Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique

• Published in: Clinical microbiology and infection Vol. 21; no. 5; pp. 470.e9 - 470.e16
• Main Authors: Wang, D.N., Wu, W.J., Wang, T., Pan, Y.Z., Tang, K.L., She, X.L., Ding, W.J., Wang, H.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.05.2015
• Subjects: Bacterial L-form; Bacterial Proteins - genetics; Bacteriological Techniques - methods; Bile - microbiology; biliary diseases; Culture Media - chemistry; Gallbladder - microbiology; gene assay; Humans; L Forms - isolation & purification; Paratyphoid Fever - microbiology; Polymerase Chain Reaction; Salmonella Paratyphi A; Salmonella paratyphi A - genetics; Salmonella paratyphi A - isolation & purification; Salmonella Typhi; Salmonella typhi - genetics; Salmonella typhi - isolation & purification; Sequence Analysis, DNA; Typhoid Fever - microbiology; Virulence Factors - genetics; Bacterial L-form; gene assay; biliary diseases; Salmonella Typhi; Salmonella Paratyphi A
• ISSN: 1198-743X; 1469-0691
• DOI: 10.1016/j.cmi.2014.12.016

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.