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Abstract The resistance of foodborne pathogens to antimicrobial agents is a potential danger to human health. Hence, establishing the status of good agricultural practices (GAPs) and the antimicrobial susceptibility of major foodborne pathogens has a significant programmatic implication in planning interventions. The objective of this study was to assess the gap in attaining GAP and estimate the prevalence and antimicrobial susceptibility profile of Salmonella in vegetable farms fertilized with animal manure in Addis Ababa, Ethiopia. A total of 81 vegetable farms from four sub-cities in Addis Ababa were visited, and 1119 samples were collected: soil (n = 271), manure (n = 375), vegetables (n = 398), and dairy cattle feces (n = 75). Additional data were collected using a structured questionnaire. Isolation of Salmonella was done using standard microbiology techniques and antimicrobial susceptibility testing was conducted using disk diffusion assays. Carriage for antimicrobial resistance genes was tested using polymerase chain reaction (PCR). Among the 81 vegetable farms visited, 24.7% used animal manure without any treatment, 27.2% used properly stored animal manure and 80.2% were easily accessible to animals. The prevalence of Salmonella was 2.3% at the sample level, 17.3% at the vegetable farm level, and 2.5% in vegetables. The highest rate of resistance was recorded for streptomycin, 80.7% (21 of 26), followed by kanamycin, 65.4% (17 of 26), and gentamicin, 61.5% (16 of 26). Multidrug resistance was detected in 61.5% of the Salmonella isolates. Vegetable farms have a gap in attaining GAPs, which could contribute to increased contamination and the transfer of antimicrobial resistance to the vegetables. The application of GAPs, including proper preparation of compost and the appropriate use of antimicrobials in veterinary practices, are recommended to reduce the emergence and spread of antimicrobial resistance.

ABSTRACT Pseudomonas syringae pv. syringae (Pss) is an emerging phytopathogen that causes Pseudomonas leaf spot (PLS) disease in pepper plants. Pss can cause serious economic damage to pepper production, yet very little is known about the virulence factors carried by Pss that cause disease in pepper seedlings. In this study, Pss strains isolated from pepper plants showing PLS symptoms in Ohio between 2013 and 2021 (n = 16) showed varying degrees of virulence (Pss populations and disease symptoms on leaves) on 6-week-old pepper seedlings. In vitro studies assessing growth in nutrient-limited conditions, biofilm production, and motility also showed varying degrees of virulence, but in vitro and in planta variation in virulence between Pss strains did not correlate. Comparative whole-genome sequencing studies identified notable virulence genes including 30 biofilm genes, 87 motility genes, and 106 secretion system genes. Additionally, a total of 27 antimicrobial resistance genes were found. A multivariate correlation analysis and Scoary analysis based on variation in gene content (n = 812 variable genes) and single nucleotide polymorphisms within virulence genes identified no significant correlations with disease severity, likely due to our limited sample size. In summary, our study explored the virulence and antimicrobial gene content of Pss in pepper seedlings as a first step toward understanding the virulence and pathogenicity of Pss in pepper seedlings. Further studies with additional pepper Pss strains will facilitate defining genes in Pss that correlate with its virulence in pepper seedlings, which can facilitate the development of effective measures to control Pss in pepper and other related P. syringae pathovars.IMPORTANCEPseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool–moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

IntroductionControl of Campylobacter from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of Campylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows Campylobacter to colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO4).MethodsSince mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigate Campylobacter in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO4 (1 mM) in C. jejuni 81–176, a human isolate which is widely studied.ResultsFurthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1–T8) were selected for further studies. These selected eight Tat inhibitors cleared all tested Campylobacter strains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO4in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellular C. jejuni cells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO4. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; p

