Delayed lactose utilization among Shiga toxin-producing Escherichia coli of serogroup O121.

Two outbreaks of Shiga toxin-producing Escherichia coli O121:H19 associated with wheat flour, in the United States of America and Canada, involved strains with an unusual phenotype, delayed lactose utilization (DLU). These strains do not ferment lactose when initially cultured on MacConkey agar (MAC), but lactose fermentation occurs following subculture to a second plate of MAC. The prevalence of DLU was determined by examining the ß-galactosidase activity of 49 strains of E. coli O121, and of 37 other strains of E. coli. Twenty four of forty three O121:H19 and one O121:NM displayed DLU. Two strains (O121:NM and O145:H34) did not have detectable ß-galactosidase activity. ß-glucuronidase activity of O121 strains was also determined. All but six DLU strains had normal ß-glucuronidase activity. ß-glucuronidase activity was suppressed on MAC for 17 of 23 O121 non-DLU strains. Genomic analysis found that DLU strains possessed an insertion sequence, IS600 (1267 bp), between lacZ (ß-galactosidase) and lacY (ß-galactoside permease), that was not present in strains exhibiting normal lactose utilization. The insert might reduce the expression of ß-galactoside permease, delaying import of lactose, resulting in the DLU phenotype. The high probability of DLU should be considered when using lactose-containing media for the isolation of STEC O121.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase.

Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan-Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase (cst) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

Simultaneous Identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari with SmartCycler-Based Multiplex Quantitative Polymerase Chain Reaction.

BACKGROUND: Consumption of Campylobacter contaminated food or water is a leading cause of human acute gastroenteritis. Campylobacter jejuni, Campylobacter coli, and Campylobacter lari account for over 95% of total Campylobacter infections. A multiplex quantitative polymerase chain reaction (qPCR) for simultaneous identification of C. jejuni, C. coli, and C. lari was developed for use with the SmartCycler II system. MATERIALS AND METHODS: We evaluated and combined previously described primers and probes for Campylobacter detection, designed a new internal amplification control, and optimized the multiplex qPCR for the detection of C. jejuni, C. coli, and C. lari. RESULTS: This method was 100% specific when tested against a panel of 32 target Campylobacter strains and 31 non-Campylobacter reference strains. Furthermore, there was no cross-reactivity with seven strains from four nontarget Campylobacter species. The amplification efficiency of each target in this multiplex qPCR was over 90%, and each coefficient of linearity was greater than 0.99. With artificially mixed genomic DNA, this method detected as few as two, three, and two genome copies of C. jejuni, C. coli, and C. lari, respectively. This method was also able to detect these three Campylobacter species in artificially contaminated milk with a sensitivity of five spiked cells of each target per reaction. CONCLUSION: The three Campylobacter targets were simultaneously identified using artificially mixed genomic DNA and spiked raw milk. This SmartCycler-based multiplex qPCR is a rapid, specific, and sensitive method to identify C. jejuni, C. coli, and C. lari.

Genome sequencing and annotation of a Campylobacter coli strain isolated from milk with multidrug resistance.

As the most prevalent bacterial cause of human gastroenteritis, food-borne Campylobacter infections pose a serious threat to public health. Whole Genome Sequencing (WGS) is a tool providing quick and inexpensive approaches for analysis of food-borne pathogen epidemics. Here we report the WGS and annotation of a Campylobacter coli strain, FNW20G12, which was isolated from milk in the United States in 1997 and carries multidrug resistance. The draft genome of FNW20G12 (DDBJ/ENA/GenBank accession number LWIH00000000) contains 1, 855,435 bp (GC content 31.4%) with 1902 annotated coding regions, 48 RNAs and resistance to aminoglycoside, beta-lactams, tetracycline, as well as fluoroquinolones. There are very few genome reports of C. coli from dairy products with multidrug resistance. Here the draft genome of FNW20G12, a C. coli strain isolated from raw milk, is presented to aid in the epidemiology study of C. coli antimicrobial resistance and role in foodborne outbreak.

Detection and isolation of low levels of E. coli O157:H7 in cilantro by real-time PCR, immunomagnetic separation, and cultural methods with and without an acid treatment.

UNLABELLED: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. PRACTICAL APPLICATION: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.

Efficacy of a post enrichment acid treatment for isolation of Escherichia coli O157:H7 from alfalfa sprouts.

