Plant-Derived Phenolic Acids Limit the Pathogenesis of Salmonella Typhimurium and Protect Intestinal Epithelial Cells during Their Interactions.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

Effects of Metabolites of Lactobacillus casei on Expression and Neutralization of Shiga Toxin by Enterohemorrhagic Escherichia coli.

Shiga toxin (stx), produced by enterohemorrhagic Escherichia coli (EHEC) or Shigella, causes hemolytic uremic syndrome (HUS) in humans. EHEC-mediated illnesses are recommended to treat by immune supportive strategies, instead of antibiotic therapy. Widely used probiotic Lactobacillus casei produces many bioactive metabolites, i.e., conjugated linoleic acids (CLAs) which have potential to educate host immunity and control EHEC growth and expression of its virulence genes. In this study, it was found that total metabolites of L. casei exerted a protective effect on Gb3 receptor containing mammalian cells against stx exposure.

Assessment of the prevalence, serotype, and antibiotic resistance pattern of Salmonella enterica in integrated farming systems in the Maryland-DC area.

Implementation of organic/pasture farming practices has been increasing in the USA regardless of official certification. These practices have created an increasingly growing demand for marketing safe products which are produced through these systems. Products from these farming systems have been reported to be at greater risk of transmitting foodborne pathogens because of current trends in their practices. Salmonella enterica (SE) is a ubiquitous foodborne pathogen that remains a public health issue given its prevalence in various food products, but also in the environment and as part of the microbial flora of many domestic animals. Monitoring antibiotic resistance and identifying potential sources contamination are increasingly important given the growing trend of organic/pasture markets. This study aimed to quantify prevalence of SE at the pre- and post-harvest levels of various integrated farms and sites in Maryland-Washington D.C. area, as well as identify the most prevalent serovars and antibiotic resistance patterns. Samples from various elements within the farm environment were collected and screened for SE through culture and molecular techniques, which served to identify and serotype SE, using species and serovar-specific primers, while antibiotic resistance was evaluated using an antibiogram assay. Results showed a prevalence of 7.80% of SE pre-harvest and 1.91% post-harvest. These results also showed the main sources of contamination to be soil (2.17%), grass (1.28%), feces (1.42%) and unprocessed produce (1.48%). The most commonly identified serovar was Typhimurium (11.32%) at the pre-harvest level, while the only identified serovar from post-harvest samples was Montevideo (4.35%). With respect to antibiotic resistance, out of the 13 clinically relevant antibiotics tested, gentamycin and kanamycin were the most effective, demonstrating 78.93 and 76.40% of isolates, respectively, to be susceptible. However, ampicillin, amoxicillin and cephradine had the lowest number of susceptible isolates with them being 10.95, 12.36, and 9.83%, respectively. These results help inform farms striving to implement organic practices on how to produce safer products by recognizing areas that pose greater risks as potential sources of contamination, in addition to identifying serotypes of interest, while also showcasing the current state of antibiotic efficacy and how this can influence antibiotic resistance trends in the future.

Alternative approaches to therapeutics and subtherapeutics for sustainable poultry production.

The world population is growing rapidly and thus its demand for food is growing as well. To meet the demand of the ever-increasing number of consumers, the poultry industry and both of its main sectors-conventional and organic/cage-free farming-are expanding in parallel. Due to increasing demand of poultry products and higher mortality rate of chicks (an average 0.3% increase of mortality over last 5 yr), both conventional and organic poultry farming systems struggle with various issues; animal welfare, environmental sustainability, and antibiotic resistance of the prevailing zoonotic/enteric pathogens are common issues for conventional farming whereas slow growth rate, higher costs, inefficient land use, different diseases of the chicken, and cross-contamination with bacterial pathogens into the final products are the major issues for organic poultry farming. On top of these issues, the use of subtherapeutic antibiotics was recently banned in conventional farming systems and by definition the organic farming system cannot use the antibiotics/synthetic chemicals even for therapeutic use. In conventional farming system, use of therapeutic antibiotics may result in residuals antibiotics in the final products. As a result, sustainable alternatives are in demand to mitigate the prevailing issues for both conventional and organic farming. Potential alternatives may include bacteriophages, vaccination, probiotics, plant-derived prebiotics, and synbiotics. These alternatives have beneficial attributes and shortcomings of their use in both conventional and organic poultry production system. In this review, we'll discuss the scope of these potential alternatives as therapeutics and subtherapeutics in sustainable poultry production and ways to improve their efficacy.

