Comparative prevalence and diversity of Campylobacter jejuni strains in water and human beings over a 1-year period in southwestern Alberta, Canada.

Campylobacter jejuni was isolated from diarrheic people, river water (Oldman River watershed), wastewater, and drinking water over a 1-year period in southwestern Alberta (2008-2009). High rates of campylobacteriosis were observed during the study period (≥115 cases/100 K). Infections occurred throughout the year, with peaks in late summer and early autumn. Most infections occurred in people living in Lethbridge. Campylobacter jejuni was not isolated from municipal drinking water. In contrast, the bacterium was isolated from untreated and treated wastewater and river water (all sites). There were no correlations between C. jejuni recovery/detection from water and river flow rates, water turbidity, or fecal coliforms. Campylobacter jejuni recovery from water did not correspond to the peak periods of campylobacteriosis. The bacterium was most commonly isolated downstream of wastewater outfalls; waterfowl congregated at these sites, particularly during the winter months. A comparison of C. jejuni isolates from people and water revealed that most subtypes in water did not correspond to subtypes recovered from diarrheic people and were linked to waterfowl and other non-human animal sources. We conclude that waterborne C. jejuni did not contribute significantly to the high rates of campylobacteriosis observed in diarrheic people during the study period.

Glutamine, glutamate, and arginine-based acid resistance in Lactobacillus reuteri.

This study aimed to determine whether glutamine deamidation improves acid resistance of Lactobacillus reuteri, and to assess whether arginine, glutamine, and glutamate-mediated acid resistance are redundant or complementary mechanisms of acid resistance. Three putative glutaminase genes, gls1, gls2, and gls3, were identified in L. reuteri 100-23. All three genes were expressed during growth in mMRS and wheat sourdough. L. reuteri consistently over-expressed gls3 and the glutamate decarboxylase gadB. L. reuteri 100-23ΔgadB over-expressed gls3 and the arginine deiminase gene adi. Analysis of the survival of L. reuteri in acidic conditions revealed that arginine conversion is effective at pH of 3.5 while glutamine or glutamate conversion were effective at pH of 2.5. Arginine conversion increased the pHin but not ΔΨ; glutamate decarboxylation had only a minor effect on the pHin but increased the ΔΨ. This study demonstrates that glutamine deamidation increases the acid resistance of L. reuteri independent of glutamate decarboxylase activity. Arginine and glutamine/glutamate conversions confer resistance to lactate at pH of 3.5 and phosphate at pH of 2.5, respectively. Knowledge of L. reuteri's acid resistance improves the understanding of the adaptation of L. reuteri to intestinal ecosystems, and facilitates the selection of probiotic and starter cultures.

Functional characterization of sucrose phosphorylase and scrR, a regulator of sucrose metabolism in Lactobacillus reuteri.

Lactobacillus reuteri harbours alternative enzymes for sucrose metabolism, sucrose phosphorylase, fructansucrases, and glucansucrases. Sucrose phosphorylase and fructansucrases additionally contribute to raffinose metabolism. Glucansucrases and fructansucrases produce exopolysaccharides as alternative to sucrose hydrolysis. L. reuteri LTH5448 expresses a levansucrase (ftfA) and sucrose phosphorylase (scrP), both are inducible by sucrose. This study determined the contribution of scrP to sucrose and raffinose metabolism in L. reuteri LTH5448, and elucidated the role of scrR in regulation sucrose metabolism. Disruption of scrP and scrR was achieved by double crossover mutagenesis. L. reuteri LTH5448, LTH5448ΔscrP and LTH5448ΔscrR were characterized with respect to growth and metabolite formation with glucose, sucrose, or raffinose as sole carbon source. Inactivation of scrR led to constitutive transcription of scrP and ftfA, demonstrating that scrR is negative regulator. L. reuteri LTH5448 and the LTH5448ΔscrP or LTH5448ΔscrR mutant strains did not differ with respect to glucose, sucrose or raffinose utilization. However, L. reuteri LTH5448ΔscrP produced more levan, indicating that the lack of sucrose phosphorylase is compensated by an increased metabolic flux through levansucrase. In conclusion, the presence of alternate pathways for sucrose and raffinose metabolism and their regulation indicate that these substrates, which are abundant in plants, are preferred carbohydrate sources for L. reuteri.

Levansucrase and sucrose phoshorylase contribute to raffinose, stachyose, and verbascose metabolism by lactobacilli.

