Soft-Agar-Coated Filter Method for Early Detection of Viable and Thermostable Direct Hemolysin (TDH)- or TDH-Related Hemolysin-Producing Vibrio parahaemolyticus in Seafood

Bacterial strains.A total of 54 V. parahaemolyticus strains including two tdh-positive and trh-positive (tdh⁺trh⁺) strains, 35 only tdh-positive strains, 9 only trh-positive strains, and 8 tdh-negative and trh-negative strains of various serotypes (i.e., O1:K1, O1:K25, O3:K6, O3:K48, O4:K6, O4:K8, O4:K68, and O8:K41) were used to evaluate the specificity of the multiplex PCR assay described below. Among them, V. parahaemolyticus KE 10540 (a clinical isolate; serotype O3:K46, tdh⁺trh⁺) was used to determine the sensitivity of the multiplex PCR assay. Strains KE 10540, KE 10460 (an environmental isolate; O3:K56, lacking both tdh and trh) and an environmental isolate of V. alginolyticus AKO18 (motile) were used for the preliminary experiments described below. Furthermore, 23 strains of various serotypes positive for either or both tdh and trh were used for a subsequent quasi-field experiment. The strains were maintained on heart infusion agar (Difco Laboratories, Detroit, Mich.) containing 2% NaCl (final concentration) until use.

Specificity and sensitivity tests of multiplex PCR.Whole genomic DNAs of the 54 V. parahaemolyticus strains were prepared in Tris-EDTA buffer (pH 8.0) essentially as described elsewhere (1). The DNA preparations thus obtained were used as templates to evaluate the specificity of the multiplex PCR assay targeting both tdh and trh. KE 10540 was inoculated into alkaline peptone water (APW; 10 g of peptone and 10 g of NaCl in 1,000 ml of distilled water, pH 8.8) and incubated at 37°C with shaking for 10 h to obtain exponential growth (ca. 3.0 × 10⁸ CFU/ml). After incubation, 100 μl each of a series of 10-fold dilutions (10⁻¹ to 10⁻⁸ or ca. 3.0 × 10⁷ to 30 × 10⁷ CFU/ml) of the culture was dispensed into microtubes and heated at 100°C for 10 min. After centrifugation at 10,000 × g for 5 min, the supernatants were used as template DNAs to determine the sensitivity of the multiplex PCR assay.

Multiplex PCR was performed with the oligonucleotide primers 5′-CCACTACCACTCTCATATGC-3′ (sense primer) and 5′-GGTACTAAATGGCTGACATC-3′ (antisense primer) at positions 451 to 469 and 713 to 694 in tdh, respectively (positions according to Honda et al. [6]) (GenBank accession no. D90238) to yield a 251-bp fragment and with the primers 5′-TTGGCTTCGATATTTTCAGTATCT-3′ (sense primer) and 5′-CATAACAAACATATGCCCATTTCCG-3′ (antisense primer) at positions 561 to 576 and 1211 to 1196 in trh, respectively (positions according to Lin et al. [11]) (GenBank accession no. L11929) to yield a 500-bp fragment. PCR amplification was performed in a total volume of 20 μl. Two microliters of each template DNA preparation was added to the PCR master mix, which consisted of 2 μl of 10× PCR buffer (Mg²⁺ free; Promega Corporation, Madison, WI), 2.4 μl of 25 mM MgCl₂ (final concentration, 3.0 mM), 0.25 μl of a deoxynucleoside triphosphate mixture (a 0.125 mM concentration of each deoxynucleoside triphosphate), 0.125 μl of each primer (0.125 μM concentration of each primer), and 0.125 μl (0.625 U) of Taq DNA polymerase (Takara Bio Co., Shiga, Japan), with the remaining volume consisting of distilled water. A GeneAmp PCR System 2700 thermal cycler (Applied Biosystems, Foster City, Calif.) was used for PCR amplification consisting of initial denaturation at 94°C for 3 min; 25 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, and extension at 72°C for 60 s; and a final extension at 72°C for 5 min. Five microliters of the PCR products was electrophoresed on 2% agarose gels, stained with ethidium bromide (0.25 μg/ml), and photographed under UV light.

