Detection Of Virulence Genes In Shigella Flexneri By Multiplex PCR And Research On The Pic Gene Function On Shigella Flexneri2a

Shigella spp. are exquisitely fastidious gram-negative organisms. Based on biotyping, the genus is divided into four species and around48serotypes (including subgroups). In China, S.flexneri2a was the predominant subgroup.Shigellosis is usually a self-limiting infection, however when it subsides, the intestinal ulcers heal with scar tissue formation.Uncomplicated recovery is usual and the organisms rarely cause other types of infections. Adversely, in some cases, depending on the virulence of the strain, the nutritional and immune status of the host, the initial infection maybe followed by neurological complications or kidney failure.Isolation of the bacterium and confirmation of the disease by microbiological and biochemical methods remain as the "Gold Standard" which exists many shortcomings including wasting time, low sensitivities, specimen impaired by the use of antibiotics and impossible to detect the virulence. Thus, a rapid and specific multiplex PCR assay was designed for the simultaneous detection of chromosomal-and plasmid-encoded virulence genes (ipaH ial, and set IB) in S.flexneri in order to:1.Identify shigella strains from other enterobacteria;2.Identify the virulence of the shigella strains;3.Identify the serotypes of the strain.86strains of Shigella flexneri from clinical isolates were studied by multiplex PCR with3pairs of primers based on the gene sequences of these virulence genes. The virulence of amplification products of12strains to HeLa cells was determined by plaque formation test. Interestingly, one strain who missed the setlB gene but remain invasive plasmid shows obvious virulence descent by plaque formation test. The strain was named S. flexneri2a No.51, SF51. Following tests proved that the SF51misses pic gene.To prove the function and importance of pic gene in S. flexneri infection, pic deletion mutant named SF301-△pic was constructed and the virulent change of the mutant was detected by Gentamycin Protection Test and Mouse Sereny Test. No significant difference of growth rates and basic biochemical events were observed among three strains SF301, SF301-△pic and SF51. However, obvious differences in virulence ability was observed:cell invasion ratio of SF301-△pIc downs to61%and SF51downs to35%compare with standard strain SF301; Sereny test proves that typical mouse keratoconjunctivitis shows after infection at24hr and attained the maximum at48hr. Pic deletion mutants shows less symptoms than standard strain SF301. Eye sections were HE stained after48hr infection and observed under light microscope. Typical symptoms of SF301are strong features with limited invasion including corneal epithelial thickening and loss, accompanied by mild, moderate and severe ulcers. While pic deletion mutants show little pathological changes after48hr infection according to the eye-section HE stain.Using laser scanning confocal microscope, the cytoskeleton variation of Hela cell was observed after30min exposure to the3strains separately. Fewer membrane ruffles were induced by pic deletion mutants comparing with SF301.In conclusion, Shigella flexneri2a pic gene, which locates at the unstable chromosomal site, spontaneously deletes at a low frequency. Once the gene lost, the cellular virulence of Shigella flexneri2a comparable decends.