Combined effect of metabolites produced by a modified Lactobacillus casei and berry phenolic extract on Campylobacter and microbiome in chicken cecum contents.

Campylobacter is one of the most common foodborne bacterial pathogens causing illness, known as campylobacteriosis, in the United States. More than 70% of the campylobacteriosis cases have direct or indirect relation with poultry/poultry products. Currently, both conventional and organic/pasture poultry farmers are searching for sustainable alternative to antibiotics which can reduce colonization and cross-contamination of poultry products with Campylobacter and promote poultry health and growth. Probiotic and their nutritional supplement, known as prebiotic, have become consumers' preferences as alternatives to antibiotics/chemicals. In this study, we evaluated the combined effect of plant-derived prebiotic and probiotic-derived metabolites in reducing growth of Campylobacter in cecum contents, a simulated chicken gut condition. Cecum contents were collected from chickens pre-inoculated with kanamycin-resistant Campylobacter (CJRMKm), were incubated over 48 h time period, while being supplemented with either berry phenolic extract (BPE), cell free cultural supernatant from an engineered probiotic, Lactobacillus casei, or their combination. It was found that combine treatments were able to reduce both inoculated and naturally colonized Campylobacter more effectively. Microbiome analysis using 16S rRNA sequencing also revealed that combine treatments were capable to alter natural microflora positively within chicken cecum contents. Differences were observed in bacterial abundance at both phylum and genus level but did not show significant alteration in alpha diversity due to this treatment. PRACTICAL APPLICATION: The results of this study provide critical information for understanding the potential of synbiotic as an alternative in sustainable poultry farming. The outcomes of this study will lead future direction of using combination of probiotic-derived metabolites and BPE in poultry farming.

Purified Plant-Derived Phenolic Acids Inhibit Salmonella Typhimurium without Alteration of Microbiota in a Simulated Chicken Cecum Condition.

Salmonella enterica serovar Typhimurium (ST) remains a predominant zoonotic pathogen because of its colonization in poultry, survivability in the environment, and increasing antibiotic-resistance pattern. Plant-derived phenolics, gallic acid (GA), protocatechuic acid (PA), and vanillic acids (VA) have demonstrated antimicrobial activity in vitro; therefore, this study collected chicken cecal fluid and supplemented it with these phenolics to evaluate their potential for eliminating ST and mod-ulating the microbiota of complex environments. ST was quantified through plating, while micro-biome analysis was performed through pair-end 16S-rRNA gene sequencing. CFU/mL of ST in cecal fluid with GA was significantly reduced by 3.28 and 2.78 log at 24 h and 48 h, while PA only had a slight numerical decrease. VA significantly reduced ST by 4.81 and 5.20 log at 24 h and 48 h. Changes in relative abundance of major phyla were observed at 24 h for samples with GA and VA as Firmicute levels increased 8.30% and 20.90%, while Proteobacteria decreased 12.86% and 18.48%, respectively. Significant changes in major genre were observed in Acinetobacter (3.41% for GA) and Escherichia (13.53% for VA), while Bifidobacterium increased (3.44% for GA) and Lactobacillus remained unchanged. Results suggest that phenolic compounds exert different effects on certain pathogens, while supporting some commensal bacteria.

Diarrheagenic Escherichia coli and Their Antibiotic Resistance Patterns in Dairy Farms and Their Microbial Ecosystems.

