A trend towards the abandonment of obtaining pure culture isolates in frontline laboratories is at a crossroads with the ability of public health agencies to perform their basic mandate of foodborne ...disease surveillance and response. The implementation of culture-independent diagnostic tests (CIDTs) including nucleic acid and antigen-based assays for acute gastroenteritis is leaving public health agencies without laboratory evidence to link clinical cases to each other and to food or environmental substances. This limits the efficacy of public health epidemiology and surveillance as well as outbreak detection and investigation. Foodborne outbreaks have the potential to remain undetected or have insufficient evidence to support source attribution and may inadvertently increase the incidence of foodborne diseases. Next-generation sequencing of pure culture isolates in clinical microbiology laboratories has the potential to revolutionize the fields of food safety and public health. Metagenomics and other 'omics' disciplines could provide the solution to a cultureless future in clinical microbiology, food safety and public health. Data mining of information obtained from metagenomics assays can be particularly useful for the identification of clinical causative agents or foodborne contamination, detection of AMR and/or virulence factors, in addition to providing high-resolution subtyping data. Thus, metagenomics assays may provide a universal test for clinical diagnostics, foodborne pathogen detection, subtyping and investigation. This information has the potential to reform the field of enteric disease diagnostics and surveillance and also infectious diseases as a whole. The aim of this review will be to present the current state of CIDTs in diagnostic and public health laboratories as they relate to foodborne illness and food safety. Moreover, we will also discuss the diagnostic and subtyping utility and concomitant bias limitations of metagenomics and comparable detection techniques in clinical microbiology, food and public health laboratories. Early advances in the discipline of metagenomics, however, have indicated noteworthy challenges. Through forthcoming improvements in sequencing technology and analytical pipelines among others, we anticipate that within the next decade, detection and characterization of pathogens via metagenomics-based workflows will be implemented in routine usage in diagnostic and public health laboratories.
Bovine mastitis is the most common infectious disease in dairy cattle with major economic implications for the dairy industry worldwide. Continuous monitoring for the emergence of antimicrobial ...resistance (AMR) among bacterial isolates from dairy farms is vital not only for animal husbandry but also for public health.
In this study, the prevalence of AMR in 113 Escherichia coli isolates from cases of bovine clinical mastitis in Canada was investigated. Kirby-Bauer disk diffusion test with 18 antibiotics and microdilution method with 3 heavy metals (copper, zinc, and silver) was performed to determine the antibiotic and heavy-metal susceptibility. Resistant strains were assessed for efflux and ß-lactamase activities besides assessing biofilm formation and hemolysis. Whole-genome sequences for each of the isolates were examined to detect the presence of genes corresponding to the observed AMR and virulence factors.
Phenotypic analysis revealed that 32 isolates were resistant to one or more antibiotics and 107 showed resistance against at least one heavy metal. Quinolones and silver were the most efficient against the tested isolates. Among the AMR isolates, AcrAB-TolC efflux activity and ß-lactamase enzyme activities were detected in 13 and 14 isolates, respectively. All isolates produced biofilm but with different capacities, and 33 isolates showed α-hemolysin activity. A positive correlation (Pearson r = + 0.89) between efflux pump activity and quantity of biofilm was observed. Genes associated with aggregation, adhesion, cyclic di-GMP, quorum sensing were detected in the AMR isolates corroborating phenotype observations.
This investigation showed the prevalence of AMR in E. coli isolates from bovine clinical mastitis. The results also suggest the inadequacy of antimicrobials with a single mode of action to curtail AMR bacteria with multiple mechanisms of resistance and virulence factors. Therefore, it calls for combinatorial therapy for the effective management of AMR infections in dairy farms and combats its potential transmission to the food supply chain through the milk and dairy products.
Biodegradable scaffolds have been extensively studied due to their wide applications in biomaterials and tissue engineering. However, infections associated with in vivo use of these scaffolds by ...different microbiological contaminants remain to be a significant challenge. This review focuses on different sterilization techniques including heat, chemical, irradiation, and other novel sterilization techniques for various biodegradable scaffolds. Comparisons of these techniques, including their sterilization mechanisms, post-sterilization effects, and sterilization efficiencies, are discussed.
is a formidable bacterial pathogen that is responsible for infections in humans and various species of wild, companion, and agricultural animals. The ability of
to move between humans and livestock ...is due to specific characteristics of this bacterium as well as modern agricultural practices. Pathoadaptive clonal lineages of
have emerged and caused significant economic losses in the agricultural sector. While humans appear to be a primary reservoir for
, the continued expansion of the livestock industry, globalization, and ubiquitous use of antibiotics has increased the dissemination of pathoadaptive
in this environment. This review comprehensively summarizes the available literature on the epidemiology, pathophysiology, genomics, antibiotic resistance (ABR), and clinical manifestations of
infections in domesticated livestock. The availability of
whole-genome sequence data has provided insight into the mechanisms of host adaptation and host specificity. Several lineages of
are specifically adapted to a narrow host range on a short evolutionary time scale. However, on a longer evolutionary time scale, host-specific
has jumped the species barrier between livestock and humans in both directions several times.
illustrates how close contact between humans and animals in high-density environments can drive evolution. The use of antibiotics in agriculture also drives the emergence of antibiotic-resistant strains, making the possible emergence of human-adapted ABR strains from agricultural practices concerning. Addressing the concerns of ABR
, without negatively affecting agricultural productivity, is a challenging priority.