Preliminary experiments.In order to establish a reliable protocol for the method, the influence of incidental dead cells or viable avirulent strains and the effects of DNase treatment on the culture prior to DNA template preparation were evaluated as follows.

Preparation of bacterial inoculants.KE 10540 (tdh⁺trh⁺) was inoculated to 200 ml of APW and incubated at 37°C with shaking for 10 h. After incubation, a portion of the culture was diluted with sterile saline to prepare a bacterial suspension of ca. 5.0 × 10² CFU/ml to be used as “live inoculant” of KE 10540. Another portion (100 ml) of the culture was centrifuged at 8,000 × g for 10 min, and the supernatants were discarded. The bacterial pellet was resuspended in 10 ml of saline containing 200 ppm sodium hypochlorite and incubated at room temperature for 4 h to kill all viable cells. Subsequently, the bacterial cells were washed three times with sterile saline and finally suspended in 1 ml of sterile saline to be used as “dead inoculant” of KE 10540 (ca. 3.0 × 10¹⁰ dead cells/ml). In addition, KE 10460 (lacking both tdh and trh) or V. alginolyticus AKO 18 (lacking both tdh and trh) was inoculated into 100 ml of APW and incubated at 37°C with shaking for 10 h. After incubation, the culture was centrifuged at 8,000 × g for 10 min. Bacterial cells were washed three times with sterile saline and finally suspended in 1 ml of sterile saline to be used as “live inoculant” (approximately 3.0 × 10¹⁰ CFU/ml) of KE 10460 or AKO 18.

Experiment 1a.Filter papers (Whatman no. 6; pore size, 3 μm; diameter, 110 mm [Maidstone, England]) folded into a cone shape were autoclaved. After autoclaving, the sterile filter papers were submerged for 5 min in APW containing 0.5% agar (agar no. 1; Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) maintained at 50 to 60°C. After submersion, the filter papers (Fig. 1a) were removed from the medium and placed in an upright position on a sterile dish in a clean bench for 30 min to produce soft-agar-coated filters.

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FIG. 1.

Photographs of the soft-agar-coated filter method. (a) A Whatman no. 6 filter paper is folded into a cone shape and coated with 0.5% agar. (b) A sterile polypropylene funnel is placed over an alkaline peptone water container. (c) Ten milliliters of APW is added to the cone. (d) A lid is loosely fitted over the container.

Two hundred microliters of viable inoculant of KE 10540 (tdh⁺trh⁺) was added to APW (190 ml) in a sterile plastic container (BC2200; outside diameter, 73 mm; internal diameter, 63 mm; length, 90.5 mm; equipped with a screw lid) (Eiken Kizai Co. Ltd., Tokyo, Japan) in which the total cell number in the medium was calculated to be ca. 100 viable cells. A sterile polypropylene funnel (NL4252-0065; maximum diameter, 65 mm; minimum diameter, 16 mm; length, 67 mm) (Nalgene Co., Rochester, N.Y.) was placed over the container (Fig. 1b). The soft-agar-coated filter paper was then fitted onto the funnel, and 10 ml of APW was added into the cone (Fig. 1c); finally, a lid was loosely fitted over the container (Fig. 1d). The containers were then incubated at 37°C for 6, 8, 12, 16, or 20 h.

After incubation, 1 ml of the culture in the cone was transferred to a microtube and centrifuged at 10,000 × g for 5 min. After centrifugation, the supernatant was discarded, and the bacterial pellet was suspended in 100 μl of sterile saline. The suspension was placed in another microtube and mixed with 26 μl of DNase solution containing 25 U of DNase (DNase I from bovine pancreas in 50% glycerol solution with 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM CaCl₂; Sigma Chemical Co., St. Louis, Mo.) and 14 μl of buffer solution (400 mM Tris-HCl, pH 7.5, 80 mM MgCl₂, 50 mM dithiothreitol in ultra-purified water) and incubated at 37°C for 1 h.

