Study On L-form Induction Of Salmonella Typhimurium By Bile In Vitro And Determination Of The L-forms Derived From Salmonella Typhimurium In Pig Gallbladders

Objective:(1)To investigate the influence of bile on the formation of Salmonella Typhimurium L-form and develop an assay for detecting S. Typhimurium L-form.(2)To understand the situations of pig gallbladders carrying S. Typhimurium L-forms and explore the assay for detecting S. Typhimurium L-forms in gallbladders. Methods:(1)S.Typhimurium was inoculated into pig bile to be cultured at 37 ℃aerobicly.Bile-induced cultures were cultured by the routine bacteriological methods during 1h to 60 d.(2)Meanwhile,bile-induced cultures, filtered by germtight filters, were inoculated into PG medium for the isolation and culture of L-forms pure cultures by the nonhigh osmotic isolation technique.L-form isolates were identified by morphological character, culture characteristics,Gram stain,cell wall stain, carbohydrate fermentation tests and serologic tests.(3)Then L-form isolates were detected by the duplex PCR assay for inv A gene specific for the genus Salmonella and STM4495 gene specific for S. Typhimurium,and amplification products were sequenced and analyzed.(4)284 pig gallbladder specimans( 142 gallbladder tissues and 142 biles), collected from a slaughterhouse in Guiyang,were detected for S. Typhimurium by the routine bacteriological methods.(5)And they were also cultured and isolated for bacteria L-forms by the nonhigh osmotic isolation technique.Then the L-form isolates were determined by the duplex PCR assay for inv A and STM4495 genes of S.Typhimurium L-form. Parts of PCR products of inv A and STM4495 genes were sequenced and analyzed.Results:(1)The bacterial species could be isolated from 1h to 60 d bile-induced cultures by the routine bacteriological methods,and they were indentified as S.Typhimurium.(2)And meanwhile,the L-forms could be isolated by the nonhigh osmotic isolation technique.The L-form cells,sphere or oval,could not grow on blood agar,Ma Conkey agar and LEM plate.Their Gram stain,cell wall stain and serologic tests were neagtive.Results of carbohydrate fermentation tests could not be determined.(3)284bp and 915 bp amplification products of the L-forms isolates were the same as amplification products of the bacterial form of S.Typhimurium by the duplex PCR assay for inv A and STM4495 genes.Compared with S.Typhimurium from Gene Bank,the coincidence ratio of their nucleotide sequences was, respectively,100% and 99%.(4)There were 45 bile and 104 gallbladder tissue specimens carrying bacteria among 284 gallbladder specimens,and the positive rate was 52.46%(149/284).By Gram stain and biochemical reactions,there were 17 species,200 strains of bacteria,including 184 strains of gram negative bacteria and 16 strains of gram positive bacteria.The commonest organisms in negative bacteria was Escherichi a coli(32.75%,93/284),followed by Aeromon as,Enterobacte r cloacae,Proteus mirabilis,Pantoea ananas,Citrobacter freundii,Edwardsiella tarda,Pseudomonas,Alcaligenes,Salmonella,Proteus vulgaris,Pasteurella,Klebsiella pneumonia,Citrobacter diversus,Klebsiella oxytoca.There were 8 strains of Salmonella by the serological identification,including 4 strains of Salmonella Kottbus and 4 strains of Salmonella London.All of gram positive bacteria were Streptococcus(5.63%,16/284),of which Streptococcus bovis(81.25%,13/16) was the dominant speciesand the rest was Streptococcus mitis.(5)There were 90 bile and 67 gallbladder tissue specimens carrying bacteria L-forms among 284 gallbladder specimens by the nonhigh osmotic isolation technique,and the positive rate was 55.%(157/284).9 gallbladder specimens(3 biles and 6 gallbladder tissues) carried Salmonella L-forms by the detection of the duplex PCR assay for inv A and STM4495 genes,and the positive rate was 3.17%(9/284),of which 4 gallbladder specimens carried S.Typhimurium L-forms and 5 gallbladder specimens carried other Salmonella L-forms.Among 9 gallbladder specimens carrying Salmonella L-forms, 5 gallbladder specimens(2 biles and 3 gallbladder tissues) were negative by the routine bacteriological methods,but carried 2 strains of S.Typhimurium L-forms and 3 strains of other Salmonella L-forms by the nonhigh osmotic isolation technique.Parts of PCR products of inv A and STM4495 genes were sequenced and analyzed,the results showed that the coincidence ratio of their nucleotide sequences was both 99% by Compared with S.Typhimurium from Gene Bank.Conclusion:(1)S.Typhimurium could occour cell wall defcient and differentiate into S.Typhimurium L-form under pig bile;(2)S.Typhimurium L-form could not be isolated,cultured and identified by the routine bacteriological methods;(3)S.Typhimurium stable L-form could be identified by the the duplex PCR assay for inv A and STM4495 genes;(4)There were Salmonella and lots of other bacterial species in pig gallbladders by the routine bacteriological methods,of which the gram negative bacteria dominated,and the determinative rate of E.coli was the highest;(5)S.Typhimurium L-forms in pig gallbladder could be isolated by the nonhigh osmotic isolation technique;(6)S.Typhimurium L-form could exist in pig gallbladder,and it made the host become a latent reservoir of S.Typhimurium.