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

Listeria monocytogenes infiltrated the reticulate structureof a membrane filter and passed through a filter with pore sizesof 0.45 µm and 0.2 µm in 6 to 24 h and 5 to 6 days,respectively. Flagellar motility and expansive pressure generatedby the growing bacterial population were indicated as the drivingforces of infiltration.

INTRODUCTION

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Introduction

Listeria monocytogenes is a serious threat to humans becauseof food-borne infections (2, 11). The organism causes generalizedinfections by exhibiting a remarkable ability to disseminateto the brain, spleen, liver, and other organs (4). This invasivevirulence of the organism has been investigated by focusingon the ability of L. monocytogenes to invade and multiply withinhost cells such as macrophages, hepatocytes, and endothelialcells (3-5). Recently it has been shown that several bacterialspecies (e.g., L. monocytogenes) have the distinct ability toinfiltrate into the reticulate structures of membrane substrates(thickness, 150 µm), without artificial pressure, allowingthe bacteria to pass through a membrane filter placed on agarmedia (7). In the present study, we examined the smallest poresize through which L. monocytogenes could pass, the time requiredfor the pass-through, and other influencing factors. Then, thebacterial traits contributing to the ability to pass throughmembrane filters were analyzed by addressing flagellar motilityand flagellum-independent infiltration forces.

Bacterial strains and examination of pass-through activity.

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Bacterial strains and...

L. monocytogenes strain EGD was described previously (10). StrainEGDe (DH-L478), its isogenic mutants DH-L975 (Tn917 insertionin flaA [flagellin-encoding gene]), DH-L1042 (flaA in-framedeletion) and the vector pCON1 (6) were supplied by D. E. Higgins,Department of Microbiology and Molecular Genetics, Harvard MedicalSchool. DH-L1042/pCON1FlaA is a Fla⁺ revertant obtained by electroporationof DH-L1042 with pCON1 carrying flaA DNA (PCR product by primers5'-GGGGTACCCCCGCACAAGTAAGTAAGCCG-3' and 5'-CGGGATCCCGTAACATTGGCTCTGTGCCCC-3').Leifson's stain (8) was carried out to confirm flagellationof 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 usedroutinely 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 ofbacteria, logarithmic phase bacteria grown in LB broth werecentrifuged, washed with sterile saline (0.15 M NaCl) and thensuspended in saline (approximately 5 x 10⁸ CFU/ml). Ten microlitersof the suspension was placed on the center of the autoclavedmembrane filter, which was made of mixed esters of cellulose(diameter, 25 mm) and placed on LB agar medium, unless otherwisementioned. MF-Millipore membrane filters (pore sizes, 0.45,0.3, and 0.22 µm; thickness, 150 µm; Bedford) andADVANTEC filters (pore size, 0.2 µm; thickness, 133 µm;and pore size, 0.1 µm; thickness, 110 µm; Tokyo)were used. After absorbance of the 10-µl droplet (horizontaldiameter, <6 mm) into the filter, the plate was invertedand incubated at 30°C or 37°C. At a 6- or 12-h timeinterval, the filters were removed, and the remaining agar platewas incubated for 24 h and then examined for bacterial growtharound the area where the original 10-µl droplet had beenplaced (Fig. 1). Then, the growing bacteria on the agar mediumwere examined for their Listeria-specific characteristics usingstandard techniques (1). The filter was determined to be intactby using the bubble point test as described previously (7).Pass-through times were recorded in two ways, that is, as theshortest time recorded in the four test filters (in two independentexperiments with two filters) and as the absolute time neededfor pass-through of bacteria in all of the nine test filters(in three independent experiments with three filters). Filterswith the same lot number were used in the repeated experiments.

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FIG. 1. Membrane filter pass-through activity of L. monocytogenes. (A) A membrane filter (pore size, 0.45 µm) placed on LB agar medium was point-inoculated with strain EGD and incubated at 30°C for 24 h. (B) Bacterial growth on the agar medium after removal of the membrane filter. Arrows indicate the growing bacterial population on the filter and the agar medium under the filter. Ring-shaped characteristic bacterial growth corresponding to the circular margin of the inoculated area on the filter is visible on the agar surface.

The influence of filter pore size and incubation temperature on the ability of Listeria strains to pass through a filter.

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The influence of filter...

Eight L. monocytogenes strains with swimming activity at 30°Cand not at 37°C and one L. innocua strain with swimmingactivity at 30°C and 37°C were examined for the shortesttime required to pass through a membrane filter with a poresize ranging from 0.2 to 0.45 µm on LB medium at 30°Cand 37°C (Table 1). All strains were able to pass througha membrane filter with a pore size of 0.2 µm. Generally,there were no remarkable differences among the examined strains.More time was required for the strains to pass through a membranefilter with a smaller pore sizes (e.g., with the EGD strainat 30°C, it took 18 h with a pore size of 0.45 µmand 120 h with a pore size of 0.2 µm). Pass-through ofa membrane filter with a pore size of 0.1 µm was examinedwith L. monocytogenes EGD. No pass-through was seen, even aftera 21-day incubation at 30°C (data not shown). It was noteworthythat the pass-through time for a 0.45-µm-pore-size filterwas shorter at 30°C than at 37°C (Table 1); however,this was not the case with 0.22- or 0.2-µm-pore-size filters.No differences in growth rates of EGD between 30°C- and37°C-LB broth cultures were recognized (data not shown).Since L. monocytogenes is known to be active even at low temperatures,the filter pass-through activity of an EGD strain at 15°Cwas examined. The shortest time required to pass through at15°C was 48 h with a 0.45-µm-pore-size filter, 168h with a 0.3-µm filter, and 456 h with a 0.22-µmfilter.

