Survival and Expression of rpoS and grxB of Cronobacter sakazakii in Powdered Infant Formula Under Simulated Gastric Conditions of Newborns.

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96-1.05, 1.02-1.28 and 1.11-1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn's stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns.

Whole genome sequence of Vibrio cholerae NB-183 isolated from freshwater in Ontario, Canada harbors a unique gene repertoire.

OBJECTIVE: Vibrio cholerae is an enteric pathogen that poses a significant threat to global health. It causes severe dehydrating diarrheal disease cholera in humans. V. cholerae could be acquired either from consuming contaminated seafood or direct contact with polluted waters. As part of a larger program that assesses the microbial community profile in aquatic systems, V. cholerae strain NB-183 was isolated and characterized using a combination of culture- and whole-genome sequencing-based approaches. DATA DESCRIPTION: Here we report the assembled and annotated whole-genome sequence of a V. cholerae strain NB-183 isolated from a recreational freshwater lake in Ontario, Canada. The genome was sequenced using short-read Illumina systems. The whole-genome sequencing yielded 4,112,549 bp genome size with 99 contigs with an average genome coverage of 96× and 47.42% G + C content. The whole genome-based comparison, phylogenomic and gene repertoire indicates that this strain harbors multiple virulence genes and biosynthetic gene clusters. This genome sequence and its associated datasets provided in this study will be an indispensable resource to enhance the understanding of the functional, ecological, and evolutionary dynamics of V. cholerae.

Draft genome sequences of two Salmonella Uzaramo isolates from poultry in South Africa.

Salmonella enterica is a zoonotic pathogen and a leading cause of foodborne gastroenteritis in humans. Here, we report the draft genome sequences of two Salmonella Uzaramo isolates, which were isolated from poultry organs during routine post-mortem examination in South Africa. Currently, whole-genome sequences on Salmonella Uzaramo are scanty.

Evaluation of the Immunoprotective Capacity of Five Vaccine Candidate Proteins against Avian Necrotic Enteritis and Impact on the Caecal Microbiota of Vaccinated Birds.

Avian necrotic enteritis is an enteric disease of broiler chickens caused by certain pathogenic strains of Clostridium perfringens in combination with predisposing factors. A vaccine offering complete protection against the disease has not yet been commercialized. In a previous study, we produced five recombinant proteins predicted to be surface-exposed and unique to necrotic enteritis-causing C. perfringens and the immunogenicity of these potential vaccine candidates was assessed in broiler chickens. In the current work, the relative contribution of the antibodies raised by these putative antigens to protect broiler chickens was evaluated using an experimental necrotic enteritis induction model. Additionally, the link between the immune response elicited and the gut microbiota profiles in immunized birds subjected to infection with virulent C. perfringens was studied. The ELISA results showed that the IgY antibody titers in vaccinated birds on days 21 and 33 were significantly higher than those on days 7 and 14 and those in birds receiving the adjuvant alone, while the relative contribution of the specific immunity attributed to these antibodies could not be precisely determined using this experimental necrotic enteritis induction model. In addition, 16S rRNA gene amplicon sequencing showed that immunization of birds with recombinant proteins had a low impact on the chicken caecal microbiota.

Draft genome sequences of two Proteus mirabilis isolates recovered from a municipal wastewater treatment plant in Ontario, Canada.

Proteus mirabilis is a Gram-negative bacterium that is frequently implicated in urinary tract infections in humans and companion animals and has also been associated with foodborne infections in several countries. Here, we report the draft genome sequences of two P. mirabilis isolates recovered from municipal wastewater.

Survival and predictive modeling of Listeria monocytogenes under simulated human gastric conditions in the presence of bovine milk products.