Ruminants are the largest reservoir for all types of Escherichia coli, including the pathogenic ones, which can potentially be transmitted to humans via the food chain and environment. A longitudinal study was performed to estimate the prevalence and antibiotic-resistant pattern of pathogenic E. coli (pE.coli) strains in dairy farm environments. A total of 846 environmental samples (water, lagoon slurry, bedding, feed, feces, soil, and compost) were collected in summer over two years from five dairy farms in Maryland, USA. An additional 40 soil samples were collected in winter and summer seasons for evaluating microbiome composition. Collected environmental samples were screened for the presence of pE.coli, which was isolated using a selective culture medium, for later confirmation and virotyping using PCR with specific primers. The overall prevalence of pE.coli in dairy farms was 8.93% (71/846), with the most common virotype identified in isolates being ETEC, followed by STEC. The highest pE.coli prevalence were recorded in lagoon slurry (21.57%) while the lowest was in compost heap (2.99%). Among isolates, 95.87% of the virotypes were resistant to 9 classes of antibiotics whereas only 4.12% were sensitive. The highest proportion (68.04%) of resistance was found for quinolones (e.g., ciprofloxacin). The resulting metagenomic analysis at the phylum and genus levels of the grazing land soil suggests that climatic conditions actively influence the abundance of bacteria. Proteobacteria, which contains many Gram-negative foodborne pathogens (including pE.coli), was the most predominant phylum, accounting for 26.70% and 24.93% of soil bacteria in summer and winter, respectively. In addition to relative abundance, there was no significant difference in species diversity between seasons when calculated via Simpson (D) and Shannon (H) index. This study suggests that antibiotic-resistant E. coli virotypes are present in the dairy farm environment, and proper steps are warranted to control its transmission irrespective of seasonality.

A Highly Effective Bacteriophage-1252 to Control Multiple Serovars of Salmonella enterica.

Salmonella enterica (S. enterica) is the most common foodborne pathogen worldwide, leading to massive economic loss and a significant burden on the healthcare system. The primary source of S. enterica remains contaminated or undercooked poultry products. Considering the number of foodborne illnesses with multiple antibiotic resistant S. enterica, new controlling approaches are necessary. Bacteriophage (phage) therapies have emerged as a promising alternative to controlling bacterial pathogens. However, the limitation on the lysis ability of most phages is their species-specificity to the bacterium. S. enterica has various serovars, and several major serovars are involved in gastrointestinal diseases in the USA. In this study, Salmonella bacteriophage-1252 (phage-1252) was isolated and found to have the highest lytic activity against multiple serovars of S. enterica, including Typhimurium, Enteritidis, Newport, Heidelberg, Kentucky, and Gallinarum. Whole-genome sequencing analysis revealed phage-1252 is a novel phage strain that belongs to the genus Duplodnaviria in the Myoviridae family, and consists of a 244,421 bp dsDNA, with a G + C content of 48.51%. Its plaque diameters are approximately 2.5 mm to 0.5 mm on the agar plate. It inhibited Salmonella Enteritidis growth after 6 h. The growth curve showed that the latent and rise periods were approximately 40 min and 30 min, respectively. The burst size was estimated to be 56 PFU/cell. It can stabilize and maintain original activity between 4 °C and 55 °C for 1 h. These results indicate that phage-1252 is a promising candidate for controlling multiple S. enterica serovars in food production.

Intracellular autolytic whole cell Salmonella vaccine prevents colonization of pathogenic Salmonella Typhimurium in chicken.