The enrichment, detection and isolation procedure in the current US FDA BAM have been shown effective for Escherichia coli O157:H7 in a wide variety of foods. Recently reported modifications to the enrichment protocol, including post-enrichment immunomagnetic separation (IMS) procedures have improved sensitivity of the method for alfalfa sprouts. However, cultural isolation on selective agar plates still presents a challenge in this food matrix. The focus of this study was to reduce levels of competing microflora and enhance isolation of E. coli O157:H7 on selective agars. We optimized the use of a short acid treatment after enrichment and with post-enrichment IMS beads. The optimized acid treatments were then evaluated in alfalfa sprouts artificially contaminated with E. coli O157:H7. After 5h enrichment of alfalfa sprouts contaminated at 0.2cfu/g, there was significant improvement in isolation on the selective plates following acid treatment of two types of IMS beads. Following 24h enrichment of alfalfa sprouts contaminated at low levels, E. coli O157:H7 was only recovered from 8/25 samples when no IMS or acid treatments were used. The use of only the acid treatment improved recovery to 19/25 samples. Following IMS of the enrichment broths, acid treatment increased isolation to 23/25 for Pathatrix™ and 24/25 for BeadRetriever™ IMS. Acid treatment (pH 2) of the enrichment broth for 30min or IMS beads for 7min is a simple and rapid way to greatly improve isolation of E. coli O157 from alfalfa sprout enrichments by reducing the interfering microflora on the selective media.

Evaluation of shiga toxin-producing Escherichia coli (STEC) method for the detection and identification of STEC O104 strains from sprouts.

Protocols for Shiga toxin-producing Escherichia coli (STEC) typically focus on the detection and recovery of E. coli O157:H7; however, the prevalence of outbreaks associated with non-O157 STEC are increasing and the efficacy of current testing strategies have not been fully evaluated. Non-O157 STEC are a very diverse group whose pathogenic characteristics and clinical significance have not been well established. During an outbreak situation, the rapid dissemination of specific strain characteristics can provide information needed to verify standard laboratory methodology and identify potentially effective tests to detect and recover the outbreak strain from foods. This study validated the use of a standard method to detect and recover two strains of E. coli O104 STEC at a level of approximately 1 CFU/g from sprouts. The use of additional serotype-specific real-time PCR assays and supplemental chromogenic media to assist the detection and recovery of these organisms were also evaluated.

Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes.

Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx(1d) and stx(2f), and some also missed stx(2b) and stx(2g). Most of the serological assays examined did not detect Stx2c, Stx2e, Stx2f, and Stx2g, and some strain-to-strain variation in reactivity was observed for Stx2b.

Optimization and evaluation of a modified enrichment procedure combined with immunomagnetic separation for detection of E. coli O157:H7 from artificially contaminated alfalfa sprouts.

Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37°C for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix.

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods.

Detection of Escherichia coli O157:H7 by conventional cultural methods can be difficult in food matrices with a high background flora such as raw ground beef. Raw ground beef samples, artificially contaminated separately with five strains of E. coli O157:H7 at low (~0.2 cfu/g) and high (~2 cfu/g) levels, were enriched by two enrichment protocols; buffered peptone water (BPW) at 37 °C for 5h and 24h and modified buffered peptone water with pyruvate (mBPWp) for 5h at 37 °C followed by adding selective agents and incubating at 42 °C to 24h. Detection of added E. coli O157:H7 by real-time PCR (RTiPCR) and recovery on isolation agars was performed before and after PATHATRIX™ immunomagnetic separation (IMS). RTiPCR detection and cultural recovery of inoculated E. coli O157:H7 after 5h enrichment were poor at 0.21-0.24 cfu/g. The addition of IMS after 5h enrichment did not improve RTiPCR detection but markedly improved recovery by culturing. By extending enrichment to 24h, RTiPCR detection improved to 76% for either enrichment protocol without IMS. When 24h enrichment was followed by IMS, RTiPCR detection was also further improved. Cultural recovery after 24h enrichment was 56% and 84% without IMS and 100% and 92% after IMS for BPW and mBPWp respectively. Extended enrichment to 24h followed by IMS was found to be sensitive and reliable for detection and cultural recovery from raw ground beef using either enrichment method.

Multiplex real-time PCR detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli.