Raffinose family oligosaccharides (RFOs) in food are considered anti-nutritional factors. This study elucidated the role of α-galactosidase (α-Gal), levansucrase, and sucrose phosphorylase for conversion of RFOs by lactobacilli. Quantification of gene expression by reverse-transcriptase quantitative PCR revealed that expression of levansucrase and sucrose phosphorylase by Lactobacillus reuteri is increased more than 100 fold when sucrose or raffinose are available. Fava bean (Vicia faba) or field pea (Pisum sativum) flours were fermented with α-Gal positive L. reuteri or α-Gal negative Lactobacillus sanfranciscensis. Isogenic strains lacking levansucrase activity, a L. reuteri ftfA mutant and a L. sanfranciscensis levS mutant, were used for comparison. During growth in pulse flours, L. sanfranciscensis accumulated melibiose and α-galactooligosaccharides (α-GOSs); the levansucrase-negative strain did not grow. L. reuteri metabolized raffinose, stachyose, and verbascose by levansucrase activity and accumulated α-GOSs as metabolic intermediates. Oligosaccharide metabolism in the levansucrase-negative mutant was slower, and accumulation of α-GOSs was not observed. The use of sorghum sourdough fermented with L. reuteri LTH5448 and bean flour in gluten-free baking converted RFOs to α-GOSs by levansucrase and invertase activities. In conclusion, the elucidation of the role levansucrase in RFO metabolism by lactobacilli allowed the conversion or hydrolysis of RFOs in food fermentations.

Whole-Genome Sequences of Vibrio Species from Warm-Water Shrimps Imported into Canada: Detection of Genetic Elements Associated with Antimicrobial Resistance and Potential Mobilizing Capacities.

We present draft genome sequences of Vibrio species (Vibrio alginolyticus, Vibrio cholerae, and two Vibrio parahaemolyticus strains) that were isolated from warm-water shrimps imported into Canada. All four isolates harbor genetic elements associated with antimicrobial resistance (AMR), including mobile genetic elements that can promote horizontal transfer of AMR genes.

Draft Genomes of 92 Vibrio Isolates from Warm-Water Shrimps Imported into Canada between 2009 and 2019.

Vibrio spp. were isolated from raw shrimps imported into Canada (2009 to 2019). A total of 92 isolates with various multidrug resistance profiles were sequenced, including 59 V. parahaemolyticus, 12 V. diabolicus, 10 V. cholerae, 7 V. alginolyticus, 1 V. campbellii, 1 V. harveyi, 1 V. owensii, and 1 V. vulnificus isolate.

Draft Genomes of 83 Vibrio Isolates from Fresh Canadian Mollusks.

A subset of Vibrio spp. isolated from fresh Canadian mollusks (2014 to 2018) were selected for sequencing based on antimicrobial resistance profiles. The resulting de novo draft genomes include 38 Vibrio alginolyticus, 32 V. diabolicus, 10 V. parahaemolyticus, 1 V. cholerae, 1 V. ordalii, and 1 Vibrio sp. isolate.

Draft Genomes of 75 Vibrio Isolates from Retail Mollusks Collected in Canada between 2014 and 2018.

Vibrio spp. isolated from fresh retail mollusk samples were selected for sequencing based on their antimicrobial resistance burden. The de novo genomes include those for Vibrio alginolyticus (n = 48), V. diabolicus (n = 15), V. parahaemolyticus (n = 3), V. cholerae (n = 2), V. metoecus (n = 1), V. vulnificus (n = 1), V. fluvialis (n = 1), and unidentified Vibrio spp. (n = 4).

Molecular Epidemiological Evidence Implicates Cattle as a Primary Reservoir of Campylobacter jejuni Infecting People via Contaminated Chickens.

The study aimed to determine the relative contribution of cattle to the burden of illness in a model agroecosystem with high rates of human campylobacteriosis (≥ 115 cases/100 K), and high densities of cattle, including large numbers of cattle housed in confined feeding operations (i.e., in southwestern Alberta, Canada). To accomplish this, a large-scale molecular epidemiological analysis of Campylobacter jejuni circulating within the study location was completed. In excess of 8000 isolates of C. jejuni from people (n = 2548 isolates), chickens (n = 1849 isolates), cattle (n = 2921 isolates), and water (n = 771 isolates) were subtyped. In contrast to previous studies, the source attribution estimates of clinical cases attributable to cattle vastly exceeded those attributed to chicken (i.e., three- to six-fold). Moreover, cattle were often colonized by C. jejuni (51%) and shed the bacterium in their feces. A large proportion of study isolates were found in subtypes primarily associated with cattle (46%), including subtypes infecting people and those associated with chickens (19%). The implication of cattle as a primary amplifying reservoir of C. jejuni subtypes in circulation in the study location is supported by the strong cattle association with subtypes that were found in chickens and in people, a lack of evidence indicating the foodborne transmission of C. jejuni from beef and dairy, and the large number of cattle and the substantial quantities of untreated manure containing C. jejuni cells. Importantly, the evidence implicated cattle as a source of C. jejuni infecting people through a transmission pathway from cattle to people via the consumption of chicken. This has implications for reducing the burden of campylobacteriosis in the study location and elsewhere.

Exploring the Potential of the Microbiome as a Marker of the Geographic Origin of Fresh Seafood.

