An international outbreak of salmonellosis associated with raw almonds contaminated with a rare phage type of Salmonella enteritidis.

During the winter of 2000 to 2001, an outbreak due to Salmonella Enteritidis (SE) phage type 30 (PT30), a rare strain, was detected in Canada. The ensuing investigation involved Canadian and American public health and food regulatory agencies and an academic research laboratory. Enhanced laboratory surveillance, including phage typing and pulsed-field gel electrophoresis, was used to identify cases. Case questionnaires were administered to collect information about food and environmental exposures. A case-control study with 16 matched case-control pairs was conducted to test the hypothesis of an association between raw whole almond consumption and infection. Almond samples were collected from case homes, retail outlets, and the implicated processor, and environmental samples were collected from processing equipment and associated farms for microbiological testing. One hundred sixty-eight laboratory-confirmed cases of SE PT30 infection (157 in Canada, 11 in the United States) were identified between October 2000 and July 2001. The case-control study identified raw whole almonds as the source of infection (odds ration, 21.1; 95% confidence interval, 3.6 to infinity). SE PT30 was detected in raw whole natural almonds collected from home, retail, distribution, and warehouse sources and from environmental swabs of processing equipment and associated farmers' orchards. The frequent and prolonged recovery of this specific organism from a large agricultural area was an unexpected finding and may indicate significant diffuse contamination on these farms. Identification of almonds as the source of a foodborne outbreak is a previously undocumented finding, leading to a North American recall of this product and a review of current industry practices.

Prevalence of Yersinia enterocolitica shedding and bioserotype distribution in Ontario finisher pig herds in 2001, 2002, and 2004.

We investigated characteristics of Yersinia enterocolitica infection in Ontario finisher pig herds. Our specific objectives were to estimate or test: prevalence of Y. enterocolitica shedding in finisher pigs, bioserotype distribution, agreement between the herd-level tests based on sampling pig and pooled fecal samples, whether bioserotypes cluster by farms, and whether Y. enterocolitica-positive herds cluster spatially. In total, 3747 fecal samples were collected from 100 farms over the years 2001, 2002, and 2004 (250 total herd visits). Fecal samples were tested by culture and positive isolates were biotyped and serotyped. Apparent pig-level prevalence of Y. enterocolitica was 1.8%, 3.2%, and 12.5% in 2001, 2002, and 2004, respectively. Estimated true pig-level prevalence of Y. enterocolitica was 5.1%, 9.1%, and 35.1% in 2001, 2002, and 2004, respectively. Herd-level prevalence was 16.3%, 17.9%, and 37.5% in 2001, 2002, and 2004, respectively. In all years, the most common bioserotype was 4, O:3, followed by bioserotype 2, O:5,27. Kappa between herd-level status based on pig and pooled samples ranged between 0.51 and 0.68 for biotype 1A and bioserotype 4, O:3, respectively. For 4, O:3, a significant bias in discordant pairs was detected, indicating that pig samples were more sensitive than pooled samples in declaring a herd as positive. Farms tended to be repeatedly positive with the same bioserotype, but positive study farms did not cluster spatially (suggesting lack of between herd transmission and lack of a common geographic risk factor).

Collaborative Evaluation of the Plate Loop Technique for Determining Viable Bacterial Counts in Raw Milk.

A collaborative evaluation of the plate loop count technique was undertaken involving 13 laboratories with 29 technologists and 27 plate loops, The log variance of the Plate Loop Count (PLC) was 0.007, compared with a log variance of 0.001 for the reference Standard Plate Count (SPC), both of which fell within the tolerance limit of 0.012 considered acceptable for reproducibility by Standard Methods. The overall log10 mean PLC (1.975) compared favourably with the log10 mean SPC (1.960). Despite this overall image of precision and accuracy of the PLC, comparative data between individual technicians and/or loops was significantly different. Factors which contributed to this inaccuracy and imprecision of the PLC included improper loop calibration and significant variation in technique not only between analysts but also by individual analysts between loops.

Effect of heat treatment on the performance of tryptose-sulfite-cycloserine agar for enumeration of Clostridium perfringens.

Dissolving dehydrated tryptose-sulfite-cycloserine agar by only boiling or microwaving was found to inhibit Clostridium perfringens colony development in pour plates when compared with C. perfringens recovery in tryptose-sulfite-cycloserine agar prepared by autoclaving.

