Pathogen Characterization of Fresh and Stored Mesophilic Anaerobically Digested Biosolids.

Culturable bacterial pathogens (Campylobacter, Salmonella, Listeria, Yersinia) and indicators (E. coli, enterococci, Clostridium perfringens) were quantified at six water resource recovery facilities that land apply anaerobically digested biosolids in Ontario, Canada. Cryptosporidium parvum and Giardia lamblia were also quantified by polymerase chain reaction (PCR). Salmonella and Listeria were frequently detected in sludge and liquid biosolids (70-100% of samples) but less often in fresh dewatered cake biosolids (50-60%); with low levels in fresh cake (<100 cells/g dw). Yersinia were in 20 to 30% of samples, typically at very low levels (<10 cell/g dw). Giardia and Cryptosporidium were detected in 80 and 20% of cake biosolids at geometric means of 270 cysts/g dw and 70 oocysts/g dw, respectively. E. coli reduction was typically >2-log10 while pathogen reduction was variable. "Sudden increase" of pathogens was not observed, however, Salmonella and E. coli showed regrowth (at 1 to 3 orders of magnitude) after 2- to 3-day storage at 30 °C.

Yersinia enterocolitica-Specific Infection by Bacteriophages TG1 and ϕR1-RT Is Dependent on Temperature-Regulated Expression of the Phage Host Receptor OmpF.

UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.

Complete genome sequence of bacteriophage vB_YenP_AP5 which infects Yersinia enterocolitica of serotype O:3.

BACKGROUND: Bacteriophage vB_YenP_AP5 is a lytic bacteriophage capable of infecting Yersinia enterocolitica strains of serotype O:3, an epidemiologically significant serotype within this bacterial species that causes yersiniosis in humans. This work describes the complete genome sequence of this phage. RESULTS: The genome consists of linear double-stranded DNA of 38,646 bp, with direct terminal repeats of 235 bp in length, and a GC content of 50.7%. There are 45 open reading frames which occupy 89.9% of the genome. Most of the proteins encoded by this virus exhibit sequence similarity to Yersinia phage φYeO3-12 and Salmonella phage φSG-JL2 proteins. CONCLUSIONS: Genomic and morphological analyses place the bacteriophage vB_YenP_AP5 in the T7likevirus genus of the subfamily Autographivirinae within the family Podoviridae.

Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods.

A study was conducted to evaluate the performance of the ALOA (chromogenic media) in combination with immunomagnetic separation (IMS) for the detection of Listeria monocytogenes in ready-to-eat food products. IMS-ALOA method was found to be equivalent to Health Canada's reference culture method as well as comparable to BAX-PCR method in terms of the sensitivity of the methods for the detection of L. monocytogenes in ready-to-eat foods such as turkey roast, beef roast, mixed vegetable salads, potato and egg salad, soft cheese and smoked salmon. The IMS-ALOA method gave 100% sensitivity in the inclusivity tests with 42 pure L. monocytogenes strains. Exclusivity testing with five other species of Listeria genus and 29 pure non-L. monocytogenes strains from 21 different genera showed 97% specificity. The method was able to detect L. monocytogenes at levels near or below 1cfu/25g regulatory limit in ready-to-eat food matrices after 24h enrichment, with a turnaround time of 3days compared to 7-8days for culture method. IMS-ALOA method is a valuable alternate test method for the screening of L. monocytogenes in a variety of foods especially ready-to-eat foods.

Application of an automated immunomagnetic separation-enzyme immunoassay for the detection of Salmonella enterica subspecies enterica from poultry environmental swabs.

An automated immunomagnetic separation (IMS) and enzyme immunoassay (EIA) was applied to the detection of Salmonella enterica subspecies enterica serotypes from poultry environmental samples. The analytical sensitivity and specificity of the IMS-EIA for 46 S. enterica serotypes and 33 non-salmonellae isolates belonging to 21 different genera were 91.3% and 90.9%, respectively. From post enrichment S. enterica cultures, the limit of detection of the assay was estimated at 10(4)-10(6) CFU/mL. Application of IMS-EIA on 850 naturally contaminated poultry environmental samples achieved 98.4% sensitivity and 96.8% specificity, as compared with a standard culture reference method performed concurrently on the same set of samples. The IMS-EIA described, allows for the identification of suspect positive samples within 48 h of testing versus 4-6 days required by standard culture methods while significantly reducing the materials and labour required for the detection of S. enterica serotypes in poultry environmental samples.