After DNase treatment, half (50 μl) of the mixture was placed in a microtube and heated at 100°C for 10 min and used as a source of template DNA for the multiplex PCR assay. In addition, we performed the following PCR assays on the template DNA in order to ensure the validity of any negative result of the multiplex PCR assay: PCR assay targeting the 16S rRNA sequence universal to nearly any bacterium with the oligonucleotide primers 5′-CAGGCCTAACACATGCAAGTC-3′ (sense primer) and 5′-GGGCGGWGTGTACAAGGC-3′ (antisense primer) to yield a ca. 1,300-bp fragment (13), and an assay targeting the ToxR gene sequence species-specific to V. parahaemolyticus with the oligonucleotide primers 5′-GTCTTCTGACGCAATCGTTG-3′ (sense primer) and 5′-ATACGAGTGGTTGCTGTCATG-3′ (antisense primer) to yield a 368-bp fragment (9) in order to confirm the presence of any bacterial cells and V. parahaemolyticus cells in the culture, respectively (collectively referred to as “positive control” PCR assays hereafter).

Meanwhile, the remaining mixture (50 μl) was placed in a microtube and centrifuged at 10,000 × g for 5 min. After centrifugation, 20 μl of the supernatant was placed in a microtube and heated at 100°C for 10 min and then used as a source of “template DNA” for the multiplex PCR assay in order to confirm that any free-ranging DNA fragments containing tdh or trh had been completely digested through DNase treatment (referred to as “negative control” PCR assay hereafter).

Experiment 1b.Two hundred microliters of viable inoculant of KE 10540 or 200 μl of the dead inoculant of KE 10540 was added to APW (190 ml) in a sterile plastic container, in which the total number of cells in the medium was calculated to be ca. 100 viable cells or ca. 6.0 × 10⁸ dead cells, respectively. A soft-agar-coated filter paper with a sterile funnel was placed over the container. Ten milliliters of APW was then added to the bottom of the cone, and finally the lid was loosely fitted over the container. The container was then incubated at 37°C for 20 h. After incubation, the culture was treated with or without DNase, and template DNAs thus prepared were used for the multiplex PCR assay and the positive and negative control PCR assays.

Experiment 2a.Two hundred microliters of the dead inoculant of KE 10540 and 200 μl of the viable inoculant of KE 10460 or AKO 18 (combinations 1 and 2, respectively) were added to APW (190 ml) in a sterile plastic container, in which the initial cell number in the medium was calculated to be ca. 6.0 × 10⁸ dead cells for KE 10540 and ca. 6.0 × 10⁸ viable cells for KE 10460 or AKO 18. As described for experiment 1, a sterile funnel with a cone-shaped filter paper coated with 0.5% soft agar was placed over the container, and 10 ml of APW was added to the bottom of the cone; finally, a lid was loosely fitted over the container. The container was then incubated at 37°C for 20 h. After incubation, template DNA from the culture that had been pretreated with DNase was assayed by the multiplex PCR and the positive and negative control PCR assays. The experiment was repeated in triplicate.

Experiment 2b.Two hundred microliters of the viable inoculant of KE 10540 and 200 μl of the live inoculant of KE 10460 or AKO 18 (combinations 3 and 4, respectively) were added to APW (190 ml) in a sterile plastic container, in which the initial cell number in the medium was calculated to be ca. 100 viable cells of KE 10540 and ca. 6.0 × 10⁸ viable cells of KE 10460 or AKO 18. The samples thus prepared were then processed and assayed in the same manner as described for experiment 2a. The experiment was repeated in triplicate.

