Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada.

An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.

Assessment of Listeria sp. Interference Using a Molecular Assay To Detect Listeria monocytogenes in Food.

Detection of Listeria monocytogenes in food is currently based on enrichment methods. When L. monocytogenes is present with other Listeria species in food, the species compete during the enrichment process. Overgrowth competition of the nonpathogenic Listeria species might result in false-negative results obtained with the current reference methods. This potential issue was noted when 50 food samples artificially spiked with L. monocytogenes were tested with a real-time PCR assay and Canada's current reference method, MFHPB-30. Eleven of the samples studied were from foods naturally contaminated with Listeria species other than those used for spiking. The real-time PCR assay detected L. monocytogenes in all 11 of these samples; however, only 6 of these samples were positive by the MFHPB-30 method. To determine whether L. monocytogenes detection can be affected by other species of the same genus due to competition, an L. monocytogenes strain and a Listeria innocua strain with a faster rate of growth in the enrichment broth were artificially coinoculated at different ratios into ground pork meat samples and cultured according to the MFHPB-30 method. L. monocytogenes was detected only by the MFHPB-30 method when L. monocytogenes/L. innocua ratios were 6.0 or higher. In contrast, using the same enrichments, the real-time PCR assay detected L. monocytogenes at ratios as low as 0.6. Taken together, these findings support the hypothesis that L. monocytogenes can be outcompeted by L. innocua during the MFHPB-30 enrichment phase. However, more reliable detection of L. monocytogenes in this situation can be achieved by a PCR-based method mainly because of its sensitivity.

Rapid Detection of Salmonella in Foods Using EF-18 Agar in Conjunction with the Hydrophobic Grid Membrane Filter.

A new selective and differential culture medium for Salmonella , EF-18 agar, was developed for use with the hydrophobic grid membrane filter (HGMF). The new medium was designed to be both more highly selective and more specific in its differential reactions than its predecessor, selective lysine agar. The HGMF/EF-18 agar method was evaluated against the conventional cultural method (AOAC/BAM) using a total of 954 samples comprising 25 product categories. The HGMF/EF-18 method detected 653 Salmonella -positive samples and the AOAC/BAM method detected 654. The HGMF/EF-18 method with an overall false-negative rate of 2% was determined to be equivalent in sensitivity to the AOAC/BAM procedure. The presumptive false-positive rates were 0.3% for HGMF/EF-18 and 7.9% for AOAC/BAM.

Use of Fast Green FCF with Tryptic Soy Agar for Aerobic Plate Count by the Hydrophobic Grid Membrane Filter.

A hydrophobic grid membrane filter (HGMF) method for aerobic plate count using Tryptic Soy Agar with fast green FCF was evaluated against a conventional pour plate method on 250 food samples, representing 25 product categories. The HGMF method yielded counts equivalent to or significantly higher than the pour plate method for 24 of the 25 product categories (t-test for paired data).

Overnight Enumeration of Vibrio parahaemolyticus In Seafood by Hydrophobic Grid Membrane Filtration.

A method was developed for direct enumeration of Vibrio parahaemolyticus in foods by hydrophobic grid membrane filter. The method consisted of a 4-5 h resuscitation step to recover injured cells, followed by overnight incubation at 42°C on V. parahaemolyticus Sucrose (VPS) agar, a new selective and differential medium. The confirmation rate of typical colonies on VPS agar was greater than 98%. The new method produced significantly higher counts of V. parahaemolyticus than the FDA method (P<0.01) when tested with chill-, freeze- or heat-stressed samples, and was equivalent to the FDA method (P>0.05) for recovery of osmotically stressed V. parahaemolyticus .