Marleen M. Wekell

Recovery of Aeromonas hydrophila from oysters implicated in an outbreak of foodborne illness.

Potentially pathogenic Aeromonas hydrophila organism were isolated from oysters frozen at -72°C for 1-1/2 years. The oysters which had been associated with 472 cases of gastroenteritis in Louisiana in November 1982, were examined and found negative for Salmonella , pathogenic Vibrio parahaemolyticus , and diarrhetic shellfish poison. In 1983, oysters from the same shellfish growing area in Louisiana were implicated in seven cases of gastroenteritis caused by A. hydrophila . The oysters collected in 1982 were reexamined and found to contain A. hydrophila (MPN 9.3/100 g). Twenty-three of 28 strains identified by the MICRO-IS and API-20E systems were positive for at least one of the tests for virulence which included the suckling mouse test, the adrenal Y-1 mouse cell test, and hemolysin assays. Of five strains tested, all showed activity in the rabbit ileal loop. Although these results do not prove that A. hydrophila caused the outbreak in 1982, they suggest that in cases of foodborne illness involving oysters, A. hydrophila should be included in the screening tests.

Template preparation for PCR and RFLP of amplification products for the detection and identification of Cyclospora sp. and Eimeria spp. Oocysts directly from raspberries.

Raspberries were epidemiologically associated with cyclosporiasis outbreaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanensis and several species of a closely related genus, Eimeria, were sequenced and primers for a nested PCR developed in a previous study. The ability to distinguish amplified products of Cyclospora sp. from those of Eimeria spp. is important for testing food and environmental samples. Therefore, an RFLP analysis of amplified products was used to differentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make template preparation for PCR challenging. Several approaches for PCR template preparation from raspberry samples were evaluated. Template preparation methods using various washing and concentration steps, oocyst disruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified primers were evaluated first with oocysts of Eimeria tenella then refined with oocysts of C. cayetanensis. Approximately 10 E. tenella oocysts per PCR or approximately 19 C. cayetanensis oocysts per PCR were detected with the optimized template preparation method. The addition of 20 microliters of raspberry wash sediment extract and nonfat dried milk solution did not inhibit the amplification of DNA from as few as 10 E. tenella and 25 C. cayetanensis oocysts in a 100-microliter PCR. The nucleotide sequences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar in the amplified region, but the amplification products from the two genera were distinguished using an RFLP analysis with the restriction enzyme MnlI.

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.

An oligonucleotide-ligation assay for the differentiation between Cyclospora and Eimeria spp. polymerase chain reaction amplification products.

An oligonucleotide-ligation assay (OLA) was developed and compared to a restriction fragment length polymorphism (RFLP) test for distinguishing between 294-bp polymerase chain reaction (PCR) amplification products of the 18S rRNA gene from Cyclospora and Eimeria spp. The PCR/OLA correctly distinguished between three Cyclospora, three E. tenella, and one E. mitis strains and the ratio of positive to negative spectrophotometric absorbance (A490) values for each strain ranged from 4.086 to 15.280 (median 9.5). PCR/OLA provides a rapid, reliable, spectrophotometric alternative to PCR/RFLP.

Campylobacter jejuni in a Washington State shellfish growing bed associated with illness

Consumption of raw Pacific oysters ( Crassotea gigas ) harvested from a Washington State recreational shellfish bed were associated with illness. Illness occurred within 2 d of ingestion of a half-dozen shellstock oysters. Each oyster consist of approximately 20 g of meat. The duration of illness lasted 2 d. Routinely, Campylobacter species have been found in several shellfish beds in the Puget Sound Bay. Its presence in the marine environment appears to be incidental and primarily, comes from wild birds, farm runoff, and sewage bypasses. This paper describes the first reported case of Campylobacter gastroenteritis associated with raw oyster consumption in the State of Washington.

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.

Incidence of Motile Aeromonads From United States West Coast Shellfish Growing Estuaries

The distribution of motile Aeromonas species in marine and tributary waters, sediment, and shellfish from 12 major estuarine areas in Washington, Oregon, and California with commercial or sport shellfish harvest was determined during the summer months. Aeromonas spp. were found in half of the total of 400 samples analyzed. Two enrichment broths, tryptic soy ampicillin broth (TSBA) and alkaline peptone water (APW), were compared for recovery of Aeromonas from Washington and Oregon samples. More Aeromonas were isolated using TSBA. For Washington and Oregon samples, recoveries using TSBA were 82 and 77% respectively compared to 31 and 50% using APW. For California samples, only APW was used with 28% samples positive. Of 767 isolates tested, 93.5% were positive for hemolysis, a trait reported to correlate with enterotoxin production and pathogenicity. Of the hemolysis positive strains, 59.5% were toxic to Y-1 adrenal cells.

Enumeration of Vibrio species, including V. cholerae, from samples of an oyster growing area, Grays Harbor, Washington.

Water, shellfish, and sediment samples from Grays Harbor, a major commercial oyster producing estuary in the State of Washington, were examined for levels of Vibrio species. Non-01 V. cholerae was found at low levels in 37.8% of the samples. While V. parahaemolyticus was found in all samples, levels were low. V. mimicus and V. fluvialis were found infrequently and at low levels. Potentially pathogenic strains of non-01 V. cholerae and Kanagawa positive V. parahaemolyticus were isolated from oysters suggesting a potential for human illness.

Differentiation of Clostridium perfringens from related clostridia in iron milk medium

The stormy fermentation reaction of Clostridium perfringens in iron milk medium was compared to that of several C. perfringens -like strains. These clostridia, C. barati , C. perenne , C. absonum , and C. paraperfringens are very similar to C. perfringens on the basis of certain biochemical reactions and, consequently, are often difficult to distinguish from C. perfringens . Furthermore, these related clostridia may also be present in foods. Results of this study demonstrate that after 18 h of incubation at 45°C, only C. perfringens gave a positive reaction in iron milk with inocula as low as 22 cells/g. Some of the other strains began to show only gas production at 18 h. After 24 to 42 h some strains gave positive results and after 72 h all were positive. Enumeration of C. perfringens from food samples in iron milk medium by a 3-tube most probable number (MPN) technique gave similar results to enumeration by plate count using Shahidi-Ferguson Perfringens (SFP) agar. Furthermore, a rapid positive response occurred after only 2 and 3 h incubation of iron milk inoculated with 108 and 107 cells/ml, respectively. The high selectivity, ease of identification and rapid growth of C. perfringens in iron milk make the iron milk MPN procedure a valuable assay for accurate enumeration and differentiation of C. perfringens from related Clostridia in food products.

Aeromonas hydrophila in shellfish growing waters: incidence and media evaluation

Levels of Aeromonas hydrophila determined for the shellfish growing area of Grays Harbor, Washington, ranged from 3 to 4600/100 g in oysters and from 3 to 2400/100 ml in water. Of isolates tested, 80% produced a hemolysin, a trait reported to correlate with enterotoxin production and pathogenicity. Two enrichment broths, Tryptic Soy Broth with ampicillin (TSBA) and Modified Rimler Shotts Broth (MRSB) were compared in combination with three solid agar media: Rimler Shotts (RS), Peptone Beef Extract Glycogen (PBG), and MacConkeys (MCA) agars. TSBA was far superior to MRSB in isolating this species from the environmental samples tested.