Understanding the functional role of bacterial genes in the persistence of Salmonella in plant organs can facilitate the development of agricultural practices to mitigate food safety risks associated with the consumption of fresh produce contaminated with Salmonella spp. Our study showed that Salmonella enterica subsp. enterica serotype Typhimurium (strain MDD14) persisted less in inoculated tomato plants than other Salmonella Typhimurium strains tested (JSG210, JSG626, JSG634, JSG637, JSG3444, and EV030415; P < 0.01). In-vitro assays performed in limited-nutrient conditions (growth rate, biofilm production, and motility) were inconclusive in explaining the in-planta phenotype observed with MDD14. Whole-genome sequencing combined with non-synonymous single nucleotide variations analysis was performed to identify genomic differences between MDD14 and the other Salmonella Typhimurium strains. The genome of MDD14 contained a truncated version (123 bp N-terminal) of yicC and a mutated version of rpoS (two non-synonymous substitutions, i.e., G66E and R82C), which are two stress-induced proteins involved in iron acquisition, environmental sensing, and cell envelope integrity. The rpoS and yicC genes were deleted in Salmonella Typhimurium JSG210 with the Lambda Red recombining system. Both mutants had limited persistence in tomato plant organs, similar to that of MDD14. In conclusion, we demonstrated that YicC and RpoS are involved in the persistence of Salmonella in tomato plants in greenhouse conditions and, thus, could represent potential targets to mitigate persistence of Salmonella spp. in planta. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Agriculture, and particularly livestock and animal source foods, has been closely linked to improvements in human nutrition. Production, income, and women’s empowerment improve household food security and child nutritional outcomes in interacting ways. Khat production in Eastern Ethiopia is changing the economic and livelihood landscape for communities that have traditionally relied upon small-scale mixed agriculture and livestock production. How this shifting livelihood landscape and the empowerment of women in these communities are affecting nutritional outcomes has not been investigated. Using cross-sectional data collected during formative research for the Campylobacter Genomics and Environmental Enteric Dysfunction (CAGED) project, we developed models to examine the roles of livelihood activities, including livestock production, staple crop production, and khat production, and women’s empowerment in child nutrition outcomes. Survey participants were randomly selected mothers of children aged 10–15 months from Haramaya district, Eastern Hararghe, Oromia, Ethiopia. Nested logistic regression models were performed for each nutrition outcome: children’s animal source food consumption, children’s dietary diversity, and child stunting, wasting, and underweight. Explanatory variables included those for livelihood (tropical livestock unit, crop production, and khat production ladder) and women’s empowerment (as indicated by domains of the Women’s Empowerment in Agriculture Index), and covariates including child sex, mother’s age, mother’s education, assets, income, and kebele. Results indicated that khat production and tropical livestock units were not significantly associated with any of the child nutrition outcomes. However, results did indicate that the odds of reporting child animal source food consumption in households where the mother was empowered in the leadership domain was 3.33 times that in households where the mother wasn’t (p

Salmonella is the leading cause of death associated with foodborne illnesses in the USA. Difficulty in treating human salmonellosis is attributed to the development of antimicrobial resistance and the pathogenicity of Salmonella strains. Therefore, it is important to study the genetic landscape of Salmonella, such as the diversity, plasmids, and presence antimicrobial resistance genes (AMRs) and virulence genes. To this end, we isolated Salmonella from environmental samples from small specialty crop farms (SSCFs) in Northeast Ohio from 2016 to 2021; 80 Salmonella isolates from 29 Salmonella-positive samples were subjected to whole-genome sequencing (WGS). In silico serotyping revealed the presence of 15 serotypes. AMR genes were detected in 15% of the samples, with 75% exhibiting phenotypic and genotypic multidrug resistance (MDR). Plasmid analysis demonstrated the presence of nine different types of plasmids, and 75% of AMR genes were located on plasmids. Interestingly, five Salmonella Newport isolates and one Salmonella Dublin isolate carried the ACSSuT gene cassette on a plasmid, which confers resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline. Overall, our results show that SSCFs are a potential reservoir of Salmonella with MDR genes. Thus, regular monitoring is needed to prevent the transmission of MDR Salmonella from SSCFs to humans.