Salmonella enterica (SE) is a major foodborne bacterial pathogen in the United States, commonly found as the normal flora of various animals that is attributed to causing at least 1.2 million infections annually. Poultry plays a major role in disseminating SE through direct contact with live animals and consumption of contaminated products. Vaccinating poultry against SE is a sustainable approach that can reduce SE in the host, preventing future infections in humans. An intracellular autolytic SE serovar Typhimurium vaccine (STLT2+P13+19) was developed by integrating genes 13 (holin) and 19 (lysozyme) of bacteriophage P22 into the bacterial chromosome. These were inserted downstream of sseA, an SPI-2 chaperone in SE that expresses during the intracellular phase of SE. Intracellular viability of STLT2+P13+19 reduced by 94.42% at 24 hr compared to the wild type in chicken macrophage cells (HD-11), whereas growth rate and adhesion ability remained unchanged. Inoculating STLT2+P13+19 in HD-11 significantly enhanced the relative log fold expression of genes associated to production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-10, IL-12 p40, IL-18, and GM-CSF) and Toll-like-receptors (TRL-3 and 7). Vaccination of an in vivo chicken model demonstrated significant changes in secretion of iNOS, IL-6, IL-8, IL-12, and TNF-α, as well as a reduction in the intestinal colonization of SE serovar Typhimurium. Microbiome analysis of cecal fluid using 16S rRNA gene sequencing also showed modulation of intestinal microbial composition, specifically a decrease in relative abundance of Proteobacteria and increasing Firmicutes. This study provides insight into a novel vaccine design that could make food products safer without the use of synthetic compounds.

Influence of Manure Application on the Soil Bacterial Microbiome in Integrated Crop-Livestock Farms in Maryland.

As a traditional agricultural system, integrated crop-livestock farms (ICLFs) involve the production of animals and crops in a shared environment. The ICLFs in the mid-Atlantic region of the United States practice sustainable manure aging or composting processes to provide an on-farm source of soil amendment for use as natural fertilizer and soil conditioner for crop production. However, crop fertilization by soil incorporation of aged manure or compost may introduce different microbes and alter the soil microbial community. The aim of this study was to characterize the influence of aged or composted manure application on the diversity of soil bacterial community in ICLFs. Soil samples from six ICLFs in Maryland were collected before (pre-crop) and during the season (2020-2021) and used to analyze soil bacterial microbiome by 16S rDNA sequencing. Results showed that both phylum- and genus-level alterations of soil bacterial communities were associated with amendment of aged or composted manure. Particularly, Proteobacteria and Actinobacteria were enriched, while Acidobacteria, Bacteroidetes, Planctomycetes, Firmicutes, and Chloroflexi were reduced after manure product application. Meanwhile, the relative abundance of Bacillus was decreased, while two zoonotic pathogens, Salmonella and Listeria, were enriched by manure amendments. Overall, animal manure amendment of soil increased the phylogenetic diversity, but reduced the richness and evenness of the soil bacterial communities. Although manure composting management in ICLFs benefits agricultural sustainable production, the amendments altered the soil bacterial communities and were associated with the finding of two major zoonotic bacterial pathogens, which raises the possibility of their potential transfer to fresh horticultural produce crops that may be produced on the manured soils and then subsequently consumed without cooking.

Effectiveness of probiotics, prebiotics, and prebiotic-like components in common functional foods.

The bioactive ingredients in commonly consumed foods include, but are not limited to, prebiotics, prebiotic-like components, probiotics, and postbiotics. The bioactive ingredients in functional foods have also been associated with beneficial effects on human health. For example, they aid in shaping of gut microflora and promotion of immunity. These functional components also contribute in preventing serious diseases such as cardiovascular malfunction and tumorigenesis. However, the specific mechanisms of these positive influences on human health are still under investigation. In this review, we aim to emphasize the major contents of probiotics, prebiotics, and prebiotic-like components commonly found in consumable functional foods, and we present an overview of direct and indirect benefits they provide on human health. The major contributors are certain families of metabolites, specifically short-chain fatty acids and polyunsaturated fatty acids produced by probiotics, and prebiotics, or prebiotic-like components such as flavonoids, polyphenols, and vitamins that are found in functional foods. These functional ingredients in foods influence the gut microbiota by stimulating the growth of beneficial microbes and the production of beneficial metabolites that, in turn, have direct benefits to the host, while also providing protection from pathogens and maintaining a balanced gut ecosystem. The complex interactions that arise among functional food ingredients, human physiology, the gut microbiota, and their respective metabolic pathways have been found to minimize several factors that contribute to the incidence of chronic disease, such as inflammation oxidative stress.

