Evaluation and single-laboratory verification of a proposed modification to the U.S. Food and Drug Administration method for detection and identification of Campylobacter jejuni or Campylobacter coli from raw silo milk.

The current U.S. Food and Drug Administration (FDA) methodology for detection of Campylobacter, a leading source for foodborne illness, is outdated. The purpose of this study, therefore, was to improve and update the cultural and identification methods found in the FDA/Bacteriological Analytical Manual (BAM). Raw silo milk samples containing typical and atypical strains of Campylobacter jejuni and Campylobacter coli at different levels (5 CFU/25 g, 50 CFU/25 g, and 125 CFU/25 g) were analyzed. Valid results were obtained from 240 test portions. Six inoculated (at the levels described above) and two uninoculated samples were sent to a participating laboratory to mimic a "real-world" scenario. These combined data indicated that the use of sheep blood in combination with enrichment is not necessary. R & F Campylobacter jejuni/Campylobacter coli Chromogenic Plating Medium is significantly (P < 0.05) more sensitive for detection of C. jejuni or C. coli at low inoculation levels than the modified Cefoperazone Charcoal Deoxycholate Agar used in the BAM. The quantitative PCR method described demonstrated rapid confirmation and identification of C. jejuni or C. coli. It reduced the time to isolate C. jejuni or C. coli, and increased the sensitivity compared to the current BAM protocol.

AOAC International methods committee guidelines for validation of qualitative and quantitative food microbiological official methods of analysis.

Responding to a need for a guide for conducting Official Method validation studies of microbiological methods, AOAC utilized the experience of three microbiologists who have been active in the field of method validation. In collaboration, a document was prepared which covered the following areas: terms and their definitions associated with the Official Methods program (e.g., reference methods, alternative methods, and ruggedness testing), protocols and validation requirements for qualitative methods versus those for quantitative methods, the concept of the precollaborative study, ruggedness testing, tests for significant differences, performance indicators, and the approval process. After its preparation, this document was reviewed by the members of the Methods Committee on Microbiology and Extraneous Materials and by members of the Official Methods Board. Herein is presented the approved version of that document.

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.

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.

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.

Microbiological Quality of Oysters ( Crassostrea gigas ) and Water of Live Holding Tanks in Seattle, WA Markets.

Oyster ( Crassostrea gigas ) and water samples from Live Holding Tanks at five different Seattle area retail markets were analyzed for microbiological quality indicators and for potential pathogens monthly from March to September, 1987. Aeromonas hydrophilia was the most frequently isolated potential pathogen in this study with a higher incidence in oysters (78%) compared to water (53%). Vibrio cholerae non 01 and V. fluvialis were isolated from oyster samples from two different markets but not from water. V. alginolyticus was isolated from 53% of the water samples but was not found in any of the oysters. One oyster sample had a non-pathogenic Yersinia entercolitica . Yersinia spp. were isolated from oyster samples from one tank at two sampling periods. Salmonella typhimurium was isolated from one oyster sample. Samples were examined for Listeria spp. during the August sampling period and none were detected. The aerobic plate count was similar for both oyster and water samples and averaged 2000 CFU/gm. Total coliform levels were significantly higher (P<.05) for oysters (525MPN/100gm) compared to water (11MPN/100ml). The degree of water turbidity, crowding and species diversity varied between markets and sampling periods.

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 MacConkey's (MCA) agars. TSBA was far superior to MRSB in isolating this species from the environmental samples tested.

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.

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.

Bacteriological Quality of Fresh Seafood Products from Seattle Retail Markets.

A microbiological survey of 287 (fresh) seafood products from Puget Sound retail markets was conducted over a period of 1 year. The microbiological quality of fresh seafood was high, with only 2.1 % of the samples exceeding the maximum limit for acceptability as suggested by the International Commission on Microbiological Specifications for Foods (ICMSF). The overall microbiological data of positive units given as arithmetic means were: coliforms MPN/g, 199; Escherichia coli MPN/g, 21; coagulase-positive Staphylococcus aureus MPN/g, 66; enterococci/g, 9121; Clostridium perfringens /g, 18; Bacillus cereus /g, 100; and Vibrio parahaemolyticus MPN/g, 3.7. The standard plate count means 1.0 × 103 to 2.5 × 107 colony-forming units (CFU)/g, giving a mean value of 2.0 × 105 CFU/g. The percentages of seafood samples positive for pathogens were S. aureus , 37.6; Yersinia enterocolitica , 3.8; V. parahaemolyticus , 2.8; C. perfringens , 2.4; and B. cereus , 0.7. Vibrio cholerae , Clostridium botulinum , Salmonella and Shigella species were not isolated.