Draft genome sequences of 148 Klebsiella pneumoniae species complex members from bovine and human hosts.

Klebsiella pneumoniae species complex members, particularly K. pnemoniae sensu stricto, are common bovine clinical mastitis pathogens and often the cause of hospital- and community-acquired infections in humans. Here, we present 148 draft genome assemblies and annotations of K. pneumoniae species complex members from bovine and human hosts in Canada.

Screening of Lactic Acid Bacteria Strains for Potential Sourdough and Bread Applications: Enzyme Expression and Exopolysaccharide Production.

Twenty-eight strains of lactic acid bacteria (LAB) were characterized for the ability to express enzymes of interest (including protease, xylanase, α-amylase, laccase, and glucose oxidase) as well as the ability to produce exopolysaccharide (EPS). The screening of enzyme capability for all LAB strains proceeded in a progressive 3-stage manner that helps to profile the efficiency of LAB strains in expressing chosen enzymes (Stage 1), highlights the strains with affinity for flour as the substrate (Stage 2), and discerns strains that can adapt well in a simulated starter environment (Stage 3). The theoretical ability of LAB to express these enzymes was also assessed using Basic Local Alignment Search Tool (BLAST) analysis to identify the underlying genes in the whole genome sequence. By consolidating both experimental data and information obtained from BLAST, three LAB strains were deemed optimal in expressing enzymes, namely, Lb. delbrueckii subsp. bulgaricus (RBL 52), Lb. rhamnosus (RBL 102), and Lb. plantarum (ATCC 10241). Meanwhile, EPS-producing capabilities were observed for 10 out of 28 LAB strains, among which, Lactococcus lactis subsp. diacetylactis (RBL 37) had the highest total EPS yield (274.15 mg polysaccharide/L culture) and produced 46.2% polysaccharide with a molecular mass of more than 100 kDa.

Draft genome sequences of 13 Salmonella enterica subsp. enterica isolates from chickens, cows, and Canadian Salmonella outbreaks.

Salmonella enterica is the etiological agent responsible for salmonellosis. Here, we report the draft whole genome sequences of 13 S. enterica subsp. enterica isolates from chickens and cows, as well as from previous Canadian Salmonella outbreaks investigated by the Canadian Food Inspection Agency.

Genomic and phenotypic profiling of Staphylococcus aureus isolates from bovine mastitis for antibiotic resistance and intestinal infectivity.

BACKGROUND: 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. RESULTS: 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 log10 reductions of intracellular S. aureus. CONCLUSIONS: 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.

A longitudinal census of the bacterial community in raw milk correlated with Staphylococcus aureus clinical mastitis infections in dairy cattle.

BACKGROUND: Staphylococcus aureus is a common cause of clinical mastitis (CM) in dairy cattle. Optimizing the bovine mammary gland microbiota to resist S. aureus colonization is a growing area of research. However, the details of the interbacterial interactions between S. aureus and commensal bacteria, which would be required to manipulate the microbiome to resist infection, are still unknown. This study aims to characterize changes in the bovine milk bacterial community before, during, and after S. aureus CM and to compare bacterial communities present in milk between infected and healthy quarters. METHODS: We collected quarter-level milk samples from 698 Holstein dairy cows over an entire lactation. A total of 11 quarters from 10 cows were affected by S. aureus CM and milk samples from these 10 cows (n = 583) regardless of health status were analyzed by performing 16S rRNA gene amplicon sequencing. RESULTS: The milk microbiota of healthy quarters was distinguishable from that of S. aureus CM quarters two weeks before CM diagnosis via visual inspection. Microbial network analysis showed that 11 OTUs had negative associations with OTU0001 (Staphylococcus). A low diversity or dysbiotic milk microbiome did not necessarily correlate with increased inflammation. Specifically, Staphylococcus xylosus, Staphylococcus epidermidis, and Aerococcus urinaeequi were each abundant in milk from the quarters with low levels of inflammation. CONCLUSION: Our results show that the udder microbiome is highly dynamic, yet a change in the abundance in certain bacteria can be a potential indicator of future S. aureus CM. This study has identified potential prophylactic bacterial species that could act as a barrier against S. aureus colonization and prevent future instances of S. aureus CM.