Competitive reduction of poultry-borne enteric bacterial pathogens in chicken gut with bioactive Lactobacillus casei.

In this study, the effect of sustainable probiotics on Campylobacter jejuni colonization and gut microbiome composition was evaluated using chicken as a model organism. Chickens were given Lactobacillus casei over-expressing myosin-cross-reactive antigen (LC+mcra). LC+mcra can generate bioactive compounds in larger quantity including conjugated linoleic acid. A total of 120 chickens were used in duplicate trials to investigate the effectiveness of LC+mcra in decreasing C. jejuni colonization by means of kanamycin resistant strain compared to the control group. We observed that LC+mcra can efficiently colonize various parts of the chicken gut and competitively reduce colonization of natural and challenged C. jejuni and natural Salmonella enterica. LC+mcra was found to reduce C. jejuni colonization in cecum, ileum and jejunum, by more than one log CFU/g when compared to the no-probiotic control group. Furthermore, 16S rRNA compositional analysis revealed lower abundance of Proteobacteria, higher abundance of Firmicutes, along with enriched bacterial genus diversity in gut of LC+mcra fed chicken. Decreased contamination of drinking water by C. jejuni and S. enterica was also observed, suggesting a potential function of reducing horizontal transfer of enteric bacteria in poultry. Outcomes of this study reveal high potential of LC+mcra as sustainable approach to decrease colonization of C. jejuni and S. enterica in poultry gut along with other beneficial attributes.

Limiting the pathogenesis of Salmonella Typhimurium with berry phenolic extracts and linoleic acid overproducing Lactobacillus casei.

The growing threat of emergent multidrug-resistant enteric bacterial pathogens, and their adopted virulence properties are directing to find alternative antimicrobials and/or development of dietaries that can improve host gut health and/or defense. Recently, we found that modified Lactobacillus casei (Lc + CLA) with increased production of conjugated linoleic acid has antimicrobial and other beneficial properties. Further, prebiotic alike products such as berry pomace extracts (BPEs), increase the growth of probiotics and inhibit the growth of certain bacterial pathogens. In this study, we evaluated the antibacterial effect of genetically modified Lc + CLA along with BPEs against major enteric pathogen Salmonella enterica serovar Typhimurium (ST). In mixed culture condition, the growth of ST was significantly reduced in the presence of Lc + CLA and/or BPEs. Bacterial cell-free cultural supernatant (CFCS) collected from wild-type Lc or modified Lc + CLA strains also inhibited the growth and survival of ST, and those inhibitory effects were enhanced in the presence of BPEs. We also found that the interaction of the pathogen with cultured host (HD-11 and INT-407) cells were also altered in the presence of either Lc or Lc + CLA strain or their CFCSs significantly. Furthermore, the relative expression of genes related to ST virulence and physicochemical properties of ST was altered by the effect of CFCSs of either Lc or Lc + CLA. These findings indicate that a diet containing synbiotic, specifically linoleic acid, over-produced Lc + CLA and prebiotic product BPEs, might have the potential to be effective in controlling ST growth and pathogenesis.

Growth Inhibition and Alternation of Virulence Genes of Salmonella on Produce Products Treated with Polyphenolic Extracts from Berry Pomace.