A multiplex real-time PCR method was developed for detection of heat-labile and heat-stable toxin genes in enterotoxigenic Escherichia coli. Approximately 10 CFU per reaction mixture could be detected in rinsates from produce samples. Several foods representative of varieties previously shown to have caused enterotoxigenic E. coli outbreaks were spiked and enriched for 4 or 6 h. Both heat-labile and heat-stable toxin genes could be detected in the foods tested, with the exception of hot sauce, with threshold cycle values ranging from 25.2 to 41.1. A procedure using membrane filtration which would allow enumeration of the enterotoxigenic E. coli population in a food sample in less than 28 h by real-time PCR analysis of colonies picked from media highly selective for E. coli was also developed.

Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR.

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the beta-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA (multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, degrees C) for PCR products were 85.42--stx1, 81.93--stx2, and 88.25--uidA in simplex assays and 85.20--stx1, 81.20--stx2, and 88.16--uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.

Optimization of a 3'-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR.

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA (gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5' nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non-E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.

Multiplex real-time PCR method to identify Shiga toxin genes stx1 and stx2 and Escherichia coli O157:H7/H- serotype.

A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H- uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were >/= 1.89, and as few as 6 CFU/reaction could be detected.

Evaluation and interlaboratory validation of a selective agar for phosphatidylinositol-specific phospholipase C activity using a chromogenic substrate to detect Listeria monocytogenes from foods.

Phosphatidylinositol-specific phospholipase C (PI-PLC) activity is a potential virulence factor and is exhibited only by the Listeria species Listeria monocytogenes and Listeria ivanovii. A chromogenic substrate for the direct detection of PI-PLC activity is available in a new medium (BCM L. monocytogenes plating agar). The use of a chromogenic substrate offers a mechanism with which to directly screen for L. monocytogenes and L. ivanovii other than the esculin used in Oxford (OXF) and Palcam (PAL) agars, which screen for all Listeria species. The specificity levels of BCM plating agar and of BCM confirmation and rhamnose agars were evaluated with 107 Listeria and 10 Bacillus species isolates. In addition, BCM L. monocytogenes plating agar was compared with standard Listeria selective agars (OXF and PAL agars) with regard to the recovery of L. monocytogenes from 2,000 food and environmental samples obtained from eight participating laboratories. A Listeria species was isolated from at least one of the agars in 209 analyses, and L. monocytogenes was isolated in 135 of these analyses. In 27 of the analyses in which L. monocytogenes was isolated, one or more of the selective differential agars used failed to isolate L. monocytogenes, and therefore the results of these analyses were discrepant. Relative to a reference method involving the use of all three agars (OXF, PAL, and BCM agars), the OXF-BCM, PAL-BCM, and OXF-PAL combinations had sensitivities of 99.3, 99.2, and 90.2%, respectively. In statistical analyses of the different combinations of agars, the OXF-BCM and BCM-PAL combinations were found to be superior to the OXF-PAL combination for the detection of L. monocytogenes.

An Improved Rapid Technique for Isolation of Escherichia coli O157:H7 from Foods.

A newly revised enrichment and agar-plating system was tested for selectivity and sensitivity in recovery of unstressed and cold-stressed Escherichia coli O157:H7 from foods. Various foods inoculated with known levels of enterohemorrhagic E. coli O157:H7 (EHEC) were tested by enrichment for 6 h at 37°C in modified tryptic soy broth (mTSB) base supplemented with vancomycin, cefsulodin and cefixime, referred to as EHEC enrichment broth (EEB). Subsequently, portions were spread-plated on sorbitol-MacConkey agar supplemented with tellurite and cefixime (TCSMAC). Further selective enrichment was also examined using immunomagnetic separation (IMS) from the EEB prior to spread-plating on TCSMAC agar. These methods were compared to a procedure of enrichment in mTSB (supplemented with novobiocin) at 37°C for 24 h followed by spread-plating of decimal dilutions on hemorrhagic colitis 4-methylumbelliferyl-B-D-glucuronide (HC-MUG) agar. The new enrichment isolation technique was found to be sensitive at a level of one EHEC organism per 10 g of food in four food types. This represents an approximate l00-fold to 1,000-fold enhancement in sensitivity over the comparative method for foods with high levels of competitive microflora. These enrichment-isolation protocols also were compared in analysis of naturally contaminated raw or undercooked ground beef samples implicated in foodborne illness. EEB-TCSMAC with and without IMS were combined with rapid biochemical tests, and with O157 latex agglutination and confirmation of toxin genes by polymerase chain reaction (PCR) to provide a completed test within 30 h of initiating testing. The new system was successful in 15 of 17 samples, where only 6 of 17 were found positive by the comparative technique.