Nanoparticle-Enabled Combination Therapy Showed Superior Activity against Multi-Drug Resistant Bacterial Pathogens in Comparison to Free Drugs.

The emergence of multidrug-resistant (MDR) bacterial pathogens in farm animals and their zoonotic spread is a concern to both animal agriculture and public health. Apart from antimicrobial resistance (AMR), bacterial pathogens from the genera of Salmonella and Staphylococcus take refuge inside host cells, thereby demanding intervention strategies that can eliminate intracellular MDR pathogens. In this study, seven clinical isolates of Salmonella and Staphylococcus from swine farms were characterized for antibiotic (n = 24) resistance, resistance mechanisms, and virulence characteristics. All isolates showed resistance to one or more antibiotics and S. enterica ser. Typhimurium isolate had the highest resistance to the panel of antibiotics tested. Major resistance mechanisms identified were efflux pump and beta-lactamase enzyme activities. Staphylococcus isolates showed complete hemolysis and strong biofilm formation, while Salmonella isolates caused partial hemolysis, but showed no or weak biofilm formation. MDR isolates of S. aureus M12 and S. enterica ser. Typhimurium bacteria were subsequently tested against combinations of antibiotics and potentiating adjuvants for improved antibacterial efficacy using a checkerboard assay, and their fractional inhibitory concentration index (FICI) was calculated. A combination of chitosan and silica nanoparticles containing tetracycline (TET) and efflux pump inhibitor chlorpromazine (CPZ), respectively, was characterized for physicochemical properties and effectiveness against MDR Salmonella enterica ser. Typhimurium isolate. This combination of nano-encapsulated drugs improved the antibacterial efficacy by inhibiting AMR mechanisms (efflux activity, beta-lactamase enzyme activity, and hydrogen sulfide (H2S) production) and reducing intracellular pathogen load by 83.02 ± 14.35%. In conclusion, this study sheds light on the promising applicability of nanoparticle-enabled combination therapy to combat multidrug-resistant pathogens encountered in animal agriculture.

Comparative genomic analysis of Staphylococcus aureus isolates associated with either bovine intramammary infections or human infections demonstrates the importance of restriction-modification systems in host adaptation.

Staphylococcus aureus is a major etiological agent of clinical and subclinical bovine mastitis. The versatile and adaptative evolutionary strategies of this bacterium have challenged mastitis control and prevention globally, and the high incidence of S. aureus mastitis increases concerns about antimicrobial resistance (AMR) and zoonosis. This study aims to describe the evolutionary relationship between bovine intramammary infection (IMI)-associated S. aureus and human pathogenic S. aureus and further elucidate the specific genetic composition that leads to the emergence of successful bovine IMI-associated S. aureus lineages. We performed a phylogenomic analysis of 187 S. aureus isolates that originated from either dairy cattle or humans. Our results revealed that bovine IMI-associated S. aureus isolates showed distinct clades compared to human-originated S. aureus isolates. From a pan-genome analysis, 2070 core genes were identified. Host-specific genes and clonal complex (CC)-specific genes were also identified in bovine S. aureus isolates, mostly located in mobile genetic elements (MGEs). Additionally, the genome sequences of three apparent human-adapted isolates (two from CC97 and one from CC8), isolated from bovine mastitis samples, may provide an snapshot of the genomic characteristics in early host spillover events. Virulence and AMR genes were not conserved among bovine IMI-associated S. aureus isolates. Restriction-modification (R-M) genes in bovine IMI-associated S. aureus demonstrated that the Type I R-M system was lineage-specific and Type II R-M system was sequence type (ST)-specific. The distribution of exclusive, virulence, and AMR genes were closely correlated with the presence of R-M systems in S. aureus, suggesting that R-M systems may contribute to shaping clonal diversification by providing a genetic barrier to the horizontal gene transfer (HGT). Our findings indicate that the CC or ST lineage-specific R-M systems may limit genetic exchange between bovine-adapted S. aureus isolates from different lineages.