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples.

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.

Effect of environmental stresses on the mean and distribution of individual cell lag times of Escherichia coli O157:H7.

The effect of starvation, heat or acid stress on duration of individual cell lag time (tau) and standard deviation (SD) of tau was investigated using Escherichia coli O157:H7. Cells were stressed by exposure to acid (pH 3.5), heat (50 degrees C), or starvation in either glucose-free mineral medium (MOPS), tryptic soy broth (TSB) or Luria broth (LB). Stressed cells were then diluted into wells of a Bioscreen plate to obtain single cells per well. Replicate time to detection (td) values were obtained using the Bioscreen and used to calculate the tau and SD. Significant (P< or =0.05) increases in tau over untreated controls were found for the following treatments: 14 days in acid; 2 h of heating; 3 days starvation in MOPS; and 2 days starvation in either TSB or LB. The largest increase in tau was >2-fold from 2.5 to 5.6 h observed with the heat treatment. MOPS starvation was more detrimental to the cells than was acid treatment over the same time period. A significant increase in SD was found with 21 days acid treatment, and 2 days starvation in either TSB or LB. No significant increase in SD was found for MOPS starvation or heat treatment. Lognormal, Gamma, ExtremeValue and Weibull distributions were fitted to the tau data using BestFit. The results suggest that the Lognormal distribution is suitable for fitting tau data from either stressed or unstressed cells.

Evaluation of an automated immunomagnetic separation method for the rapid detection of Salmonella species in poultry environmental samples.

Automated immunomagnetic separation (AIMS), using Dynabeads anti-Salmonella (Dynal, Oslo), was evaluated for its ability to detect Salmonella spp. in poultry environmental samples in comparison with standard, culture-based method (Health Canada, Health Protection Branch, MFHPB-20). AIMS was found to be more reliable in detecting Salmonella from artificially inoculated enrichment broths at low levels and exhibited a 15.5% higher sensitivity value than the culture method.

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples.

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples.

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.

Evaluation of the MicroFoss system for enumeration of total viable count, Escherichia coli and coliforms in ground beef.

This study was conducted to evaluate the performance of the MicroFoss system (Biosys, Ann Arbor, MI) for enumeration of total viable organisms, Escherichia coli and coliforms in ground beef. The system performance was compared to that of the USDA Bacteriological Analytical Method (BAM) reference culture methods. The correlation coefficients for the regression lines comparing the MicroFoss system detection times to the results of plate count methods for the total viable counts, coliform counts and the most probable number (MPN) method for E. coli were -0.95, -0.96 and -0.97, respectively. Tests comparing the reproducibility of data generated independently by two technicians on the same batch of samples showed no significant differences (P>0.05) in the MicroFoss detection times and culture results. The plate count methods for the total viable counts and coliform counts, and the MPN method for E. coli required 10, 11 and 22 times, respectively, the amount of time to complete tests compared to the length of time required to perform these tests using the MicroFoss system. The MicroFoss system produced reproducible data and provided a rapid and cost-efficient alternative method for enumeration of TVC, coliforms and E. coli in ground beef.

Detection of Bacillus cereus Diarrheal Enterotoxin in Raw and Pasteurized Milk.

Raw and pasteurized milk samples submitted for routine quality analysis were screened for the presence of Bacillus cereus diarrheal enterotoxin (BDE) using the TECRA BDE Visual Immunoassay (VIA) kit. BDE was not detected in 298 raw milk samples tested by the TECRA VIA. B. cereus was isolated from 2 of 298 (0.7%) raw milk samples cultured. Culture supernatants from these isolates were positive for BDE in the TECRA VIA but negative in the Reverse Passive Latex Agglutination (RPLA) test for BDE. Forty-three of 112 (38.4%) pasteurized milk samples incubated at 10°C until their expiry dates were positive for BDE by the TECRA VIA. The same number of samples incubated at 4°C had no detectable levels of enterotoxin. B. cereus in the range of 103 to 106 CFU/ml was isolated from all BDE-positive pasteurized milk samples. BDE was detected in the culture supernatants of all the 43 isolates by TECRA VIA and in 30 of these isolates by RPLA. These results demonstrate that moderate temperature abuse of pasteurized milk may allow the growth of B. cereus and BDE production.