Improved medium for recovery and enumeration of Pseudomonas aeruginosa from water using membrane filters.

A modified mPA medium, designated mPA-C, was shown to recover Pseudomonas aeruginosa from a variety of water sources with results comparable to those with mPA-B and within the confidence limits of a most-probable-number technique. Enumeration of P. aeruginosa on mPA-C was possible after only 24 h of incubation at 41.5 degrees C, compared with 72 h of incubation required for mPA-B and 96 h of incubation for a presumptive most probable number.

Novobiocin-brilliant green-glucose agar: new medium for isolation of salmonellae.

A new medium, called novobiocin-brilliant green-glucose (NBG) agar, was developed for the isolation of Salmonella spp. and evaluated against other conventionally used media including bismuth sulfite, xylose-lysine decarboxylase, brilliant green-sulfa, hektoen enteric, and salmonella-shigella agars. NBG had recovery rates comparable to the other enteric media tested with pure cultures as well as with naturally contaminated amphibian and reptile waters and fecal specimens. However, NBG, hektoen enteric, and salmonella-shigella agars failed to differentiate Salmonella typhi from a fecal specimen even after enrichment in selenite F. Although Citrobacter freundii could grow and resembled salmonellae on NBG, at no time was the recovery of Salmonella spp. colonies jeopardized by the presence of C. freundii in either seeded or naturally contaminated samples. Confirmation rates of typical colonies from NBG agar also compared favorably to the other media tested; however, bismuth sulfite, although selective, was found to have varied differential characteristics for Salmonella spp. As a result, many more colonies had to be picked, which caused bismuth sulfite agar to have the lowest confirmation rate of the media tested. The distinct advantage that NBG agar offers over the conventional method tested, including bismuth sulfite, is the consistent differential reaction of all Salmonella subgroups including biochemically atypical strains. The medium is inexpensive, easy to prepare, and can be stored for at least 2 weeks at 4 degrees C without loss of selective or differential properties.

A Critical Evaluation of Automatic Bacterial Colony Counters.

Four automatic colony counters (ACC), 3M Model 620 and Artek Models 480, 870 and 880, were evaluated for their precision and accuracy in counting bacterial colonies in pour-plates prepared using raw and pasteurized milk samples. The automatic colony counters were precise, labor saving devices. but not one of the ACC units approached our acceptability criterion that 90% of the ACC counts fall within ± 10% of the corresponding manual count. Some parameters of experimental design and instrument calibration which may significantly influence the response and performance of the automatic counters are discussed.

Simple bacterial preservation medium and its application to proficiency testing in water bacteriology.

A medium composed of nutrient broth, 1.8% boric acid, and 1% sodium chloride at pH 7.0 was shown to maintain the stability of Escherichia coli cultures for up to 10 days at room temperature. By using this preservation medium for preparing a simulated sample a successful proficiency test survey in water bacteriology was conducted.

Automated Counting of Bacterial Colonies on Spread Agar Plates and Non-Gridded Membrane Filters.

The Biotran II automated colony counter was compared with a manual procedure for accuracy in counting bacterial colonies using both spread agar plate and membrane filter techniques. Comparative total bacterial counts of 250 samples (14 food, 124 water and 112 raw milk) were analyzed using the spread agar plate technique. Compared to manual enumeration, the Biotran II was found to be inaccurate for counting bacterial colonies on spread agar plates. Only 60 (24%) and 79 (31.6%) Biotran II counts fell within 10 and 20%, respectively, of the corresponding manual counts. Two samples from each of three river and four effluent sources were analyzed for total aerobic, total coliform, fecal coliform, fecal streptococci and total staphylococci bacterial counts using non-gridded membrane filters. A yellow acetate filter was used to mask the background growth and enhance the target colonies on the membrane filter. However, the method had limited success. Only 12 (20.7%) and 20 (34.5%) Biotran II counts fell within 10 and 20%, respectively, of the corresponding manual counts. Until the effect of background growth can be eliminated, the Biotran II cannot be relied upon to accurately count bacterial colonies on membrane filters.

Evaluation of Millipore Swab-Membrane Filter Kits.