ABSTRACT: Organic farming, including integrated crop-livestock farms and backyard farming, is gaining popularity in the United States, and products from these farms are commonly sold at farmers' markets, local stores, and roadside stalls. Because organic farms avoid using antibiotics and chemicals and because they use composted animal waste and nonprofessional harvesting and packaging methods, their products have an increased risk of cross-contamination with zoonotic pathogens. This study sets out to evaluate the efficiency of new postharvest disinfection processes using natural berry pomace extracts (BPEs) as a means to reduce the bacterial load found in two common leafy greens, spinach and celery. Spinach and celery were inoculated with a fixed bacterial load of Salmonella Typhimurium and later were soaked in BPE-supplemented water (wBPE) for increasing periods of time, at two different temperatures (24 and 4°C). The remaining live bacteria were quantified (log CFU per leaf), and numbers were compared with those on vegetables soaked in water alone. The relative expression of virulence genes (hilA1/C1/D1, invA1/C1/E1/F1) of wBPE-treated Salmonella Typhimurium was determined. For spinach, there was a significant (P < 0.05) reduction of Salmonella Typhimurium: 0.2 to 1.2 log CFU/mL and 0.5 to 5 log CFU/mL at 24 and 4°C, respectively. For celery, there was also a significant (P < 0.05) reduction of Salmonella Typhimurium at either 24 or 4°C. The changes in relative expression of virulence genes of Salmonella Typhimurium isolated from spinach and celery varied depending on the treatment conditions but showed a significant down-regulation of inv genes when treated at 24°C for 1,440 min (P < 0.05). After seven uses, the total polyphenolic compounds in wBPE remained at an effective concentration. This research suggests that soaking these vegetables with BPE-containing water at lower temperatures can still reduce the Salmonella Typhimurium load enough to minimize the risk of infection and alter virulence properties.

Prevention of enteric bacterial infections and modulation of gut microbiota with conjugated linoleic acids producing Lactobacillus in mice.

Probiotics are recognized for outcompeting pathogenic bacteria by competitive receptor-mediated colonization and secretion of functional metabolites which are antimicrobial against certain microbes as well as improving host's gut health and immunity. Recently, we have constructed a bioactive Lactobacillus casei (LC) strain, LC+mcra , by inserting mcra (myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the modulation of gut microbiome and protective roles of LC+mcra against pathogenic Salmonella enterica serovar Typhimurium (ST) and enterohemorrhagic E. coli (EHEC) infections in BALB/cJ mice. We observed that LC+mcra colonized efficiently in mice gut intestine and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum (p < 0.05). Positive modulation of the cecal microbiota, for example, higher relative abundances of Firmicutes, lower relative abundances of Proteobacteria, and increased bacterial species diversity/richness, was detected in ST-challenged mice pretreated with LC+mcra based on 16S metagenomic sequencing. Cytokine gene expression analysis indicated that mice pretreated with LC+mcra associated with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed daily with LC+mcra for one week could protect themselves from the impairments caused by enteric infections with ST or EHEC. These impairments include weight loss, negative hematological changes, intestinal histological alterations, and potential death. This in vivo study suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic effectively improves intestinal microbiota composition and prevents/combats foodborne enteric bacterial infections with pathogenic Salmonella and diarrheagenic E. coli.

Synbiotic-like effect of linoleic acid overproducing Lactobacillus casei with berry phenolic extracts against pathogenesis of enterohemorrhagic Escherichia coli.

