Changes detected in the genome sequences of Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Salmonella enterica after serial subculturing.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.

Diversity and Dynamics of the Canadian Coastal Vibrio Community: an Emerging Trend Detected in the Temperate Regions.

Vibrio species are indigenous to the marine and estuarine environments around the world and are the leading cause of water- and seafood-borne illnesses due to conditions favoring the transmission and growth of the species. Horizontal gene transfer, recombination, and mutation enable Vibrio spp. to adapt rapidly to environmental challenges from biotic and abiotic parameters, including temperature, salinity, and nutrient status of the coastal waters. This surveillance study provides evidence of Vibrio cholerae emerging in the temperate estuaries of Canada, thereby redefining the diversity and dynamics of its coastal Vibrio population. The presence of the pathogenic context in Vibrio parahaemolyticus was also detected with an increasing trend during the study period.IMPORTANCE Proliferation and abundance of the harmful biotypes of Vibrio spp. in the estuaries of Canada indicate the possibility of producing contaminated seafood for human consumption. The findings of this surveillance study may lead to awareness which may help efforts to reduce the occurrence of illnesses or outbreaks caused by Vibrio spp. in seafood.

Trend and Pattern of Antimicrobial Resistance in Molluscan Vibrio Species Sourced to Canadian Estuaries.

The emergence of antimicrobial resistance (AMR) in foodborne bacteria is a growing concern worldwide. AMR surveillance is a key element in understanding the implications resulting from the use of antibiotics for therapeutic as well as prophylactic needs. The emergence and spread of AMR in foodborne human pathogens are indirect health hazards. This surveillance study reports the trend and pattern of AMR detected in Vibrio species isolated from molluscs harvested in Canada between 2006 and 2012 against 19 commonly used antibiotics. Five common antibiotics, ampicillin, cephalothin, erythromycin, kanamycin, and streptomycin, predominantly contributed to AMR, including multidrug resistance (MDR) in the molluscan Vibrio spp. isolated in 2006. A prospective follow-up analysis of these drugs showed a declining trend in the frequency of MDR/AMR Vibrio spp. in subsequent years until 2012. The observed decline appears to have been influenced by the specific downturn in resistance to the aminoglycosides, kanamycin, and streptomycin. Frequently observed MDR/AMR Vibrio spp. in seafood is a potential health concern associated with seafood consumption. Our surveillance study provides an indication of the antibiotics that challenged the marine bacteria, sourced to Canadian estuaries, during and/or prior to the study period.

Phenotypic and genotypic characterization of Canadian clinical isolates of Vibrio parahaemolyticus collected from 2000 to 2009.

Vibrio parahaemolyticus is the leading bacterial cause of food-borne illness due to the consumption of contaminated seafood. The aim of the present study was to determine the population of its subtypes and establish a better understanding of the various types of V. parahaemolyticus strains that are causing human illness in Canada. The subtypes for 100 human clinical isolates of V. parahaemolyticus collected between 2000 and 2009 were determined by performing serotyping, ribotyping, pulsed-field gel electrophoresis, and multilocus sequence typing. Within this panel of strains, there was a high level of diversity (between 22 and 53 subtypes per method), but the presence of predominant clones with congruent subtypes between the various methods was also observed. For example, all 32 isolates belonging to sequence type 36 (ST36) were from serogroup O4, while 31 of them were ribotype EcoVib235-287, and 24 of the 32 were SfiI pulsed-field gel electrophoresis (PFGE) pattern VPSF1.0001. With regard to the presence of known virulence genes, 74 of the 100 isolates were PCR positive for the presence of the thermostable direct hemolysin (tdh); and 59 of these 74 strains also contained the second virulence marker, the tdh-related hemolysin (trh). The detection of trh was more predominant (81%) among the clinical isolates, and only four (4%) of the clinical isolates tested negative for the presence of both tdh and trh. This database, comprising 100 clinical isolates of V. parahaemolyticus strains from Canada, forms a baseline understanding of subtype diversity for future source attribution and other epidemiologic studies.

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 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).

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.

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.

Genome Sequence of a Canadian Vibrio parahaemolyticus Isolate with Unique Mobilizing Capacity.

Vibrio parahaemolyticus is a clinically significant marine bacterium implicated in gastroenteritis among consumers of raw or undercooked seafood. This report presents the whole-genome sequence of a unique strain of V. parahaemolyticus isolated from oysters harvested in Canada.

Detection, Enumeration, and Isolation of Vibrio parahaemolyticus and V. vulnificus from Seafood: Development of a Multidisciplinary Protocol.