Smart traceability for food safety.

Current food production faces a tremendous challenge due to the growing human population. The global population is estimated to reach 9 billion by 2050 with 70% more food being required. Safe food is an important dimension of food security, and food traceability across the supply chain is a key component of this. However, current food traceability systems are challenged by frequent occurrences of food safety incidents and food recalls that have damaged consumer confidence, caused huge economic loss, and put pressure on food safety agencies. This review focuses on smart food traceability that has the potential to significantly improve food safety in global food supply chains. The basic concepts and critical perspectives for various detection strategies for food safety are summarized, including portable detection devices, smart indicators and sensors integrated on food packages, and data-assisted whole-genome sequencing. In addition, new digital technologies, such as Internet-of-things (IoTs) and cloud computing, are discussed with the aim of providing readers with an overview of the exciting opportunities in smart food traceability systems.

Bacillus pumilus and Bacillus subtilis Promote Early Maturation of Cecal Microbiota in Broiler Chickens.

Mature and stable intestinal microbiota in chickens is essential for health and production. Slow development of microbiota in young chickens prolongs the precarious period before reaching mature configuration. Whether probiotics can play a role in the early maturation of intestinal microbiota is unknown. To address this, day-old chicks were assigned into six groups: NC (basal diet), PC (virginiamycin), low (BPL) and high-dose (BPH) of Bacillus pumilus, and low (BSL) and high-dose (BSH) of Bacillus subtilis. Cecal contents at days 7, 14, 28 and 42 were used to analyze the treatment and time effects on the diversity and composition of microbiota. Overall, the alpha diversity was significantly decreased in the NC group between days 7 and 14, while this decline was prevented in the Bacillus subtilis probiotic (BSL and BSH) and even reversed in the BPH group. The beta-diversity showed significant responses of microbial communities to probiotics in first two weeks of life. Analyses of the abundance of microbiota reflected that members of the family Ruminococcaceae (Ruminnococcus, Oscillospira, Faecalibacterium, Butyricicoccus, and Subdoligranulum), which were dominant in mature microbiota, were significantly higher in abundance at day 14 in the probiotic groups. Conversely, the abundance of genera within the family Lachnospiraceae (Ruminococcus, Blautia, and Coprococcus) was dominant in early dynamic microbiota but was significantly lower in the probiotic groups at day 14. The Lactobacillus and Bifidobacterium abundance was higher, while the Enterobacteriaceae abundance was lower in the probiotic groups. In summary, the probiotics efficiently helped the cecal microbiota reach mature configuration earlier in life. These results could be used for the future manipulation of microbiota from the perspective of improving poultry performance.

Relative virulence of Staphylococcus aureus bovine mastitis strains representing the main Canadian spa types and clonal complexes as determined using in vitro and in vivo mastitis models.

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of ß-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1ß and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

Prevalence and mechanisms of antibiotic resistance in Escherichia coli isolated from mastitic dairy cattle in Canada.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparative genomic analysis of Escherichia coli isolates from cases of bovine clinical mastitis identifies nine specific pathotype marker genes.