Survey of Ontario Bulk Tank Raw Milk for Food-Borne Pathogens.

Raw (unpasteurized) milk can be a source of food-borne pathogens. Raw milk consumption results in sporadic disease outbreaks. Pasteurization is designed to destroy all bacterial pathogens common to raw milk, excluding spore-forming bacteria and possibly Mycobacterium paratuberculosis , but some people continue to drink raw milk, believing it to be safe. Current methods for assessing the bacteriological quality of raw milk, such as aerobic plate counts, are not usually designed to detect specific pathogens. The objective of this study was to estimate the proportion of pick-ups (loads of raw milk from a single farm bulk tank) from Ontario farm bulk tanks that contained Listeria monocytogenes . Salmonella spp., Campylobacter spp., and/or verotoxigenic Escherichia coli (VTEC). Samples from 1,720 pick-ups of raw milk were tested for the presence of these pathogens, and 47 L. monocytogenes , three Salmonella spp., eight Campylobacter spp., and 15 VTEC isolates were detected, representing 2.73, 0.17, 0.47, and 0.87% of milk samples, respectively. Estimates of the proportion of theoretical tanker truck loads of pooled raw milk contaminated with pathogens ranged from a low of 0.51 % of tankers containing raw milk from 3 bulk tanks being contaminated with Salmonella spp. to a high of 34.41 % of tankers containing raw milk from 10 bulk tanks being contaminated with at least one of the pathogens. Associations between the presence of pathogens and raw milk sample characteristics were investigated. The mean somatic cell count was higher among VTEC- or L. monocytogenes -positive samples, and the mean aerobic plate count was found to be higher among L. monocytogenes -positive samples. These results confirm the presence of bacterial food pathogens in raw milk and emphasize the importance of continued diligence in the application of hygiene programs within dairies and the separation of raw milk from pasteurized milk and milk products.

Assessment of the Microbiological Quality of Ready-To-Use Vegetables for Health-Care Food Services.

The microbiological quality of ready-to-use (RTU) vegetables, including chopped lettuce, salad mix, carrot sticks, cauliflower florets, sliced celery, coleslaw mix, broccoli florets, and sliced green peppers was determined before and after processing. Microbial profiles were obtained 24 h after processing and on days 4, 7, and 11 after storage at 4 and 10°C to simulate temperature abuse. In addition, the microbial profiles of four RTU vegetables, coleslaw mix, salad mix, cauliflower florets, and sliced green peppers were determined 7 days after distribution to a select group of Ontario hospitals. RTU vegetables, with the exception of green peppers, showed up to a 1-log decrease in aerobic colony counts after processing. These counts increased to preprocessing levels after 4 days of storage at both 4 and 10°C. RTU vegetables stored at temperature abuse conditions (10°C)had significantly higher counts (P < 0.001) on days 4 to 11 as compared to those stored at 4°C. Green peppers had the highest bacterial counts while cauliflower and chopped lettuce had the lowest counts at both storage temperatures (P < 0.05). Increased levels of Listeria monocytogenes in RTU vegetables were associated with temperature abuse. Levels of >100 MPN/g for L. monocytogenes were detected in 8 of 120 (6.7%) samples stored at 10°C but not in 175 samples stored at 4°C after 7 days (P < 0.05). Overall, L. monocytogenes was detected in 13 of 120 (10.8%) RTU vegetables stored for up to 11 days at 10°C and 5 of 176 (2.8%) samples stored at 4°C (P < 0.05). E. coli was detected in 2 of the 120 (1.7%) processed RTU vegetables after day 7 of storage at 10°C and 1 of the 65 (1.5%) unprocessed vegetables from the same batches of vegetables used for processing. This indicator organism was not detected in RTU vegetable samples stored at 4°C or in any of the RTU vegetable samples obtained from hospital coolers. Other pathogenic bacteria, such as Salmonella spp., Campylobacter spp., Yersinia enterocolitica (serotype O:3) and verocytotoxigenic E. coli (VTEC) were not detected in any of the RTU vegetables tested, Recommendations regarding processing, distribution, and storage of these products are presented.