Vibrio parahaemolyticus and V. vulnificus are bacterial foodborne pathogens that can cause illnesses in humans after ingestion or exposure to contaminated seafood or coastal waters. A procedure that combines microbiological, biochemical, and molecular methods was designed and optimized for the detection, enumeration, isolation, and characterization of these clinically significant Vibrio spp. Initially, microbiological culturing is used to resuscitate and isolate presumptive Vibrio spp. from chilled seafood samples. Biochemical tests are then used to analyze and select presumptive isolates at the species level, and, lastly, molecular methods, such as PCR targeting species-specific hemolysin genes, are used to confirm identification and assess the potential pathogenicity of presumptive isolates. By using artificially contaminated molluscan homogenates with known numbers of V. parahaemolyticus, this method yielded, on average, 90% recovery on complete agar media and 88% recovery on selective media. For V. vulnificus, the recovery rates were 86% (complete media) and 84% (selective media). Linearity of recovery of Vibrio spp. from artificially contaminated seafood homogenates supported the applicability of this method. Overall, this performance-tested protocol is easy to use, cost-effective, and fit-for-purpose, with potential for routine use in basic microbiological facilities.

Emerging Seafood Preservation Techniques to Extend Freshness and Minimize Vibrio Contamination.

Globally, the popularity of seafood consumption is increasing exponentially. To meet the demands of a growing market, the seafood industry has increasingly been innovating ways to keep their products fresh and safe while increasing production. Marine environments harbor several species of indigenous microorganisms, some of which, including Vibrio spp., may be harmful to humans, and all of which are part of the natural microbiota of the seafood. After harvest, seafood products are often shipped over large geographic distances, sometimes for prolonged periods, during which the food must stay fresh and pathogen proliferation must be minimized. Upon arrival there is often a strong desire, arising from both culinary and nutritional considerations, to consume seafood products raw, or minimally cooked. This supply chain along with popular preferences have increased challenges for the seafood industry. This has resulted in a desire to develop methodologies that reduce pathogenic and spoilage organisms in seafood items to comply with regulations and result in minimal changes to the taste, texture, and nutritional content of the final product. This mini-review discusses and compares several emerging technologies, such as treatment with plant derived natural compounds, phage lysis, high-pressure processing, and irradiation for their ability to control pathogenic vibrios, limit the growth of spoilage organisms, and keep the desired organoleptic properties of the seafood product intact.

Draft Genome Sequences of Four Vibrio parahaemolyticus Isolates from Clinical Cases in Canada.

Vibrio parahaemolyticus is a leading cause of bacterial gastroenteritis following ingestion of contaminated seafood. This report presents the draft genome sequences of four clinical strains of V. parahaemolyticus isolated in Canada. All four strains lack traditional pathogenic markers and possess uniquely individual characteristics identified using other typing criteria.

Prevalence and molecular characterization of Vibrio cholerae from ice and beverages sold in Jakarta, Indonesia, using most probable number and multiplex PCR.

Vibrio cholerae is well recognized as the causative agent of cholera, an acute intestinal infection characterized by watery diarrhea that may lead to dehydration and death in some cases. V. cholerae is a natural inhabitant of the aquatic environment in the tropical regions. Jakarta has the highest percentage of individuals affected by sporadic diarrheal illness compared with other areas in Indonesia. Inadequate safety measures for drinking water supplies, improper sanitation, and poor hygiene can increase the risk of cholera outbreaks. Few studies have been conducted on the prevalence of these bacteria in ice and beverages that are popularly sold and consumed in Jakarta. In this study, we detected and quantified V. cholerae from ice and beverages collected from several areas in five regions of Jakarta. Levels of V. cholerae in both ice and beverages were determined with the three-tube most-probable-number (MPN) method and ranged from < 0.3 to > 110 MPN/ml. The presence of regulatory and virulence gene sequences was determined by using uniplex and multiplex PCR assays. Of 110 samples tested, 33 (30%) were positive for V. cholerae; 21 (64%) were ice samples and the remaining 12 (36%) were beverages. A total of 88 V. cholerae strains were isolated, based on the presence of the toxR gene sequence identified by PCR. Other genetic markers, such as hlyA (59%), ompU (16%), and ctxA (19%), also were found during the search for potential pathogenic strains. The detection and isolation of potentially harmful V. cholerae from ice and beverages in Jakarta indicate that these products pose a health risk from choleragenic vibrios, particularly because of the emergence of classical biotypes of V. cholerae O1 and potentially harmful non-O1 serovars of this species.