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.

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.

Impact of Lactobacillus-originated metabolites on enterohemorrhagic E. coli in rumen fluid.

Rumen is one of the richest microbial ecosystems naturally harboring many zoonotic pathogens. Controlling the colonization of cattle originated zoonotic pathogens in rumen, particularly enterohemorrhagic Escherichia coli (EHEC), is critical in reducing foodborne enteric diseases in humans. In this study, we aimed to inhibit the growth of EHEC in a simulated rumen system with collected rumen fluids (RFs) using live probiotics, synbiotics, and their metabolites. EHEC inoculated RF was treated with live wild type Lactobacillus casei (LCwt), LCwt with 0.5% peanut flour (LCwt+PF), an engineered LC capable of overexpressing linoleate isomerase (LCCLA), and their metabolites collected in cell-free culture supernatants (CFCSwt, CFCSwt+PF, and CFCSCLA) at various time points. A growth stimulatory effect toward Lactobacillus spp. was exerted by all CFCS, while the EHEC was suppressed. Among other treatments only LCwt+PF reduced EHEC by 2.68 logs after 72 h. This observation was also supported by metataxonomic analysis. A reduction in Bacteroidetes and Proteobacteria while increase in Firmicutes was observed at 48 h by the presence of CFCSs as compared to the control. Our observation implies probiotic-originated metabolites modulate rumen microbiota positively which can be deployed to control the transmission of cattle-borne pathogens specifically EHEC.

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.

Antagonistic Effects of Conjugated Linoleic Acids of Lactobacillus casei against Foodborne Enterohemorrhagic Escherichia coli.

ABSTRACT: Probiotics in fermented foods or commercially available supplements benefit the host by providing metabolites and peptides. The production of these metabolites varies with the available substrates or prebiotics present in the system and their concentration. In this study, 0.5% peanut flour (PF) was used to stimulate the growth and production of metabolites of wild-type Lactobacillus casei (LCwt) and compare with an engineered L. casei (LCCLA) capable of converting a higher amount of conjugated linoleic acid (CLA). The total extracellular metabolites present in the cell-free cultural supernatant (CFCS) of LCwt (without peanut), LCwt+PF (with peanut), and LCCLA were collected after 24 and 48 h of incubation, and their antagonistic activities against enterohemorrhagic Escherichia coli (EHEC EDL933) growth and pathogenesis were evaluated. All collected metabolites exhibited varying efficiency in restraining EHEC EDL933 growth, whereas supplementing a low concentration of CLA to the 48-h CFCS from LCwt showed augmented antagonism toward EHEC EDL933. A downregulation of key virulence genes was observed from metabolites collected at the 48-h time point. These observations indicate that the presence of metabolites in CFCSs-including CLA, which is produced by Lactobacillus and was identified by gas chromatography-mass spectrometry-plays a critical role. This study demonstrates the potential applicability of Lactobacillus-originated CLA in the prevention of EHEC EDL933-mediated illnesses.

Genotypic distribution and prevalence of human papillomavirus infection in an apparently healthy female population in Bangladesh.

Objective: Human papillomavirus (HPV) comprises around 120 genotypically related viruses, classified into low- and high-risk HPVs, which are capable of replicating inside the keratinocytes of skin or mucous membranes. Studies suggest that infections with HPV-16 or HPV-18 have a higher rate of developing cancer. The aim of our study was to detect HPV early, and to estimate the genotype-specific prevalence of HPV in apparently healthy and asymptomatic females in Bangladesh. Method: After cervical swab specimen collection, a VIA test was performed to identify any type of abnormality in the cervix. A multiplex PCR amplification of HPV DNA, using L1 consensus primer systems, was performed with type-specific primers, followed by sequencing to detect HPV genotypes. Result: Of the 417 females, 121 were found to be HPV positive. The most prevalent high-risk HPV genotypes were found to be HPV-16 and HPV-18. Different patient demographic parameters, such as age, socioeconomic status, education, and history of first intercourse, were also studied to establish correlations with HPV infection. Conclusion: Our results might provide some insights into factors that influence the development of cervical cancer. They might also help in guiding better patient management, increased public health awareness, further testing, and the implementation of existing vaccines.

Antagonistic Mechanism of Metabolites Produced by Lactobacillus casei on Lysis of Enterohemorrhagic Escherichia coli.

Enhancing extracellular metabolic byproducts of probiotics is one of the promising strategies to improve overall host health as well as to control enteric infections caused by various foodborne pathogens. However, the underlying mechanism of action of those metabolites and their effective concentrations are yet to be established. In this study, we determined the antibacterial potential of the metabolites in the cell-free culture supernatant (CFCS) collected from wild-type Lactobacillus casei (LCwt) and genetically modified LC to overexpress linoleate isomerase (LCCLA). We also evaluated the mechanism of action of CFCSs collected from the culture of LCwt in the presence or absence of 0.5% peanut flour (CFCSwt and CFCSwt+PF, respectively) and LCCLA alone (CFCSCLA) against enterohemorrhagic Escherichia coli (EHEC). The metabolites present in CFCSwt+PF and CFCSCLA eliminated EHEC within 24 and 48 h, respectively. Whereas CFCSwt failed to eliminate EHEC but reduced their growth by 6.7 logs (p < 0.05) as compared to the control. Significant downregulation of the expression of cell division gene, ftsZ, supported the observed degree of bactericidal and bacteriostatic properties of the collected CFCSs. Upregulation of EHEC genes related to maintaining cell membrane integrity, DNA damage repair, and molecular chaperons indicated an intensive stress condition imposed by the total metabolites present in CFCSs on EHEC growth and cellular structures. A range of deviated morphological features provoked by the metabolites indicated a membrane-targeted action, in general, to compromise the membrane permeability of EHEC. The information obtained from this study may contribute to a more efficient prevention of EHEC related infections.

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.