphi A.With the new optimized protocols,106 S.Paratyphi A strains from various years(1998-2010)and 12 provinces in China were analyzed restropectively.The results showed that Spel,XbaI,XhoI and BlnI are appropriate for the subtyping of Salmonella paratyphi A.Through the classification and comparative analysis of similarity and discrimination of 33 strains,Spel-PFGE produced more patterns(23 patterns)and had a higher discriminatory power(0.964)than those of XbaI-PFGE and XhoI-PFGE,so SpeI-PFGE was considered as the first choice of enzymes.XbaI-PFGE was slightly less discriminatory than XhoI-PFGE,but the number of the patterns was more than XhoI-PFGE.We recommend Xbal as the secondary enzyme and XhoI as the third enzyme.BlnI-PFGE generated the least patterns than the above three enzymes,so it was discarded for further analysis.In addition we also found that Xbal-PFGE with electrophoresis parameter of 1.5-29s,20h;SpeI-PFGE with electrophoresis parameter of 1-20s,20h;and XhoI-PFGE with electrophoresis parameter of 2.2-29s,20h,had the best discrimination,which could divided into 17,23 and 16 patterns each at the most.106 S.Paratyphi A strains were analyzed with the new optimized protocols,the results showed some predominant highly clonal of S.paratyphi A existed and spreaded in different epidemic years and areas in China.However,accumulation of scattered variation has been observed with temporal change and the spread of outbreaks.Multilocus variable-number tandem repeat analysis(MLVA)based PCR molecular typing methods had been developed in recent years.Because of its proved highly discriminatory ability,good reproducibility and simple operation,it has been used for the subtyping of a variety of bacterial pathogens,including multiple Salmonella serotypes and has made great progress.So in the study,we tried to establish the MLVA method for the analysis of Salmonella Paratyphi A and evaluate its application in epidemiological investigation.TRF and TRD were used to scan the whole nucleotide sequence of Salmonella paratyphi A strains ATCC9150 published in the GenBank.In total,196 TR were found in it.34 S.Paratyphi A strains isolated from various years,provinces,and PFGE patterns were selected as the panel and were used to evaluate these obtained TR and look for the potential VNTR.Then 209 Salmonella paratyphi A isolated from 1998-2010 in 12 provinces in China were selected randomly to carry out MLVA and compare with Spel-PFGE.The results showed that 7 out of 196 TR were polymorphic.With these 7 VNTRs,209 S.Paratyphi A strains were analyzed by MLVA.The discriminatory power of MLVA is lower than that of SpeI-PFGE(optimized in this study).21 and 65 different patterns were observed in MLVA and PFGE respectively.This indicated that the copy number of VNTR is not prone to change in LS.Paratyphi A strains,especially in an outbreak.We conclude that MLVA is not suitable for the molecular surveillance and epidemiology investigation in a limited region(country or area).However it is suitable for the population genetics.MLVA is simple,rapid and easy to standardization.S.Paratyphi A strains with the same PFGE pattern could be further analyzed with MLVA.When combined with PFGE and MLVA could supply more discriminatory power,so it could be used as a supplement of PFGE which could be used in disease investigation caused by S.Paratyphi A strains and could also be used in the strains with the same PFGE pattern.