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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

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.

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.

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.

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.

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.

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. HIGHLIGHTS

Modulation of the gut microbiota with feed and/or feed additives has become a promising and important strategy for the improvement of animal/human health and performance in recent years. Indeed, host genetics, age, infection/inflammation, exposure to antibiotics, and diet are the main factors involved in the composition of the host intestinal microbiota and intestinal environment. Sustainable feed additives can also be safe, consumer-friendly, and effective, and commonly have a beneficial effect on host health. Sustainable feed additives include plant by-products and/or prebiotics, probiotics, and animal-derived feed additives. Recent studies have reported that many natural plant by-products are not only rich in polyphenolic compounds but they are also antioxidant, anti-inflammatory, anticancer, and act as antimicrobial, antiviral, cardioprotective, neuroprotective, and hepatoprotective compounds. Probiotics can also be a handful in maintaining the homeostasis in host gut in the presence or absence of prebiotics. In this chapter, we aim to focus on the factors involved and the mechanism by which the growth and the gut microflora modulation are done and ultimately improve the animal/host health and performance. Furthermore, the effect of various factors, specifically feed additives, on growth promotions of various animal food industries such as poultry, cattle, and swine will be also discussed.

Probiotics, prebiotics, or a combination of these two referred to as synbiotics, have emerged as a promising natural and alternative approach to make the sustainable animal farming. Previously, we reported that in the presence of prebiotic like components such as peanut flour, Lactobacillus produced more metabolites and inhibited several enteric pathogens. In this study, we tested a genetically modified lactic acid-producing bacterial strain Lactobacillus casei (LC), that produced large amounts of bioactive compounds including conjugated linoleic acid (CLA), in inhibiting enteric bacterial pathogens and improving host immune systems. The genetically engineered LC strain, LC+mcra (overexpressed mcra gene in LC) effectively eliminated Campylobacter jejuni (CJ) in co-culture condition without any stimulation with prebiotic like components. LC+mcra alone inhibited the growth of CJ completely by 48 h (P

This study was designed to determine the effects of bioactive phenolics (BPE) extracted from blackberry ( Rubus fruticosus ) and blueberry ( Vaccinium corymbosum ) byproducts/pomaces on Campylobacter jejuni colonization in broiler cecum. We raised a total of 120 Cobb-500 broiler chicks in duplicate trials up to 3 weeks to determine the effect of BPE on the reduction of colonization using a C. jejuni RM1221 marker strain in broiler cecum. We observed that, as a water supplement, 1.0 g Gallic Acid Equivalent (GAE)/L of BPE reduced C. jejuni colonization level by 1 log in three weeks old broiler cecum compared to the control group. 1 g GAE/L of BPE also resulted a complete inhibition of the C. jejuni marker strain in drinking water with a potential for reduced horizontal transfer in poultry flocks. In a separate experiment, we also raised a total of 200 Cobb-500 broilers in duplicate trials up to 6 weeks to investigate natural colonization of Campylobacter in presence of BPE and observed that 1.0 g GAE/L of BPE reduced natural colonization level of Campylobacter by 2 logs in broiler cecum as a water supplement. Relative expression of several C. jejuni stress response genes, including rod shape determining protein ( mreB ) were down-regulated in the presence of sub-lethal concentration of BPE. Prolonged exposure of C. jejuni to BPE resulted the minimum inhibitory concentration (MIC) of BPE to increase from 0.5 to 1.0 mg GAE/mL; however, after single sub-culture in BPE-free broth, the MIC value revived to 0.5 mg GAE/mL. Findings from this study reveal the high potential of berry phenolics as green antimicrobials against enteric pathogen Campylobacter and application in the reduction of pre-harvest colonization level of Campylobacter in poultry gut.

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

Probiotics are recognized to outcompete pathogenic bacteria by receptor-mediated colonizing and secreting functional metabolites which have direct antimicrobial activities towards pathogens and/or improving host’s gut health and immunity. We have constructed aLactobacillus casei(LC) probiotic strain, LC+mcra, by insertingmcra(myosin cross-reactive antigen) gene, which stimulates the conversion of conjugated linoleic acids. In this study, we evaluated the protective roles of LC+mcraagainst pathogenicSalmonella entericaserovar Typhimurium (ST) and enterohaemorrhagicE. coli(EHEC) infection in BALB/cJ mice. Through a series ofin vivoinvestigation, we observed that LC+mcracolonized efficiently in mice gut and competitively reduced the infection with ST and EHEC in various locations of small and large intestine, specifically cecum, jejunum, and ileum (p+mcraprotection were positively modulated with higher relative abundances Firmicutes but lower Proteobacteria plus increased bacterial species diversity/richness based on 16S metagenomic sequencing. Based on cytokine gene expression analysis by qRT-PCR, mice pretreated with LC+mcrawere found with attenuated bacterial pathogen-induced gut inflammation. Furthermore, mice fed LC+mcradaily 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. Thisin vivostudy suggests that daily consumption of novel conjugated linoleic acids over-producing probiotic might be efficient in improving gut intestinal microbiome composition and preventing/combating foodborne enteric bacterial infections with pathogenicSalmonellaand diarrheagenicE. coli.Author summaryNumerous bacteria colonize throughout the gastrointestinal tract and form a complex microbial ecosystem known as gut microbiota. A balanced microbial composition is crucial for maintaining proper gut health and host defense against pathogenic microbes. However, enteric bacterial infections could cause illness and even lead to death of host when foodborne pathogens likeSalmonellaand enterohaemorrhagicE. coli(EHEC) invade gut intestine and cause imbalance of gut microbiota. Beneficial microbes in gastrointestinal tract such asLactobacillusand their secreted bio-active metabolites, are potential bio-agents to improve gut immunity and outcompete bacterial pathogens. In this study, to evaluate roles of novelLactobacillusstrain LC+mcrawhich produce higher amount of a group of beneficial secondary metabolites called conjugated linoleic acids, we have shown that daily oral administration of this LC+mcrafor one-week in mice lead to higher proportion of beneficial bacterial colonization in different locations of intestine and a significant reduction of pathogenicSalmonellaand EHEC colonization. Furthermore, mice fed with LC+mcrarestore and modulateSalmonellainfection-induced negative impact on gut microbiota composition and protect themselves from various levels of physiological damage.

Organic farming practitioners are paving their path to grab the space of retail store shelves with the growing trend of various negative consequences of antibiotic and chemical residue in food and the scariness of antibiotic resistance. To cope with the current demand of food and to survive in the current food market, farmers are also desperately looking for guidance and regulation for improving the growth of products, particularly prevention of diseases and promotion of growth of farm animals such as poultry, pig, and cattle. As antibiotics or many other synthetic chemicals are not allowed for use in the organic farming scheme according to the rules regulatory bodies, natural and organic alternatives for growth promotion and control of diseases are the only apparent options. Bacteria or microbes and their derivatives, and plant-derived products are the potential alternatives for promoting growth and improving the health condition of farm animals and to control diseases. But the mechanism of action and the best effectiveness of these alternatives are not confirmed yet. In this chapter, we aim to discuss the urgency about the need of alternative growth promoters, potential alternatives, and their fate in food animal farming.

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.

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 &lt, 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 &lt, 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 &lt, 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.

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.