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

Abstract

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.

FIG. 1.

Structural organization of internalin and partial map of inlA (20). The horizontal arrows indicate the positions of primers seq01 and seq02 used to amplify an inlA fragment with genetic heterogeneity. The solid circle indicates the position of the mutation which created a nonsense codon in inlA of noninvasive reference strains H1, H17, H32, and H34. The solid diamond indicates the position of the deletion which created a nonsense codon in inlA of noninvasive reference strain LO28.

FIG. 2.

PCR-RFLP profiles of different strains obtained after restriction with endonucleases AluI (A) and Tsp509I (B). Lanes 1 and 18, molecular weight ladder; lane 2, noninvasive reference strain H32; lane 3, food isolate 1S; lane 4, food isolate 2S; lane 5, food isolate 1F; lane 6, food isolate 2F; lane 7, food isolate NV4; lane 8, noninvasive reference strain LO28; lane 9, invasive reference strain EGD-e; lane 10, human fecal carriage isolate H2; lane 11, sporadic patient isolate H21; lane 12, isolate 1E from food-processing facilities; lane 13, compost isolate C9; lane 14, invasive reference strain Scott A; lane 15, sporadic patient isolate H4; lane 16, rook fecal isolate 97; lane 17, isolate 6E from food-processing facilities.