A laboratory performance evaluation was done on the Millipore Total-Count (TC) and SPC swab-membrane filter kits which are designed to monitor sanitary conditions on food contact surfaces and eating utensils. Hand-shaking was as effective as vortexing for dislodging bacteria from the cotton swab in vials of Millipore buffer, but bacteria were unstable in the Millipore buffer even when refrigerated at 4°C. These results indicate that the Millipore swab-membrane filter kits could be used by public health officials but both swabbing and sampling should be completed in the field and the samplers returned to the laboratory for incubation, enumeration and proper disposal. Compared to the standard pour plate, recovery of unstressed Escherichia coli cells by the TC sampler was accurate provided not more than 125 colonies were on the membrane filter of the sampler. In comparing the two kits for recovery of stressed bacteria, the Millipore TC sampler was able to recover chlorine-stressed but not heat-stressed cells, whereas the opposite results were obtained with the SPC sampler. Hence, the availability of two different Millipore kits, neither of which recovers all types of stressed bacterial cells, indicates the need for designing a newer media formulation that could accomplish this effectively using just one sampler. Once these criteria have been met, the Millipore swab-membrane filter kit could replace the more tedious and time-consuming standard method currently used for monitoring sanitary conditions related to public health.

Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water.

Two media used to detect fecal coliforms in water by membrane filtration, m-FC and m-TEC, were modified and supplemented with the chromogenic substrate 5-bromo-6-chloro-3-indoyl-beta-D-glucuronide (BCIG) and were compared for quantitative recovery of Escherichia coli. Student's t test of data from 181 water samples of sewage, rivers, lakes, and wells did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli with these media. Target colonies were confirmed to be E. coli at rates of 98.6 and 97.3% by using FC-BCIG and TEC-BCIG media, respectively. Glucuronidase-negative isolates of E. coli were encountered at the same frequency (6.0%) on both media. This collaborative study demonstrated that either modified basal medium could be used successfully for detection of E. coli in various nontreated waters within 24 h.

Illness outbreak associated with Escherichia coli O157:H7 in Genoa salami. E. coli O157:H7 Working Group.

BACKGROUND: An outbreak of Escherichia coli O157:H7 infection was identified in the spring of 1998, with a 7-fold increase in the number of laboratory-confirmed E. coli O157:H7 cases in southern Ontario. This prompted an intensive investigation by local, provincial and federal public health officials. METHODS: Case interviews of 25 people from southern Ontario were conducted using a broad food history and environmental exposure survey. Laboratory investigations involved both case and food sampling. Specimens of foods sold locally and reportedly consumed by those affected were tested. Common suppliers of suspected foods were identified by cross-referencing suppliers' lists with stores frequented by those who fell ill. A case-control study involving 25 cases and 49 age-matched controls was conducted. This was followed by a comprehensive environmental investigation of the meat processing plant identified as the source of the E. coli. RESULTS: Thirty-nine outbreak-related cases occurred between April 3 and June 2, 1998. Of the 36 case specimens tested all were positive for E. coli O157:H7. The case-control study identified Genoa salami as the most probable (odds ratio 8 [confidence interval 2-35]) source of the outbreak. Samples of Genoa salami produced by the most commonly identified supplier later tested positive for E. coli O157:H7, and the pathogen matched the same pulsed-field gel electrophoresis pattern and phage type of the case specimens. INTERPRETATION: Our investigation, which led to a national recall of the brand of dry fermented Genoa salami identified as the source of the outbreak, supports an adherence to stringent manufacturing requirements for fermented meat products. A review of the Canadian standards for fermented meat processing and the effectiveness of their implementation is warranted.

Enumeration of total coliforms, fecal coliforms, and Escherichia coli in foods by hydrophobic grid membrane filter: collaborative study.

A collaborative study was conducted in 18 laboratories to assess the performance of the hydrophobic grid membrane filter method against that of the AOAC official first action method 46.013-46.016 for enumerating total and fecal coliforms and Escherichia coli. The study was carried out on frozen breaded fish, raw comminuted poultry, unroasted walnut pieces, ground black pepper, and cheddar cheese. The hydrophobic grid membrane filter method recovered significantly larger numbers of target bacteria in 7 of the food/analysis combinations: fecal coliforms in fish; E. coli in poultry; fecal coliforms and E. coli in walnuts; and total coliforms, fecal coliforms and E. coli in black pepper. Random error (Sr2) associated with the hydrophobic grid membrane filter method was significantly lower than that of the reference method in over 30% of the paired sample series. The hydrophobic grid membrane filter method for total coliform, fecal coliform, and E. coli enumeration in foods has been adopted official first action.