Factors Influencing the Ability of Listeria monocytogenes To Pass through a Membrane Filter by Active Infiltration

Abstract

Listeria monocytogenes infiltrated the reticulate structure of a membrane filter and passed through a filter with pore sizes of 0.45 m and 0.2 m in 6 to 24 h and 5 to 6 days, respectively. Flagellar motility and expansive pressure generated by the growing bacterial population were indicated as the driving forces of infiltration.Listeria monocytogenes is a serious threat to humans because of food-borne infections (2, 11). The organism causes generalized infections by exhibiting a remarkable ability to disseminate to the brain, spleen, liver, and other organs (4). This invasive virulence of the organism has been investigated by focusing on the ability of L. monocytogenes to invade and multiply within host cells such as macrophages, hepatocytes, and endothelial cells (3-5). Recently it has been shown that several bacterial species (e.g., L. monocytogenes) have the distinct ability to infiltrate into the reticulate structures of membrane substrates (thickness, 150 m), without artificial pressure, allowing the bacteria to pass through a membrane filter placed on agar media (7). In the present study, we examined the smallest pore size through which L. monocytogenes could pass, the time required for the pass-through, and other influencing factors. Then, the bacterial traits contributing to the ability to pass through membrane filters were analyzed by addressing flagellar motility and flagellum-independent infiltration forces.Bacterial strains and examination of pass-through activity. L. monocytogenes strain EGD was described previously (10). Strain EGDe (DH-L478), its isogenic mutants DH-L975 (Tn917 insertion in flaA [flagellin-encoding gene]), DH-L1042 (flaA in-frame deletion) and the vector pCON1 (6) were supplied by D. E. Higgins, Department of Microbiology and Molecular Genetics, Harvard Medical School. DH-L1042/ pCON1FlaA is a Fla ϩ revertant obtained by electroporation of DH-L1042 with pCON1 carrying flaA DNA (PCR product by primers 5Ј-GGGGTACCCCCGCACAAGTAAGTAAGC CG-3Ј and 5Ј-CGGGATCCCGTAACATTGGCTCTGTGC CCC-3Ј). Leifson's stain (8) was carried out to confirm flagellation of the revertant. L. monocytogenes clinical isolates CL101, CL102, CL142, and CL184, serotype reference strains F4, 1384, and 1684, and Listeria innocua 93/65 were laboratory stocks. Luria-Bertani (LB) broth and 1.5% agar medium (9) were used routinely for the growth of the above strains. Brain heart infusion (BHI) broth and agar medium (Eiken, Tokyo, Japan) and blood (sheep) agar medium (Eiken) were used in some experiments.For examination of membrane filter pass-through activity of bacteria, logarithmic phase bacteria grown in LB broth were centrifuged, washed with sterile saline (0.15 M NaCl) and then suspended in saline (approximately 5 ϫ 10 8 CFU/ml). Ten microliters of the suspension was placed on the center of the autoclaved membrane filter, which was made of mixed esters of cellulose (diameter, 25 mm) and placed on LB agar medium, unless otherwise mentioned. MF-Millipore membrane filters (pore sizes, 0.45, 0.3, and 0.22 m; thickness, 150 ...

Distinct Function of Pseudomonas aeruginosa Type IV Pili Disclosed in the Bacterial Pass‐Through of Membrane Filter with Smaller Pore Sizes

“…Scanning electron micrographs (SEM) of uninoculated filters showed broad void spaces with longer distances (Ͼ1.0 µm) between polymer net fibers composing filters (5, 11). Thus, indicated pore-size values with the membrane filters are not morphologically determined ones but are instead functional values for the ordinary pressure filtration method (11). Consistent with these findings, SEM also indicated that the bacteria in the process of infiltration were not remarkably downsizing (5,11).…”

“…Thus, indicated pore-size values with the membrane filters are not morphologically determined ones but are instead functional values for the ordinary pressure filtration method (11). Consistent with these findings, SEM also indicated that the bacteria in the process of infiltration were not remarkably downsizing (5,11). The semi-quantitative studies showed that required times for the bacterial pass-through were dependent on the indicated filter pore sizes and bacterial species and strains (5).…”

“…These pass-through-permitted filters were examined for the intactness and destructive effects due to the bacterial infiltration. The results obtained by the conventional filtration test and the bubble point test indicated intactness of the filters (5,11). Scanning electron micrographs (SEM) of uninoculated filters showed broad void spaces with longer distances (Ͼ1.0 µm) between polymer net fibers composing filters (5, 11).…”

“…For active bacterial translocation in such reticulated structures, flagella and type IV pili were supposed to work as motility organs. By mutational studies with isogenic mutants, peritrichous flagella of Listeria monocytogenes were shown to work distinctly in the pass-through of membrane filters with a 0.45-µm pore size, but not with the 0.22-µm pore size (11). With regard to the type IV pili of Pseudomonas aeruginosa, twitching motilitypositive strain PAO1 T demonstrated the pass-through of 0.22-µm pore size filter, but the twitching motilitynegative strain PAO1 C did not (5).…”

“…Thus, the limiting pore size for passing through of P. aeruginosa may be between 0.2 µm and 0.1 µm. The equal level of limiting pore size has been indicated in experiments with L. monocytogenes (11).…”