Listeria monocytogenes is an opportunistic foodborne pathogen which has been implicated in many outbreaks of foodborne diseases. This study evaluated the effects of gastric acidity and gastric digestion time of adults, L. monocytogenes strain and food type on the survival of L. monocytogenes under simulated stomach conditions of adults in in vitro gastric models with dynamic pH changes occurring throughout the exposure. Individual strains as well as a cocktail of L. monocytogenes, inoculated at 8 log CFU/mL in filtered bovine milk products, 0 % milk, 2 % milk, 2 % chocolate milk and 3.25 % milk, were introduced to the gastric models for 2 h. The survival of L. monocytogenes depended on a combination of factors, including gastric acidity and gastric digestion time of adults, L. monocytogenes strain, food type and recovery method (P < 0.05). The survival rates of L. monocytogenes inoculated in 2 % milk after a 2-h exposure to simulated gastric fluids with pH values of 1.5, 2.0 and 3.0 were 0.003 to 0.040 %, 22.7 to 43.4 % and 16.6 to 27.2 %, respectively. Fluid milk containing a higher milk fat content (3.25 % vs 0 % milk) protected L. monocytogenes from being inactivated when they were exposed to the human stomach model with a gastric acidity of pH 2.0. Compared to 0 % and 3.25 % milk, L. monocytogenes survived the best in 2 % chocolate milk, which appears to be due to the presence of milk fat (2 %) and the additional nutrients that are present in chocolate milk. A predictive mathematical model was developed that captured the population of the strains of L. monocytogenes under the in vitro conditions. This study advances our understanding of the behaviour of L. monocytogenes under various human gastric conditions and provides key parameters that can affect the survival of L. monocytogenes in the stomachs of adults. The mathematical models developed in this study can be used as a supplementary tool to help predict the survival of L. monocytogenes under similar scenarios and for relevant risk-assessment studies.

Draft Genome Sequence of Exiguobacterium sp. Strain N5, Isolated from a Recreational Freshwater Kettle Lake in Ontario.

Exiguobacterium spp. are facultative anaerobic, Gram-positive, non-spore-forming bacilli, reported to tolerate extreme environments. Here, we report the draft genome sequence of Exiguobacterium sp. strain N5, isolated from a recreational freshwater lake.

Immunization of broiler chickens with five newly identified surface-exposed proteins unique to Clostridium perfringens causing necrotic enteritis.

Since the ban or reduction on the use of antibiotic growth promoters (AGPs) in commercial broiler chickens in many countries, avian necrotic enteritis (NE) caused by Clostridium perfringens has re-emerged as one of the biggest threats for the poultry industry worldwide. While the toolbox for controlling NE in the absence of antibiotics consists of a limited number of alternatives for which the overall effectiveness has yet proven to be suboptimal, an effective vaccine would represent the best control strategy for this often-deadly disease. Using a comparative and subtractive reverse vaccinology approach, we previously identified 14 putative antigenic proteins unique to NE-causing strains of C. perfringens. In the current work, the in silico findings were confirmed by PCR and sequencing, and five vaccine candidate proteins were produced and purified subsequently. Among them, two candidates were hypothetical proteins, two candidates were prepilin proteins which are predicted to form the subunits of a pilus structure, and one candidate was a non-heme iron protein. Western blotting and ELISA results showed that immunization of broiler chickens with five of these proteins raised antibodies which can specifically recognize both the recombinant and native forms of the protein in pathogenic C. perfringens.

Draft Genome Sequence of Bacillus anthracis N1, Isolated from a Recreational Freshwater Kettle Lake in Ontario, Canada.

Bacillus anthracis is widespread in soil and a causative agent of anthrax, primarily in herbivores. Here, we report the draft genome sequence of Bacillus anthracis strain N1, which was isolated from a recreational freshwater lake and found to carry multiple antibiotic resistance genes and biosynthetic gene clusters.

Inhibition of Cronobacter sakazakii in an infant simulator of the human intestinal microbial ecosystem using a potential synbiotic.

Powdered infant formula (PIF) can be contaminated with Cronobacter sakazakii, which can cause severe illnesses in infants. Synbiotics, a combination of probiotics and prebiotics, could act as an alternative control measure for C. sakazakii contamination in PIF and within the infant gut, but synbiotics have not been well studied for their ability to inhibit C. sakazakii. Using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) inoculated with infant fecal matter, we demonstrated that a potential synbiotic, consisting of six lactic acid bacteria (LAB) strains and Vivinal GOS, can inhibit the growth of C. sakazakii in an infant possibly through either the production of antimicrobial metabolites like acetate, increasing species diversity within the SHIME compartments to compete for nutrients or a combination of mechanisms. Using a triple SHIME set-up, i.e., three identical SHIME compartments, the first SHIME (SHIME 1) was designated as the control SHIME in the absence of a treatment, whereas SHIME 2 and 3 were the treated SHIME over 2, 1-week treatment periods. The addition of the potential synbiotic (LAB + VGOS) resulted in a significant decrease in C. sakazakii levels within 1 week (p < 0.05), but in the absence of a treatment the significant decline took 2 weeks (p < 0.05), and the LAB treatment did not decrease C. sakazakii levels (p ≥ 0.05). The principal component analysis showed a distinction between metabolomic profiles for the control and LAB treatment, but similar profiles for the LAB + VGOS treatment. The addition of the potential synbiotic (LAB + VGOS) in the first treatment period slightly increased species diversity (p ≥ 0.05) compared to the control and LAB, which may have had an effect on the survival of C. sakazakii throughout the treatment period. Our results also revealed that the relative abundance of Bifidobacterium was negatively correlated with Cronobacter when no treatments were added (ρ = -0.96; p < 0.05). These findings suggest that C. sakazakii could be inhibited by the native gut microbiota, and inhibition can be accelerated by the potential synbiotic treatment.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Selection of a Potential Synbiotic against Cronobacter sakazakii.