Escherichia coli is a major causative agent of environmental bovine mastitis and this disease causes significant economic losses for the dairy industry. There is still debate in the literature as to whether mammary pathogenic E. coli (MPEC) is indeed a unique E. coli pathotype, or whether this infection is merely an opportunistic infection caused by any E. coli isolate being displaced from the bovine gastrointestinal tract to the environment and, then, into the udder. In this study, we conducted a thorough genomic analysis of 113 novel MPEC isolates from clinical mastitis cases and 100 bovine commensal E. coli isolates. A phylogenomic analysis indicated that MPEC and commensal E. coli isolates formed clades based on common sequence types and O antigens, but did not cluster based on mammary pathogenicity. A comparative genomic analysis of MPEC and commensal isolates led to the identification of nine genes that were part of either the core or the soft-core MPEC genome, but were not found in any bovine commensal isolates. These apparent MPEC marker genes were genes involved with nutrient intake and metabolism [adeQ, adenine permease; nifJ, pyruvate-flavodoxin oxidoreductase; and yhjX, putative major facilitator superfamily (MFS)-type transporter], included fitness and virulence factors commonly seen in uropathogenic E. coli (pqqL, zinc metallopeptidase, and fdeC, intimin-like adhesin, respectively), and putative proteins [yfiE, uncharacterized helix-turn-helix-type transcriptional activator; ygjI, putative inner membrane transporter; and ygjJ, putative periplasmic protein]. Further characterization of these highly conserved MPEC genes may be critical to understanding the pathobiology of MPEC.

A new, reliable, and high-throughput strategy to screen bacteria for antagonistic activity against Staphylococcus aureus.

BACKGROUND: Antibiotic-resistant Staphylococcus aureus clones have emerged globally over the last few decades. Probiotics have been actively studied as an alternative to antibiotics to prevent and treat S. aureus infections, but identifying new probiotic bacteria, that have antagonistic activity against S. aureus, is difficult since traditional screening strategies are time-consuming and expensive. Here, we describe a new plasmid-based method which uses highly stable plasmids to screen bacteria with antagonistic activity against S. aureus. RESULTS: We have created two recombinant plasmids (pQS1 and pQS3) which carry either gfpbk or mCherry under the control of a S. aureus quorum-sensing (QS) promoter (agrP3). Using this recombinant plasmid pair, we tested 81 bacteria isolated from Holstein dairy milk to identify bacteria that had growth-inhibiting activity against S. aureus and suggest potential explanations for the growth inhibition. The stability test illustrated that pQS1 and pQS3 remained highly stable for at least 24 h in batch culture conditions without selection pressure from antibiotics. This allowed co-culturing of S. aureus with other bacteria. Using the newly developed pQS plasmids, we found commensal bacteria, isolated from raw bovine milk, which had growth-inhibiting activity (n = 13) and quorum-quenching (QQ) activity (n = 13) towards both S. aureus Sa25 (CC97) and Sa27 (CC151). The pQS-based method is efficient and effective for simultaneously screening growth-inhibiting and QQ bacteria against S. aureus on agar media. CONCLUSIONS: It was shown that growth-inhibiting and QQ activity toward pQS plasmid transformants of S. aureus can be simultaneously monitored by observing the zone of growth inhibition and reporter protein inhibition on agar plates. Newly identified antagonistic bacteria and their functional biomolecules are promising candidates for future development of probiotic drugs and prophylactics/therapeutics for bacterial infections including S. aureus. Furthermore, this new approach can be a useful method to find bacteria that can be used to prevent and treat S. aureus infections in both humans and animals.

Refining Knowledge of Factors Affecting Vitamin B12 Concentration in Bovine Milk.

Milk is an excellent source of vitamin B12 (B12) for humans. Therefore, being able to guarantee a high and consistent concentration of this vitamin would enhance consumer perception of milk as a health food. The aim of the paper was to gather additional knowledge on factors that could explain B12 variation in cow milk through two observational studies: (1) to explore the relationship between milk B12 and ruminal conditions, such as pH and volatile fatty acid concentrations; and (2) to examine the impact of bedding on B12 concentrations in bulk tank milk. For study 1, a total of 72 milk and ruminal liquid samples were obtained from 45 Holstein cows fitted with ruminal cannula between 10 and 392 days of lactation. For study 2, bulk tank milk samples were obtained from 83 commercial herds; 26 herds used recycled manure solid bedding and 57 used straw bedding. Milk samples were analyzed for B12 using radioassay. Using principal component regression analysis, we observed that ruminal pH and the acetate:propionate ratio for cows receiving the early lactation ration were positively correlated with milk B12. Bedding did not influence milk B12 in bulk tanks, which averaged 4276 pg/mL. In conclusion, as B12 is synthesized by ruminal bacteria, optimizing ruminal conditions had a positive effect on milk B12, while bedding management had no influence.