Chitosan has received much attention for its role in designing and developing novel derivatives as well as its applications across a broad spectrum of biological and physiological activities, owing to its desirable characteristics such as being biodegradable, being a biopolymer, and its overall eco-friendliness. The main objective of this review is to explore the recent chemical modifications of chitosan that have been achieved through various synthetic methods. These chitosan derivatives are categorized based on their synthetic pathways or the presence of common functional groups, which include alkylated, acylated, Schiff base, quaternary ammonia, guanidine, and heterocyclic rings. We have also described the recent applications of chitosan and its derivatives, along with nanomaterials, their mechanisms, and prospective challenges, especially in areas such as antimicrobial activities, targeted drug delivery for various diseases, and plant agricultural domains. The accumulation of these recent findings has the potential to offer insight not only into innovative approaches for the preparation of chitosan derivatives but also into their diverse applications. These insights may spark novel ideas for drug development or drug carriers, particularly in the antimicrobial, medicinal, and plant agricultural fields.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome’s Simpson’s Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

ABSTRACT: Avian pathogenic E. coli (APEC), a causative agent of colibacillosis, is associated with high mortality and morbidity which results in severe economic losses to the poultry industry worldwide. APEC can be transmitted to humans through the consumption of contaminated poultry products. The limited effect of the current vaccines and the advent of drug-resistant strains have necessitated the development of alternative therapies. Previously, we identified 2 small molecules (SMs; [quorum sensing inhibitor; QSI-5] and [growth inhibitor; GI-7]) with high efficacy in vitro and in chickens subcutaneously challenged with APEC O78. Here, we optimized the oral challenge dose of APEC O78 in chickens to mimic the infection in the natural settings, evaluated the efficacy of the GI-7, QSI-5, and combination of GI-7 and QSI-5 (GI7+ QSI-5) in chickens orally infected with APEC, and compared their efficacy to sulfadimethoxine (SDM), an antibiotic currently used to treat APEC. Using the optimized dose of each SM in drinking water, GI-7, QSI-5, GI7+ QSI-5, and SDM were evaluated in chickens challenged with the optimized dose of APEC O78 (1 × 109 CFU/chicken; orally; d 2 of age) and grown on built-up floor litter. Reduction in mortality was 90, 80, 80, and 70% in QSI-5, GI-7+QSI-5, GI-7, and SDM treated groups compared to the positive control (PC), respectively. GI-7, QSI-5, GI-7+QSI-5, and SDM reduced the APEC load in the cecum by 2.2, 2.3, 1.6, and 0.6 logs and in the internal organs by 1.3, 1.2, 1.4, and 0.4 logs compared to PC (P < 0.05), respectively. The cumulative pathological lesions scores were 0.51, 0.24, 0.0, 0.53, and 1.53 in GI-7, QSI-5, GI-7+QSI-5, SDM, and PC groups, respectively. Overall, GI-7 and QSI-5 individually have promising effects as a potential antibiotic-independent approach to control APEC infections in chickens.

IntroductionWith more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States.MethodsThe goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020.ResultsA total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulence-associated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also sub-divided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3’-IIIa; 20%). Both species harbored resistance genes associated with β-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%).Discussion/ConclusionOur study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification.

ABSTRACT Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5′-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.

Introduction Undernutrition is an underlying cause of mortality in children under five (CU5) years of age. Animal-source foods have been shown to decrease malnutrition in CU5. Livestock are important reservoirs for Campylobacter bacteria, which are recognised as risk factors for child malnutrition. Increasing livestock production may be beneficial for improving nutrition of children but these benefits may be negated by increased exposure to Campylobacter and research is needed to evaluate the complex pathways of Campylobacter exposure and infection applicable to low-income and middle-income countries. We aim to identify reservoirs of infection with Campylobacter spp. of infants in rural Eastern Ethiopia and evaluate interactions with child health (environmental enteric dysfunction and stunting) in the context of their sociodemographic environment.Methods and analysis This longitudinal study involves 115 infants who are followed from birth to 12 months of age and are selected randomly from 10 kebeles of Haramaya woreda, East Hararghe zone, Oromia region, Ethiopia. Questionnaire-based information is obtained on demographics, livelihoods, wealth, health, nutrition and women empowerment; animal ownership/management and diseases; and water, sanitation and hygiene. Faecal samples are collected from infants, mothers, siblings and livestock, drinking water and soil. These samples are analysed by a range of phenotypic and genotypic microbiological methods to characterise the genetic structure of the Campylobacter population in each of these reservoirs, which will support inference about the main sources of exposure for infants.Ethics and dissemination Ethical approval was obtained from the University of Florida Internal Review Board (IRB201903141), the Haramaya University Institutional Health Research Ethics Committee (COHMS/1010/3796/20) and the Ethiopia National Research Ethics Review Committee (SM/14.1/1059/20). Written informed consent is obtained from all participating households. Research findings will be disseminated to stakeholders through conferences and peer-reviewed journals and through the Feed the Future Innovation Lab for Livestock Systems.

