title: Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique.
abstract: Bacterial L-forms have always been considered as osmotic-pressure-sensitive cell-wall-deficient bacteria and isolation culture of L-forms must use media with high osmotic pressure. However, isolation culture of stable L-forms formed in humans and animals is very difficult because they have adapted to the physiological osmotic pressure condition of the host. We use a non-high osmotic isolation technique to isolate stable L-forms of Salmonella Typhi and Salmonella Paratyphi A from bile-inducer cultures in vitro and from patients' gallbladder specimens. Multiplex PCR assay for Salmonella-specific genes and nucleotide sequencing are used to identify the Salmonella L-forms in stable L-form isolates. Using this method, we confirmed that Salmonella Paratyphi A and Salmonella Typhi cannot be isolated from bile-inducer cultures cultured for 6 h or 48 h, but the L-forms can be isolated from 1 h to 45 days. In the 524 gallbladder samples, the positive rate for bacterial forms was 19.7% and the positive rate for Salmonella spp. was 0.6% by routine bacteriological methods. The positive rate for bacterial L-forms was 75.4% using non-high osmotic isolation culture. In the L-form isolates, the positive rate of Salmonella invA gene was 3.1%. In these invA-positive L-form isolates, four were positive for the invA and flic-d genes of Salmonella Typhi, and ten were positive for the invA and flic-a genes of Salmonella Paratyphi A.
authors.affiliation: Department of Medical Microbiology and Parasitology, Institutes of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
authors.forename: D N
authors.initials: DN
authors.lastname: Wang
authors.affiliation: Department of Medical Microbiology, Guiyang Medical University, Guiyang, China; Guiyang City Maternal and Child Health Care Hospital, Guiyang, China.
authors.forename: W J
authors.initials: WJ
authors.lastname: Wu
authors.affiliation: Department of Medical Microbiology, Guiyang Medical University, Guiyang, China. Electronic address: wangtao309@com.com.
authors.forename: T
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Surgery, Affiliated Hospital of Guiyang Medical University, Guiyang, China.
authors.forename: Y Z
authors.initials: YZ
authors.lastname: Pan
authors.affiliation: Department of Surgery, Guizhou Province People's Hospital, Guiyang, China.
authors.forename: K L
authors.initials: KL
authors.lastname: Tang
authors.affiliation: Department of Medical Microbiology, Guiyang Medical University, Guiyang, China.
authors.forename: X L
authors.initials: XL
authors.lastname: She
authors.affiliation: Department of Medical Microbiology, Guiyang Medical University, Guiyang, China; Guiyang City Maternal and Child Health Care Hospital, Guiyang, China.
authors.forename: W J
authors.initials: WJ
authors.lastname: Ding
authors.affiliation: Department of Medical Microbiology, Guiyang Medical University, Guiyang, China. Electronic address: cwdbwh@126.com.
authors.forename: H
authors.initials: H
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D001646:Bile / Q000382:microbiology*; D003470:Culture Media / Q000737:chemistry; D005704:Gallbladder / Q000382:microbiology*; D006801:Humans; D007740:L Forms / Q000302:isolation & purification*; D010284:Paratyphoid Fever / Q000382:microbiology; D016133:Polymerase Chain Reaction; D012482:Salmonella paratyphi A / Q000235:genetics / Q000302:isolation & purification*; D012485:Salmonella typhi / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D014435:Typhoid Fever / Q000382:microbiology; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D037521:Virulence Factors; C075518:invA protein, Bacteria
keywords: Bacterial L-form; Salmonella Paratyphi A; Salmonella Typhi; biliary diseases; gene assay