ABSTRACT: Cronobacter sakazakii is an opportunistic foodborne pathogen that can be fatal to infants; it is commonly associated with powdered infant formula due to contamination during manufacturing processes or during preparation in hospitals or homes. This project aimed to select a potential synbiotic, a combination of probiotic strains with a prebiotic product, to inhibit the growth of C. sakazakii in an in vitro dynamic infant gut model (Simulator of the Human Intestinal Microbial Ecosystem). A total of 16 lactic acid bacteria (LAB) were tested for their inhibitory properties against four different C. sakazakii strains by a zone of inhibition test. Lactobacillus and Pediococcus species were able to inhibit the growth (>15-mm inhibition zones) of all C. sakazakii strains tested, and only one strain from the two genera exhibited atypical resistance to tetracycline. All C. sakazakii strains and the selected LAB strains, which inhibited C. sakazakii and did not exhibit atypical antibiotic resistance, were grown in Luria-Bertani or de Man Rogosa Sharpe broth, respectively, containing 1% dextrose or 1% commercial prebiotic (w/v) to compare their ability to metabolize the prebiotic product. Overall, based on the growth inhibition of C. sakazakii, antibiotic susceptibility, and prebiotic metabolism, 6 of the 16 LAB were chosen to be part of a potential synbiotic. This study has provided valuable information that will help with the development of a synbiotic that can be used in powdered infant formula to reduce the potential for C. sakazakii-related illnesses in infants.

Controlling Listeria monocytogenes Growth and Biofilm Formation Using Flavonoids.

ABSTRACT: The aim of this study was to investigate the ability of natural plant-derivate products (flavonoid compounds) to inhibit the growth and biofilm-forming ability of Listeria monocytogenes. A collection of 500 synthetic and natural flavonoids were tested individually on strains of L. monocytogenes for their antimicrobial and antibiofilm activity. The flavonoids were tested against a L. monocytogenes cocktail of five strains at a concentration of 100 µM to determine their effect on planktonic growth. The optical density was measured every hour for 24 h at 37°C, and every hour for 48 h at 22°C. A total of 17 flavonoids were chosen for further study because of their ability to significantly reduce the growth of L. monocytogenes up to 97%. An additional two flavonoids that increased planktonic growth were chosen as well to investigate whether they had the same effect on biofilm growth. A lower concentration of flavonoid compounds (50 µM) was selected to investigate the individual effects on L. monocytogenes biofilm formation using (i) stainless steel coupons to quantify biomass using crystal violet staining and (ii) glass slides using confocal laser scanning microscopic (CLSM) imaging to observe the biofilm architecture. The 19 flavonoids showed various levels of L. monocytogenes biofilm growth inhibition, ranging from 2 to 100% after 48 h of incubation at 22 or 10°C. This includes 18 of the 19 flavonoids significantly (P ≤ 0.05) inhibiting L. monocytogenes biofilm formation on stainless steel coupons under at least one of the testing conditions. However, only one flavonoid compound demonstrated significant biofilm inhibition (P ≤ 0.05) under all conditions tested. Furthermore, 8 of the selected 19 flavonoid compounds showed visible reductions through CLSM in L. monocytogenes biofilm formation. Overall, we identified five flavonoid compounds to be promising antibiofilm and antimicrobial agents against L. monocytogenes.

Putative antigenic proteins identified by comparative and subtractive reverse vaccinology in necrotic enteritis-causing Clostridium perfringens isolated from broiler chickens.