BACKGROUND: Majority of enteric infections are foodborne and antimicrobials including antibiotics have been used for their control and treatment. However, probiotics or prebiotics or their combination offer a potential alternative intervention strategy for improving the host health and preventing foodborne pathogen colonization/infections in reservoir. Further, bioengineered probiotics expressing bioactive products to achieve specific function is highly desirable. Recently, we over-expressed mcra (myosin cross-reactive antigen) gene in Lactobacillus casei (Lc) and developed a bioengineered probiotics Lc + CLA which produce higher amounts of metabolites including conjugated linoleic acid (CLA). Furthermore, we also reported that prebiotic like components such as berry pomace (byproduct) phenolic extracts (BPEs) can enhance the growth of probiotics and improved the beneficial effects of probiotics. In this study, we evaluated the antimicrobial effect of modified Lc + CLA in combination of BPEs on growth, survival and pathogenesis of enterohemorrhagic Escherichia coli (EHEC). RESULTS: In mixed culture condition, the growth of EHEC was significantly reduced in the presence Lc + CLA and/or BPEs. Cell-free cultural supernatant (CFCS) collected from Lc or Lc + CLA strain also inhibited the growth and survival of EHEC and the inhibitory effects of CFCSs against EHEC were enhanced in the presence of BPEs in concentration dependent manner. Interaction between EHEC and intestinal epithelial INT-407 cells were also altered significantly in the presence of either Lc or Lc + CLA strain or their CFCSs with or without BPEs. The expression of multiple virulence genes and physicochemical properties of EHEC were also altered when the bacterial cells were pretreated with CFCSs and/or BPEs. CONCLUSIONS: These results showed that diet containing bioactive Lc + CLA and natural prebiotic like component such as BPEs might be an effective way to prevent foodborne infections with EHEC.

Antimicrobial Effect and Probiotic Potential of Phage Resistant Lactobacillus plantarum and its Interactions with Zoonotic Bacterial Pathogens.

Development of phage-resistant probiotic particularly Lactobacillus is an alternative approach to enhance their beneficial effects as in animal feed supplements. In this study, we developed phage-resistant Lactobacillus plantarum (LP+PR) mutant and compared their antimicrobial effects and probiotic potential against zoonotic bacterial pathogens including Salmonella enterica serovar Typhimurium, enterohemorrhagic Escherichia coli (EHEC), Staphylococcus aureus, and Listeria monocytogenes with phage-sensitive L. plantarum (LP) strain. LP+PR strain showed markedly higher growth rate than wild-type LP strain. In co-culture with LP+PR and in the presence of cell-free cultural supernatants (CFCSs) of LP+PR, the growth of S. Typhimurium, EHEC, S. aureus, and L. monocytogenes were reduced significantly (P < 0.05). The adhesion ability of LP+PR was slightly higher than the LP on human epithelial INT-407 cells. Most importantly, LP+PR strain significantly inhibited the adhesive and invasive abilities of all four zoonotic pathogens to INT-407 cells (P < 0.05). Moreover, real-time qPCR revealed that in the presence of LP+PR strain or its CFCSs, expression of virulence genes of these zoonotic bacterial pathogens were suppressed significantly (P < 0.05). These findings suggest that the LP+PR strain is capable of inhibiting major zoonotic bacterial pathogens efficiently and would be a potential candidate for industrial usage in animal production or fermentation.

Effect of conjugated linoleic acid overproducing Lactobacillus with berry pomace phenolic extracts on Campylobacter jejuni pathogenesis.

Campylobacter jejuni (CJ) is one of the predominant causative agents of acute gastroenteritis in the US and other developed countries through the handling of raw chicken or the consumption of undercooked poultry and poultry products. Probiotics and their metabolites such as conjugated linoleic acids (CLAs) play a crucial role in improving host health and act as antimicrobials against enteric pathogens. Furthermore, prebiotics or prebiotic-like components such as bioactive phenolics from berry pomace can stimulate the growth of beneficial microbes including Lactobacillus casei (LC) and its metabolites, and competitively inhibit the growth of enteric bacterial pathogens. In this study, we aimed at enhancing the efficiency of antimicrobial/beneficial activities of LC and the extent of production of bioactive compounds by combining berry pomace phenolic extract (BPPE) and overproducing CLA in L. casei (LC-CLA). Under mixed culture conditions, LC-CLA in the presence of BPPE reduced the growth of CJ by more than 3 log CFU ml-1 within 48 h. The cell-free culture supernatant (CFCS) of LC-CLA in the presence of BPPE also reduced significantly the growth of CJ >3.2 log CFU ml-1 at 24 h. The interactions of CJ with cultured chicken fibroblast cells (DF-1), chicken macrophage (HD-11), and human epithelial cells (HeLa) were altered significantly. Treatments with BPPE and/or CFCS also altered the injured cell number, auto-aggregation capacity and cell surface hydrophobicity of CJ, significantly. Furthermore, combined treatments with BPPE and CFCSs of LC-CLA altered the expression of multiple virulence genes such as ciaB, cdtB, cadF, flaA, and flaB of CJ from 0.45 fold to 6.85 fold. Overall, BPPE enhanced the effect of LC-CLA in the reduction of CJ growth, survival ability, host cell-CJ interactions, and virulence gene expression. This finding indicates that a combination of BPPE and LC-CLA may be able to prevent the colonization of CJ in poultry, reduce the cross-contamination of poultry products and control poultry-borne campylobacteriosis in humans.