Comparison of Template Preparation Methods from Foods for Amplification of Escherichia coli O157 Shiga-Like Toxins Type I and II DNA by Multiplex Polymerase Chain Reaction.

Escherichia coli O157:H7 has been responsible for several recent food-borne outbreaks in the United States. To protect the public health, it is essential that rapid and sensitive methods be developed for detection of this pathogen in foods. Methods were compared for preparation of template DNA for the polymerase chain reaction (PCR) from enrichments of food homogenates seeded with E. coli O157:H7. Samples were enriched for 6 h at 37°C in modified tryptic soy broth supplemented with vancomycin, cefsulodin, and cefixime. Aliquots of the enrichments (10 ml or 1 ml) were analyzed by either washing twice with physiological saline or incubating with antibodies to O157 coupled to immunomagnetic beads (Dynal®) followed by resuspending and boiling the samples. A portion of the preparation was used in a multiplex PCR to amplify a 274-bp fragment from the sltI gene and a 364-bp fragment from the sltII gene. PCR amplification of 1-ml portions of enrichment broth was successful at inoculation levels of about 10 cells per g of food. Increasing the test sample volume to 10 ml and/or using an immunomagnetic separation step improved the PCR detection sensitivity to about 1 cell per g; the entire analysis can be completed within 12 h.

Anisakid Parasites, Staphylococcus aureus and Bacillus cereus in Sushi and Sashimi from Seattle Area Restaurants.

Samples of salmon, tuna, mackerel, and rockfish sushi were analyzed for parasites from 32 of the approximately 50 restaurants in the Seattle area that prepare sushi. The restaurants were sampled up to three times over a 19-month period. Some specialty grocery stores providing restaurants and consumers with sashimi were also sampled. Salmon sushi was most commonly affected with almost 10% of pieces infected with a maximum of 3 nematodes per piece. Only single infections were present in mackerel sushi with frequency of 5%; and tuna and rockfish sushi were free of nematodes. All nematodes were third-stage juveniles of the genus Atiisakis . Except for two moribund nematodes, all juveniles from sushi were dead, most likely the result of the practice of using fish that have been previously frozen. The two moribund nematodes were present in one salmon sushi sample, indicating that incompletely frozen product had been used. For the sashimi, no parasites were found in tuna; however, a live anisakid was found in one collection of rockfish sashimi. Efforts to detect anisakid nematodes with nondestructive methods were generally unsuccessful. Neither inspection per ultraviolet light nor by candling was effective for salmon sushi. Candling was also ineffective for mackerel but was useful for rockfish and appears to be appropriate for the analysis of tuna sushi. Results of analyses of rice from sushi samples from 19 of the restaurants indicated that the pH levels were at 4.6 or below, and no fecal coliforms were detected. Most of the aerobic plate counts were below log 6, with only 2 between log 6 and log 7. Bacillus cereus and Staphylococcus aureus were detected in rice from six restaurants each, but in no samples were these two organisms found together, and levels were well below those of public health importance.

Enumeration and Differentiation of Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA Colony Hybridization Using the Hydrophobic Grid Membrane Filtration Technique for Isolation.

We have developed a means of differentiating and enumerating Vibrio parahaemolyticus and Vibrio vuinificus by DNA-DNA colony hybridization directly on HGMF filters. V. parahaemolyticus can be detected by a tdh-3-radiolabeled gene probe and V. vuinificus detected by a specific cytotoxin-hemolysin-radiolabeled probe with enumeration directly from autoradiograms. This procedure is more rapid than current techniques allowing enumeration and identification of these two species in samples as diverse as seawater, oyster ( Crassostrea gigas ), and shrimp (Pandalidae family) within 4 d. Our method is based on a rapid technique (18 h) for isolation and enumeration of V. parahaemolyticus from food using a membrane filtration technique with hydrophobic grid filters (HGMF). With the HGMF method, however, it is not possible to differentiate V. parahaemolyticus from V. vuinificus since on the HGMF-sucrose-based agar used, the two species are indistinguishable as both species are unable to ferment sucrose. Using a combination of the HGMF and selective gene probes, these two species can be differentiated.