BACKGROUND: Avian necrotic enteritis (NE) caused by Clostridium perfringens is a disease with a major economic impact, generating losses estimated to 6 billion of dollars annually for the poultry industry worldwide. The incidence of the disease is particularly on the rise in broiler chicken flocks eliminating the preventive use of antibiotics. To date, no alternative allows for the efficient prevention of NE and a control of the disease using a vaccinal strategy would be mostly prized. For this purpose, comparative and subtractive reverse vaccinology identifying putative immunogenic bacterial surface proteins is one of the most promising approaches. RESULTS: A comparative genomic study was performed on 16 C. perfringens strains isolated from healthy broiler chickens and from broilers affected with necrotic enteritis. Results showed that the analyzed genomes were composed of 155,700 distinct proteins from which 13% were identified as extracellular, 65% as cytoplasmic and 22% as part of the bacterial membrane. The evaluation of the immunogenicity of these proteins was determined using the prediction software VaxiJen®. CONCLUSIONS: For the most part, proteins with the highest scores were associated with an extracellular localisation. For all the proteins analyzed, the combination of both the immunogenicity score and the localisation prediction led to the selection of 12 candidate proteins that were mostly annotated as hypothetical proteins. We describe 6 potential candidates of higher interest due to their antigenic potential, their extracellular localisation, and their possible role in virulence of C. perfringens.

Current and Future Perspectives on the Role of Probiotics, Prebiotics, and Synbiotics in Controlling Pathogenic Cronobacter Spp. in Infants.

Cronobacter species, in particular C. sakazakii, is an opportunistic bacterial pathogen implicated in the development of potentially debilitating illnesses in infants (<12months old). The combination of a poorly developed immune system and gut microbiota put infants at a higher risk of infection compared to other age groups. Probiotics and prebiotics are incorporated in powdered infant formula and, in addition to strengthening gut physiology and stimulating the growth of commensal gut microbiota, have proven antimicrobial capabilities. Postbiotics in the cell-free supernatant of a microbial culture are derived from probiotics and can also exert health benefits. Synbiotics, a mixture of probiotics and prebiotics, may provide further advantages as probiotics and gut commensals degrade prebiotics into short-chain fatty acids that can provide benefits to the host. Cell-culture and animal models have been widely used to study foodborne pathogens, but sophisticated gut models have been recently developed to better mimic the gut conditions, thus giving a more accurate representation of how various treatments can affect the survival and pathogenicity of foodborne pathogens. This review aims to summarize the current understanding on the connection between Cronobacter infections and infants, as well as highlight the potential efficacy of probiotics, prebiotics, and synbiotics in reducing invasive Cronobacter infections during early infancy.

Survival of Listeria monocytogenes during storage on dried apples, strawberries, and raisins at 4 °C and 23 °C.

The survival of Listeria monocytogenes was assessed during long-term storage on three dried fruits: dried apples, raisins and dried strawberries. Using sand as a carrier, the dried fruits were dry-inoculated with a four-strain cocktail of L. monocytogenes to achieve numbers of 4.0 to 4.6 log CFU/g. The inoculated foods were stored at 4 °C, 25-81% relative humidity (RH) and 23 °C, 30-35% RH for 336 days. Colonies of L. monocytogenes could not be recovered from the dried apples after inoculation, i.e., day 0. Concentrations of L. monocytogenes decreased rapidly on the raisins and dried strawberries during storage at 23 °C, with enhanced survival observed at 4 °C. Linear rates of decline for populations of L. monocytogenes during storage at 4 °C on the raisins and dried strawberries were 0.1 and 0.2 log CFU/g/month, respectively. The relative distribution of the four L. monocytogenes strains making up the cocktail was determined by multiplex PCR at the beginning of storage and after 336 days on the dried fruits. At day 0, L. monocytogenes populations were predominantly composed of the serotype 1/2a and 3a strains on both the raisins and dried strawberries. After long-term storage at 4 °C, a relative decrease in serotype 1/2a was observed on both fruits, coupled with relative increases in the serotype 3a strain during storage on both fruits, in addition to the serotype 1/2b strain on the raisins. These results demonstrate that L. monocytogenes is rapidly inactivated during storage on raisins and dried strawberries at 23 °C, but it is capable of long-term survival at 4 °C. Improved knowledge on the survival of L. monocytogenes on these commodities is important for predictive modeling and can be used to better inform microbial health risk assessments.

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called 'Pig intestinal bacterial collection' (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

SARS-CoV-2 and Risk to Food Safety.