Draft Genome Sequences of 113 Escherichia coli Strains Isolated from Intramammary Infections in Dairy Cattle.

Escherichia coli is one of the most common etiological agents responsible for clinical bovine mastitis. Here, we report the draft genome sequences and annotations of 113 E. coli strains that were isolated from Holstein cows with intramammary infections in Canada.

Staphylococcus aureus in Agriculture: Lessons in Evolution from a Multispecies Pathogen.

Staphylococcus aureus is a formidable bacterial pathogen that is responsible for infections in humans and various species of wild, companion, and agricultural animals. The ability of S. aureus to move between humans and livestock is due to specific characteristics of this bacterium as well as modern agricultural practices. Pathoadaptive clonal lineages of S. aureus have emerged and caused significant economic losses in the agricultural sector. While humans appear to be a primary reservoir for S. aureus, the continued expansion of the livestock industry, globalization, and ubiquitous use of antibiotics has increased the dissemination of pathoadaptive S. aureus in this environment. This review comprehensively summarizes the available literature on the epidemiology, pathophysiology, genomics, antibiotic resistance (ABR), and clinical manifestations of S. aureus infections in domesticated livestock. The availability of S. aureus whole-genome sequence data has provided insight into the mechanisms of host adaptation and host specificity. Several lineages of S. aureus are specifically adapted to a narrow host range on a short evolutionary time scale. However, on a longer evolutionary time scale, host-specific S. aureus has jumped the species barrier between livestock and humans in both directions several times. S. aureus 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 S. aureus, without negatively affecting agricultural productivity, is a challenging priority.

Exploring the potential of foodborne transmission of respiratory viruses.

The ongoing pandemic involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised the question whether this virus, which is known to be spread primarily though respiratory droplets, could be spread through the fecal-oral route or via contaminated food. In this article, we present a critical review of the literature exploring the potential foodborne transmission of several respiratory viruses including human coronaviruses, avian influenza virus (AVI), parainfluenza viruses, human respiratory syncytial virus, adenoviruses, rhinoviruses, and Nipah virus. Multiple lines of evidence, including documented expression of receptor proteins on gastrointestinal epithelial cells, in vivo viral replication in gastrointestinal epithelial cell lines, extended fecal shedding of respiratory viruses, and the ability to remain infectious in food environments for extended periods of time raises the theoretical ability of some human respiratory viruses, particularly human coronaviruses and AVI, to spread via food. However, to date, neither epidemiological data nor case reports of clear foodborne transmission of either viruses exist. Thus, foodborne transmission of human respiratory viruses remains only a theoretical possibility.

Concentrated sulfuric acid aqueous solution enables rapid recycling of cellulose from waste paper into antimicrobial packaging.

Waste paper is a major contributor to municipal and industrial waste, and its recycle and reuse are a current challenge. The aim of this research is to convert waste paper into value-added cellulose films through rapid dissolution in pre-cooled H2SO4 aqueous solution. Two types of waste paper, office paper and cardboard, could be dissolved within 210 s. The regenerated office paper films were transparent, and exhibited excellent mechanical properties (tensile strength: 77.55 ±â€¯6.52 MPa, elongation at break: 2.67 ±â€¯0.30 %, and Young's modulus: 5451.67 ±â€¯705.23 MPa), which were comparable to those of cellulose films prepared from spruce pulp in the same solvent. The mixed paper films showed a dramatically reduced UV transmittance due to the existence of lignin. Moreover, the regenerated films were a promising matrix to load antimicrobial compounds, and thus inhibited the growth of pathogenic bacteria. Therefore, this work provides a convenient way to directly convert waste paper into biodegradable antimicrobial packaging materials.