ABSTRACT Avian pathogenic Escherichia coli (APEC) associated with colibacillosis results in high morbidity and mortality, and severe economic losses to the poultry industry. APEC is a zoonotic pathogen and can infect humans through contaminated poultry products. Vaccination and antibiotic treatment are currently used to control APEC infections; however, the limited effect of vaccines and the emergence of antibiotic-resistant strains have necessitated the development of novel therapeutics. Here, we evaluated seven quorum sensing inhibitors (QSI) identified in our previous study, in APEC-infected chickens. QSIs were administered orally (~92 to 120 μg/bird) and chickens were challenged subcutaneously with APEC. Among them, QSI-5 conferred the best protection (100% reduction in mortality, 82% to 93% reduction in lesions [airsacculitis, perihepatitis, lung congestion, pericarditis] severity, and 5.2 to 6.1 logs reduction in APEC load). QSI-5 was further tested in chickens raised on built-up floor litter using an optimized dose (1 mg/L) in drinking water. QSI-5 reduced the mortality (88.4%), lesion severity (72.2%), and APEC load (2.8 logs) in chickens, which was better than the reduction observed with currently used antibiotic sulfadimethoxine (SDM; mortality 35.9%; lesion severity up to 36.9%; and APEC load up to 2.4 logs). QSI-5 was detected in chicken's blood after 0.5 h with no residues in muscle, liver, and kidney. QSI-5 increased the body weight gain with no effect on the feed conversion ratio and cecal microbiota of the chickens. Metabolomic studies revealed reduced levels of 5′-methylthioadenosine in QSI-5-treated chicken serum. In conclusion, QSI-5 displayed promising effects in chickens and thus, represents a novel anti-APEC therapeutic. IMPORTANCE Avian pathogenic Escherichia coli (APEC), a subgroup of ExPEC, is a zoonotic pathogen with public health importance. Quorum sensing is a mechanism that regulates virulence, biofilm formation, and pathogenesis in bacteria. Here, we identified a novel quorum sensing autoinducer-2 inhibitor, QSI-5, which showed higher anti-APEC efficacy in chickens compared to the currently used antibiotic, sulfadimethoxine at a much lower dose (up to 4,500 times). QSI-5 is readily absorbed with no residues in the tissues. QSI-5 also increased the chicken’s body weight gain and did not impact the cecal microbiota composition. Overall, QSI-5 represents a promising lead compound for developing novel anti-virulence therapies with significant implications for treating APEC infections in chickens as well as other ExPEC associated infections in humans. Further identification of its target(s) and understanding the mechanism of action of QSI-5 in APEC will add to the future novel drug development efforts that can overcome the antimicrobial resistance problem.

ABSTRACT The impact of obesity on the human microbiota, immune maturation, and influenza virus infection has not been yet established in natural host animal models of influenza. In this study, gnotobiotic (Gn) pigs were colonized with human fecal microbiota (HFM) of obese (oHFM) or healthy lean (hHFM) children and infected at different periods (2-, 3-, and 5-weeks post-transplantation) using a zoonotic influenza virus strain. The infected oHFM pigs were characterized by lower levels of Firmicutes (Lactococcus, Lactobacillus, Turicibacter, and Streptococcus) and Actinobacteria (Bifidobacterium), which was associated with higher levels of Proteobacteria (Klebsiella), Bacteroidetes, and Verrucomicrobia (Akkermansia) compared with the infected hHFM group (P