Staphylococcus aureus is one of the prevalent etiological agents of contagious bovine mastitis, causing a significant economic burden on the global dairy industry. Given the emergence of antibiotic ...resistance (ABR) and possible zoonotic spillovers, S aureus from mastitic cattle pose threat to both veterinary and public health. Therefore, assessment of their ABR status and pathogenic translation in human infection models is crucial.
In this study, 43 S. aureus isolates associated with bovine mastitis obtained from four different Canadian provinces (Alberta, Ontario, Quebec, and Atlantic provinces) were tested for ABR and virulence through phenotypic and genotypic profiling. All 43 isolates exhibited crucial virulence characteristics such as hemolysis, and biofilm formation, and six isolates from ST151, ST352, and ST8 categories showed ABR. Genes associated with ABR (tetK, tetM, aac6', norA, norB, lmrS, blaR, blaZ, etc.), toxin production (hla, hlab, lukD, etc.), adherence (fmbA, fnbB, clfA, clfB, icaABCD, etc.), and host immune invasion (spa, sbi, cap, adsA, etc.) were identified by analyzing whole-genome sequences. Although none of the isolates possessed human adaptation genes, both groups of ABR and antibiotic-susceptible isolates demonstrated intracellular invasion, colonization, infection, and death of human intestinal epithelial cells (Caco-2), and Caenorhabditis elegans. Notably, the susceptibilities of S. aureus towards antibiotics such as streptomycin, kanamycin, and ampicillin were altered when the bacteria were internalized in Caco-2 cells and C. elegans. Meanwhile, tetracycline, chloramphenicol, and ceftiofur were comparatively more effective with ≤ 2.5 log
reductions of intracellular S. aureus.
This study demonstrated the potential of S. aureus isolated from mastitis cows to possess virulence characteristics enabling invasion of intestinal cells thus calling for developing therapeutics capable of targeting drug-resistant intracellular pathogens for effective disease management.
Norovirus (NoV) is the leading cause of gastroenteritis worldwide. A robust cell culture system does not exist for NoV and therefore detailed characterization of outbreak and sporadic strains relies ...on molecular techniques. In this study, we employed a metagenomic approach that uses non-specific amplification followed by next-generation sequencing to whole genome sequence NoV genomes directly from clinical samples obtained from 8 linked patients. Enough sequencing depth was obtained for each sample to use a
assembly of near-complete genome sequences. The resultant consensus sequences were then used to identify inter-host nucleotide variations that occur after direct transmission, analyze amino acid variations in the major capsid protein, and provide evidence of recombination events. The analysis of intra-host quasispecies diversity was possible due to high coverage-depth. We also observed a linear relationship between NoV viral load in the clinical sample and the number of sequence reads that could be attributed to NoV. The method demonstrated here has the potential for future use in whole genome sequence analyses of other RNA viruses isolated from clinical, environmental, and food specimens.
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 (
) 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
and
(
< 0.05), but a lower abundance of
(
< 0.05) than those from Quebec. Alpha-diversity also differed significantly between sites. Samples harvested from Nova Scotia had greater diversity (
< 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
, 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.
Human norovirus is the leading cause of viral gastroenteritis globally, and the GII.4 has been the most predominant genotype for decades. This genotype has numerous variants that have caused repeated ...epidemics worldwide. However, the molecular evolutionary signatures among the GII.4 variants have not been elucidated throughout the viral genome.
A metagenomic, next-generation sequencing method, based on Illumina RNA-Seq, was applied to determine norovirus sequences from clinical samples.
Herein, the obtained deep-sequencing data was employed to analyze full-genomic sequences from GII.4 variants prevailing in Canada from 2012 to 2016. Phylogenetic analysis demonstrated that the majority of these sequences belong to New Orleans 2009 and Sydney 2012 strains, and a recombinant sequence was also identified. Genome-wide similarity analyses implied that while the capsid gene is highly diverse among the isolates, the viral protease and polymerase genes remain relatively conserved. Numerous amino acid substitutions were observed at each putative antigenic epitope of the VP1 protein, whereas few amino acid changes were identified in the polymerase protein. Co-infection with other enteric RNA viruses was investigated and the astrovirus genome was identified in one of the samples.
Overall this study demonstrated the application of whole genome sequencing as an important tool in molecular characterization of noroviruses.
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