The COVID-19 pandemic has brought about a consideration of our understanding of transmission of the causal agent, SARS-CoV-2 to humans and its potential effect on food safety and food security. The main routes of transmission are reported to be person-to-person, by respiratory droplets and to a lesser degree, by fomites. Concerns have been raised on the possibility of transmission via food and food packaging and whether the virus poses a risk to food safety. The current contribution provides an exposé of updated literature and reports applicable to various components of food safety and its linkage to SARS-CoV-2. The article focuses on SARS-CoV-2 survival in food, on food contact materials and food packaging, and its categorization as a foodborne vs. respiratory virus, the possibility of fecal-oral transmission and the likelihood of infection via the gastro-intestinal system. The survival and inactivation of SARS-CoV-2 in food through thermal and non-thermal inactivation methods as well as the survival and inactivation on inanimate surfaces and effective disinfection of food contact surfaces, are discussed. Ultimately, the article endeavors to add to the body of knowledge pertaining to the role of SARS-CoV-2 in food safety and thereby contribute to an agile and robust fraternity that is equipped to absorb and weather the ongoing effects of the pandemic on the food sector.

Survival and Virulence of Listeria monocytogenes during Storage on Chocolate Liquor, Corn Flakes, and Dry-Roasted Shelled Pistachios at 4 and 23°C.

ABSTRACT: The survival and virulence of Listeria monocytogenes was assessed during storage on three low-moisture foods (LMFs): chocolate liquor, corn flakes, and shelled, dry-roasted pistachios (water activity [aw] of 0.18, 0.27, and 0.20, respectively). The LMFs were inoculated with a four-strain cocktail of L. monocytogenes at 8 log CFU/g, dried, held until the aw stabilized, and then stored at 4°C and 25 to 81% relative humidity (RH) and at 23°C and 30 to 35% RH for at least 336 days. At 4°C, L. monocytogenes remained stable on the LMFs for at least 336 days. At 23°C, L. monocytogenes levels declined on the chocolate liquor, corn flakes, and pistachios at initial rates of 0.84, 0.88, and 0.32 log CFU/g/month, respectively. After 8 months at 23°C, L. monocytogenes levels on the chocolate liquor and corn flakes decreased to below the limit of detection (i.e., 0.48 log CFU/g). Relative populations of each strain were assessed before storage (i.e., day 0) and after 6 and 12 months of storage at 23 and 4°C, respectively. Generally, a decline in the relative level of the serotype 1/2a strain was observed during storage, coupled with the relative increase in other strains, depending on the LMF and storage temperature. The total viable populations of L. monocytogenes determined by the PMAxx quantitative PCR method after >12 months of storage at 4°C were significantly (1.8- to 3.7-log) higher than those obtained by plating on tryptic soy agar with yeast extract. Decreases in the culturable population of L. monocytogenes during storage on the LMFs were the result of both cellular inactivation and transition to a viable-but-nonculturable state. The surviving cells, specifically after long-term storage at 4°C on the chocolate liquor and pistachios, remained infectious and capable of intracellular replication in Caco-2 enterocytes. These results are relevant for predictive modeling used in microbial health risk assessments and support the addition of LMFs to food safety questionnaires conducted during listeriosis outbreaks.

Growth of Listeria monocytogenes Inoculated on Packaged Fresh-Cut Turnips Stored at 4 and 10°C.

ABSTRACT: The ecology of Listeria monocytogenes has been previously investigated in various whole and minimally processed raw vegetables, but not in turnips. A 2018 national Canadian recall for packaged fresh-cut turnips contaminated with L. monocytogenes raised concerns about turnips being able to support the growth of this microorganism. Thus, this study examined the growth potential of L. monocytogenes in fresh-cut turnips stored at 4 and 10°C. The bacterial microbiota of each brand of purchased turnips was also partially determined to evaluate the diversity of bacteria present on the product. Turnips were mist inoculated at an initial level of 3.0 log CFU/g using a five-strain L. monocytogenes cocktail. Samples were then stored at either 4 or 10°C for 10 days, with enumeration occurring at 0, 5, and 10 days. L. monocytogenes did grow on turnips stored at 10°C, with increases ranging from 0.87 to 1.84 log CFU/g over the 10-day storage period (P < 0.05). In contrast, L. monocytogenes was able to survive but not grow on turnips stored at 4°C for 10 days. This study reinforces the importance of strict temperature control within processing, retail, and household consumer settings. Avoiding temperature abuse conditions and storing packaged fresh-cut turnips under refrigerator conditions (≤4°C) can serve as an important hurdle to prevent and/or limit the growth of L. monocytogenes on these products.