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TABLE 1. Membrane filter pass-through activity of Listeria spp. at 30°C and 37°C

Micrograph of L. monocytogenes infiltrating into the reticulate structure of a membrane filter.

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Micrograph of l. monocytogenes...

For scanning electron microscopy, membrane specimens were fixedand vacuum dried as described previously (7). Critical pointdrying was not performed because of the incompatibility of theused filter to ethanol and isoamyl acetate. The membrane filterwas mechanically torn to see the inside structures. An S-5000Nmicroscope (Hitachi) was used. Infiltration of L. monocytogenesEGD into the reticulate structure of the membrane filter witha 0.2-µm pore size is evident in Fig. 2A, 2B, and 2C.No remarkable changes in the size of bacterial cells duringpass-through were recognized. It is noteworthy that void spacesin the polymer nets of the membrane substrate are larger thanthe diameter of the rod-shaped cell body (Fig. 2), especiallyin the 0.45-µm-pore-size filter (Fig. 2D). In other words,the pore size described by the manufacturers is not a morphologicallydetermined size, rather an estimated value from the pressuredata in mercury porosimetry and calculation based on Washburn'sequation (12).

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FIG. 2. Scanning electron micrographs of membrane filters (A through C, pore size, 0.2 µm; D, pore size, 0.45 µm). (A) The upper membrane surface inoculated with L. monocytogenes EGD. (B) Inside of the torn membrane with infiltrating EGD bacterial cells. (C) The exit side of the membrane filter permitting pass-through of EGD bacterial cells. (D) Surface of uninoculated membrane filter. Arrows indicate bacteria in the process of passing through. Each bar, 1 µm.

Effect of medium and inoculum conditions.

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Effect of medium and...

The time required for L. monocytogenes to pass through a filterwas shorter when the filter was placed on a blood agar medium.An EGD strain grown in BHI broth was point inoculated on 0.45-µm-pore-sizemembrane filters on three kinds of agar media and incubatedat 30°C. Absolute pass-through times with all nine filtersexamined in each experiment were 6 h on blood agar medium, 18h on BHI agar medium, and 24 h on LB agar medium. Differencesin growth phase (log or stationary) of the inoculated bacteriagave no remarkable differences in the pass-through times ofL. monocytogenes (data not shown). However, inoculum preparedwith the bacterial culture itself without centrifugation andsuspension into saline gave faster pass-through times with a0.45-µm filter at 30°C (12 h absolute pass-throughtime by inoculation of bacteria in culture broth, 24 h by bacteriasuspended in saline). It was noted by microscopic examinationthat the motility of saline-suspended L. monocytogenes was remarkablyreduced (data not shown).

Bacterial driving forces in pass-through of the membrane filter.

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Bacterial driving forces in...

L. monocytogenes is known to be motile and flagellated at 30°Cbut not at 37°C. Since the slower filter pass-through ofL. monocytogenes at 37°C (Table 1) seemed to reflect thedownregulated flagellation and motility of L. monocytogenesat 37°C, we compared the ability of L. monocytogenes EGDestrains with and without flagella to pass through a membranefilter. The results indicated the distinct role of the flagellain pass-through and the permissive pore size for effective flagellarmovement. As shown in Table 2, flaA gene mutants showed slowerabsolute pass-through times with a 0.45-µm filter anda revertant obtaining the plasmid-borne flaA gene passed throughat the same rate as wild-type EGDe. However, as shown in Table1, which presents the temperature-independent pass-through for0.22- and 0.2-µm pore sizes, the flagellum-dependent fasterpass-through of flagellated wild-type and revertant strainswas not seen with a 0.22-µm filter (Table 2). A pore sizeof 0.45 µm in the membrane filter seems to give enoughspace for flagellar rotation as seen in Fig. 2D, but the poresize may be too small for flagellar rotation in a 0.2-µmfilter (Fig. 2C). For slow pass-through of the membrane filterwith smaller pore sizes, expansion of the cellular populationmass by bacterial multiplication seems to be the main drivingforce. Similarly, slow pass-through behavior of L. monocytogenesin an agar gel (reticulate structure of agarose/agaropectinpolymer net) has been observed previously (10) as three-dimensionalfractal growth of the bacterial population infiltrating intohard agar medium in seven weeks. The infiltration ability ofmicroorganisms into reticulate structures seems to be an importantfactor for invasion into animal and plant tissues and in contaminationof food materials.

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TABLE 2. Role of L. monocytogenes flagella in the pass-through of membrane filters

ACKNOWLEDGMENTS

We thank D. E. Higgins for supplying the bacterial strains andthe plasmid. The Electron Microscope Core Facility at NiigataUniversity is acknowledged for the scanning electron microscopicstudies.

This study was supported by a grant from the Urakami Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan. Phone: 81 25 227 2111. Fax: 81 25 262 6360. E-mail: tohey{at}med.niigata-u.ac.jp.

REFERENCES

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