Linoleic Acids Overproducing Lactobacillus casei Limits Growth, Survival, and Virulence of Salmonella Typhimurium and Enterohaemorrhagic Escherichia coli.

Probiotics, particularly lactic acid bacteria, are biologic agents which limit the growth, virulence, and survival/colonization of various enteric bacterial pathogens and serve as potential alternatives to antibiotics. Mechanisms that contribute to this antimicrobial effect include producing bioactive metabolites/acids, increasing nutrient and receptor-mediated competition, and modulating gut microbiome ecology. However, these functions of common probiotic strains are limited due to the finite quantity of metabolites they produce and their total number in the gut ecosystem. Conjugated linoleic acids (CLAs), critical metabolites of Lactobacillus, have multiple beneficial effects on human health including anti-carcinogenesis, anti-inflammation, anti-oxidation, and anti-pathogenicity. In this study, we aim to overexpress the myosin cross-reactive antigen gene (mcra) in Lactobacillus casei (LC) to enhance the production of CLA and investigate its effectiveness against enteric bacterial pathogens, specifically Salmonella enterica serovar Typhimurium (ST) and enterohaemorrhagic Escherichia coli (EHEC). By inserting mcra in L. casei, we generated LC-CLA and found the total linoleic acid production by an individual bacterial cell was raised by 21-fold. The adherence ability of LC-CLA on human epithelial cells increased significantly and LC-CLA competitively excluded both ST and EHEC in a mixed-culture condition. Furthermore, LC-CLA significantly altered the physicochemical properties, biofilm formation abilities, interactions with host cells of both ST and EHEC, and triggered anti-inflammatory activities of host cells. These findings offer insights on applying a genetically engineered probiotic to control gut intestinal infections caused by ST and EHEC and prevent foodborne enteric illness in human.

Influence of Detection Methods in Characterizing Escherichia coli O157:H7 in Raw Goat Meat Using Conventional and Molecular Methods.

　Presence of Escherichia coli O157:H7 on fresh goat meat samples (n= 40) of Dhaka city was analyzed using conventional and molecular methods. A total of 86 presumptive E. coli O157:H7 colonies were isolated from 60% of the samples using selective agar plating method. After conventional biochemical assay followed by API 20E assay, only 11 isolates were found to be E. coli O157:H7. Further serological test identified only four isolates that has strong agglutination reaction against anti-H7 sensitized latex. The biochemically and serologically confirmed isolates were then screened for major virulence factors include eaeA, rfbE, fliC, stx1 and stx2 genes by PCR. PCR analysis of positive isolates showed, 10 isolates were eaeA and rfbE genes positive but fliC gene was only in six, indicating that these isolates were H7 positive with flagellum antigens which might not expressed or detected in serotyping tests. Multiplex PCR against eaeA, stx1 and stx2 genes of the isolates showed similar results as when done individually. These results revealed that only 7% of the primary presumptive E. coli O157:H7 was found to be stx producing E. coli O157:H7 and thus greatly influenced the detection of the pathogen in meat samples.