Geographic food fraud - misrepresenting the geographic origin of a food item, is very difficult to detect, and therefore this type of fraud tends to go undetected. This potentially negatively impacts the health of Canadians and economic success of our seafood industry. Surveillance studies have shown that up to a significant portion of commercially sold seafood items in Canada are mislabeled or otherwise misrepresented in some way. The current study aimed to determine if the microbiome of fresh shellfish could be used as an accurate marker of harvest location. Total DNA was extracted from the homogenate of 25 batches of fresh soft-shell clams (Mya arenaria) harvested in 2015 and 2018 from two locations on the East Coast of Canada and the microbiome of each homogenate was characterized using 16S rRNA targeted amplicon sequencing. Clams harvested from Nova Scotia in both years had a higher abundance of Proteobacteria and Acidobacteria (p < 0.05), but a lower abundance of Actinobacteria (p < 0.05) than those from Quebec. Alpha-diversity also differed significantly between sites. Samples harvested from Nova Scotia had greater diversity (p < 0.0001) than those from Quebec. Beta-diversity analysis showed that the microbial community composition was significantly different between the samples from Nova Scotia and Quebec and indicated that 16S rRNA targeted amplicon sequencing might be an effective tool for elucidating the geographic origin of unprocessed shellfish. To evaluate if the microbiome of shellfish experiences a loss of microbial diversity during processing and storage - which would limit the ability of this technique to link retail samples to geographic origin, 10 batches of retail clams purchased from grocery stores were also examined. Microbial diversity and species richness was significantly lower in retail clams, and heavily dominated by Proteobacteria, a typical spoilage organism for fresh seafood, this may make determining the geographic origin of seafood items more difficult in retail clams than in freshly harvested clams.

Antimicrobial activity of bioactive starch packaging films against Listeria monocytogenes and reconstituted meat microbiota on ham.

Contamination with spoilage organisms and Listeria monocytogenes are major concerns for quality and safety of cooked ready-to-eat (RTE) meat products. Thus, the objective of this study was to investigate the use of antimicrobial starch packaging films to control competitive microbiota and L. monocytogenes growth on a RTE ham product. Starch packaging films were prepared with different bioactives, gallic acid, chitosan, and carvacrol, using subcritical water technology. The viability of the incorporated strains on ham in contact with different antimicrobial starch packaging films was examined during 28-day storage period at 4 °C. Starch films with gallic acid had the least effect on ham antimicrobial activity; starch films with chitosan and carvacrol fully inhibited L. monocytogenes growth throughout 4 weeks of storage. RTE meat microbiota was more resistant to the antimicrobials than L. monocytogenes. Starch films loaded with chitosan or chitosan and carvacrol did not fully inhibit growth of RTE meat microbiota but delayed growth of RTE meat microbiota by one to two weeks. Moreover, competitive meat microbiota fully inhibited growth of L. monocytogenes. Therefore, antimicrobial starch packaging films prepared by subcritical water technology used in this study showed a promising effect on inhibiting L. monocytogenes in RTE ham.

Effect of Pressure, Reconstituted RTE Meat Microbiota, and Antimicrobials on Survival and Post-pressure Growth of Listeria monocytogenes on Ham.

Pressure treatment of ready-to-eat (RTE) meats extends the shelf life and reduces risks associated with Listeria monocytogenes. However, pressure reduces numbers of Listeria on ham by less than 5 log (CFU/g) and pressure effects on other meat microbiota are poorly documented. This study investigated the impact of pressure and RTE meat microbiota, with or without nisin and rosemary oil, on survival of Listeria after refrigerated storage. Ham was inoculated with a 5-strain cocktail of L. monocytogenes alone or with a cocktail of RTE meat microbiota consisting of Brochothrix thermosphacta, Carnobacterium maltaromaticum, Leuconostoc gelidum, and Lactobacillussakei. Products were treated at 500 MPa at 5°C for 1 or 3 min, with or without rosemary extract or nisin. Surviving cells were differentially enumerated after pressure treatment and after 4 weeks of refrigerated storage. After 4 weeks of storage, products were also analyzed by high throughput sequencing of 16S rRNA amplicons. Pressure treatment reduced counts of Listeria by 1 to 2 log (CFU/g); inactivation of RTE meat microbiota was comparable. Counts of Listeria increased by 1-3 log (CFU/g) during refrigerated storage. RTE meat microbiota did not influence pressure inactivation of Listeria but prevented growth of Listeria during refrigerated storage. Rosemary extract did not influence bacterial inactivation or growth. The combination of nisin with pressure treatment for 3 min reduced counts of Listeria and meat microbiota by >5 log (CFU/g); after 4 weeks of storage, counts were below the detection limit. In conclusion, pressure alone does not eliminate Listeria or other microbiota on RTE ham; however, the presence of non-pathogenic microbiota prevents growth of Listeria on pressure treated ham and has a decisive influence on post-pressure survival and growth.