Malnutrition refers to inadequate energy and/or nutrient intake. Malnutrition exhibits a bidirectional relationship with infections whereby malnutrition increases risk of infections that further aggravates malnutrition. Severe malnutrition (SM) is the main cause of secondary immune deficiency and mortality among children in developing countries. SM can manifest as marasmus (non-edematous), observed most often (68.6% of all malnutrition cases), kwashiorkor (edematous), detected in 23.8% of cases, and marasmic kwashiorkor, identified in ~7.6% of SM cases. Marasmus and kwashiorkor occur due to calorie-energy and protein-calorie deficiency (PCD), respectively. Kwashiorkor and marasmic kwashiorkor present with reduced protein levels, protein catabolism rates, and altered levels of micronutrients leading to uncontrolled oxidative stress, exhaustion of anaerobic commensals, and proliferation of pathobionts. Due to these alterations, kwashiorkor children present with profoundly impaired immune function, compromised intestinal barrier, and secondary micronutrient deficiencies. Kwashiorkor-induced alterations contribute to growth stunting and reduced efficacy of oral vaccines. SM is treated with antibiotics and ready-to-use therapeutic foods with variable efficacy. Kwashiorkor has been extensively investigated in gnotobiotic (Gn) mice and piglet models to understand its multiple immediate and long-term effects on children health. Due to numerous physiological and immunological similarities between pigs and humans, pig represents a highly relevant model to study kwashiorkor pathophysiology and immunology. Here we summarize the impact of kwashiorkor on children’s health, immunity, and gut functions and review the relevant findings from human and animal studies. We also discuss the reciprocal interactions between PCD and rotavirus—a highly prevalent enteric childhood pathogen due to which pathogenesis and immunity are affected by childhood SM.

Shiga toxin-producing Escherichia coli (STEC) is estimated to cause over two million cases of human disease annually. Trinidad and Tobago is one of the largest livestock producer and consumer of sheep and goat meat in the Caribbean, however, the potential role of these animals in the epidemiology of STEC infections has not been previously described. To fill this critical gap in knowledge, the prevalence of Shiga toxin genes (stx1 and stx2) shed in the faeces of healthy sheep (n = 204) and goats (n = 105) in Trinidad was investigated. Based on PCR screening, goats had a higher stx prevalence than sheep (46% vs 35%, P = 0.06). Most of the recovered STEC isolates were positive for stx1 only; and only three isolates were positive for the eae gene. None of the recovered isolates belonged to the O157 serogroup. In both species, the prevalence of stx was higher in young animals versus older animals. Sheep reared on deep litter flooring (43%) had a higher prevalence than sheep reared other flooring types, however this was not the same for goats. The presence of cows on the same premise was not an associated predictor for STEC carriage in sheep or goats. This study demonstrates that although sheep and goats in Trinidad are reservoirs for stx-positive E. coli isolates, no fecal samples tested positive for O157 STEC, harbored. Furthermore, it appears that non-O157 stx-positive isolates harbored by these animals do not pose a significant threat to human health.

ABSTRACT Avian pathogenic Escherichia coli (APEC), a subgroup of extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly implicated in urinary tract infections and meningitis in humans. A major limitation for the current ExPEC antibiotic therapy is the development of resistance, and antibacterial drugs that can circumvent this problem are critically needed. Here, we evaluated eight novel membrane-affecting anti-APEC small molecule growth inhibitors (GIs), identified in our previous study, against APEC infection in chickens. Among the GIs tested, GI-7 (the most effective), when administered orally (1 mg/kg of body weight), reduced the mortality (41.7%), severity of lesions (62.9%), and APEC load (2.6 log) in chickens. Furthermore, GI-7 administration at an optimized dose (60 mg/liter) in drinking water also reduced the mortality (14.7%), severity of lesions (29.5%), and APEC load (2.2 log) in chickens. The abundances of Lactobacillus and oleate were increased in the cecum and serum, respectively, of GI-7-treated chickens. Pharmacokinetic analysis revealed that GI-7 was readily absorbed with minimal accumulation in the tissues. Earlier, we showed that GI-7 induced membrane blebbing and increased membrane permeability in APEC, suggesting an effect on the APEC membrane. Consistent with this finding, the expression of genes essential for maintaining outer membrane (OM) integrity was downregulated in GI-7-treated APEC. Furthermore, decreased levels of lipopolysaccharide (LPS) transport (Lpt) proteins and LPS were observed in GI-7-treated APEC. However, the mechanism of action of GI-7 currently remains unknown and needs further investigation. Our studies suggest that GI-7 represents a promising novel lead compound that can be developed to treat APEC infection in chickens and related human ExPEC infections. IMPORTANCE APEC is a subgroup of ExPEC, and genetic similarities of APEC with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), have been reported. Our study identified a novel small molecule growth inhibitor, GI-7, effective in reducing APEC infection in chickens with an efficacy similar to that of the currently used antibiotic sulfadimethoxine, notably with an 8-times-lower dose. GI-7 affects the OM integrity and decreases the Lpt protein and LPS levels in APEC, an antibacterial mechanism that can overcome the antibiotic resistance problem. Overall, GI-7 represents a promising lead molecule/scaffold for the development of novel antibacterial therapies that could have profound implications for treating APEC infections in chickens, as well as human infections caused by ExPECs and other related Gram-negative bacteria. Further elucidation of the mechanism of action of GI-7 and identification of its target(s) in APEC will benefit future novel antibacterial development efforts.