Quasi-field experiment.Each of the 23 strains of the various serotypes (see Table 2) that were positive for either or both tdh and trh was inoculated into 100 ml of APW and incubated at 37°C with shaking for 10 h. After incubation, a portion of the culture was diluted with sterile saline to prepare a bacterial suspension of ca. 1 × 10² to ∼2 × 10² CFU/ml. Twenty grams of various commercially available seafood (seven packages of prawns, four packages of short-necked clams, five packages of scallops, and five packages of oysters) with or without 200 μl of the above bacterial suspension was added to APW (170 ml) in a sterile plastic container in which the initial number of artificially inoculated viable cells per 1 g of each seafood sample was calculated to be approximately 5 to ∼10. The samples thus prepared were then processed and incubated in the same manner as described for experiment 2a.

After incubation, 1 ml of the culture in the cone was transferred to a microtube and centrifuged at 10,000 × g for 5 min, and the bacterial pellet was suspended in 100 μl of sterile saline. The suspension was processed in the same manner described above to prepare sources of template DNAs to be analyzed by multiplex PCR and the positive and negative control PCR assays.

Specificity and sensitivity of the multiplex PCR assay.Assay results of multiplex PCR on the 54 V. parahaemolyticus strains were consistent with those obtained from the conventional tdh- or trh-targeted PCR assay (19). The sensitivity of multiplex PCR was determined using serial 10-fold dilutions of KE 10540 cells. Following agarose gel electrophoresis, the minimal cell concentration detectable was 3.0 × 10⁴ cells/ml (data not shown). The corresponding sensitivity was ca. 60 DNA copies in 2 μl of template preparation.

Incubation time and DNase pretreatment.The results of experiment 1a indicated that the reliable incubation time required for detecting the inoculated viable strain positive for both tdh and trh was 20 h (data not shown). The results of experiment 1b indicated that DNase pretreatment did not adversely affect the multiplex PCR assay on viable cells (Fig. 2). The assay without DNase pretreatment on the sample containing dead virulent cells yielded PCR products of tdh and trh, indicating that DNA released from dead cells into the medium had indeed diffused through the soft-agar-coated filter to the medium inside the cone. Meanwhile, the multiplex PCR assay and the negative PCR assay on the DNase-pretreated sample yielded no PCR products, indicating that the “diffused-through” DNAs were completely digested, thereby preventing any false-positive assay result.

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FIG. 2.

Results of the multiplex PCR assay with or without DNase pretreatment of cultures initially inoculated with viable or dead V. parahaemolyticus KE 10540 (tdh⁺trh⁺) in the soft-agar-coated filter method. Template DNAs were prepared from the original culture initially inoculated with viable cells (lane 1); the same culture was also treated with DNase (lane 2). Template DNAs were prepared from the original culture initially inoculated with dead cells (lane 3); the same culture was also treated with DNase (lane 4). Lane M, molecular size markers (mixture of phage λ DNA digested with HindIII and phage φX174 DNA digested with HaeIII). PCR amplicon sizes for tdh and trh are 251 bp and 500 bp, respectively.

Influences of competing bacterial cells.Results of experiment 2a (Table 1) indicated that the sample initially containing dense populations of dead tdh⁺trh⁺ cells and viable cells lacking tdh and trh (combinations 1 and 2) did not yield any positive results in the multiplex PCR assay, indicating that dead cells (without motility) of the tdh⁺trh⁺V. parahaemolyticus did not pass through the soft-agar-coated filter. In contrast, the results of experiment 2b (Table 1) indicated that the sample initially containing a very small population of viable virulent cells and a dense population of viable avirulent cells (combinations 3 and 4) yielded positive results in the multiplex PCR assay. This, in turn, indicates that concomitant bacterial cells, even densely populated, in the sample do not prevent the passage of motile, viable virulent V. parahaemolyticus cells through the filter.

Results of the quasi-field experiment.The seafood samples artificially inoculated with small numbers of viable virulent strains of various serotypes all yielded positive results in the subsequent multiplex PCR assay, whereas those without artificial inoculation did not (Table 2).