Carlos Abeyta

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.

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.

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.

Enhancement of virulence of two environmental strains of Vibrio vulnificus after passage through mice

The virulence of two environmental strains of Vibrio vulnificus to iron-loaded adult mice was enhanced by passage through mice. Estimates of 50% lethal dose values (LD50) determined by end point titration were reduced 100- and 1000-fold for the two strains. Passage through mice also selected for the opaque colony type phase variation of V. vulnificus, reported by others to be more virulent to mice than a translucent colony type.

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.

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.

Comparison of Selective Media for Primary Isolation of Campylobacters

Many different types of selective media have been developed3–5, 7, 9, 11, 12. The foremost characteristic of an effective (“high performance”) plating media is the support of the growth of a wide range of C. jejuni strains with inhibition of the growth of other unwanted bacteria (background microbial flora). Background flora associated with Campylobacter is dependent upon the sample matrix2. Campylobacter-selective, high performance media were based on the ability of the media to recover the most campylobacters (“high, target signal”) with least colonization by background contaminants (“low, competitive signal. Plate counts are the scores of distinct values for colonization by Campylobacter (“target signal” C for Campylobacter percent recovery) versus colonization by flora (“competitive signal” F for flora percent recovery). The calculated test measure differential recovery% is differential recovery %=C − F(Eqn. 1). The ratiometric recovery is ratiometric recovery = C/(C+F) (Eqn. 2). The differential recovery% value approaching 100 represents overwhelming growth of Campylobacter in the presence of little or no flora. However this test measure may not resolve situations in which there is equivalent suppression of Campylobacter growth or enhancement flora colonization. The ratiometric measure used in tandem with the differential measure resolves this redundancy. Most of the trends in variation in the data were visualized using differential recovery% side-by-side range boxplots6,10.