Use of PCR-restriction fragment length polymorphism of inlA for rapid screening of Listeria monocytogenes strains deficient in the ability to invade Caco-2 cells.
A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.
Rousseaux
S
S
Laboratoire de Microbiologie, UMR INRA 1232, ENSBANA, 21000 Dijon, France.
Olier
M
M
Lemaître
J P
JP
Piveteau
P
P
Guzzo
J
J
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018938:Caco-2 Cells; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C069761:internalin protein, Bacteria