Background Cull sows are a unique population on swine farms, often representing poor producing or compromised animals, and even though recent studies have reported that the microbiome is associated with susceptibility to diseases, the microbiome of the cull sow population has not been explored. The main objective of this study was to investigate whether there were differences in fecal and upper respiratory tract microbiota composition for groups of sows of different health status (healthy, cull, and compromised/ clinical sows) and from different farms (1 to 6). Methods Six swine farms were visited once. Thirty individual fecal samples and nasal swabs were obtained at each farm and pooled by five across health status and farm. Samples underwent 16S rRNA gene amplicon sequencing and nasal and fecal microbiota were analyzed using QIIME2 v.2021.4. Results Overall, the diversity of the nasal microbiota was lower than the fecal microbiota (p

Imaging technology can provide insight into biological processes governing plant-pathogen interactions. We created and used a bioluminescent strain of Xanthomonas hortorum pv. gardneri (Xgb) to quantify infection processes in plants using tomato as a model. An X. hortorum pv. gardneri is one of the four Xanthomonas species that causes bacterial spots in tomatoes. We used Xgb to quantify bacterial growth in planta, to assess disease severity in resistant and susceptible tomato lines, and to observe infection routes in leaves. A positive and significant linear correlation r (67) = 0.57, p ≤ 0.0001 was observed between bioluminescence signals emitted by Xgb in planta and bacterial populations determined through dilution plating. Based on bioluminescence imaging, resistant and susceptible tomato lines had significantly different average radiances. In addition, there was a positive and significant correlation r = 0.45, p = 0.024 between X. hortorum pv. gardneri-inoculated tomato lines evaluated by bioluminescence imaging and tomatoes rated in the field using the Horsfall-Barrat Scale. Heritability was calculated to compare the genetic variance for disease severity using bioluminescence imaging and classical field ratings. The genetic variances were 25 and 63% for bioluminescence imaging and field ratings, respectively. The disadvantage of lower heritability attained by bioluminescence imaging may be offset by the ability to complete germplasm evaluation experiments within 30 days rather than 90–120 days in field trials. We further explored X. hortorum pv. gardneri infection routes on leaves using spray and dip inoculation techniques. Patterns of bioluminescence demonstrated that the inoculation technique affected the distribution of bacteria, an observation verified using scanning electron microscopy (SEM). We found significant non-random distributions of X. hortorum pv. gardneri on leaf surfaces with the method of inoculation affecting bacterial distribution on leaf surfaces at 4 h postinoculation (hpi). At 18 hpi, regardless of inoculation method, X. hortorum pv. gardneri localized on leaf edges near hydathodes based on bioluminescence imaging and confirmed by electron microscopy. These findings demonstrated the utility of bioluminescent X. hortorum pv. gardneri to estimate bacterial populations in planta, to select for resistant germplasm, and to detect likely points of infection.

Colibacillosis caused by avian pathogenic E. coli (APEC), is an economically important bacterial disease of poultry. APEC are a subgroup of extra intestinal pathogenic E. coli (ExPEC) and poultry are considered potential sources of foodborne ExPEC to humans. Currently, APEC infections in poultry are controlled by antibiotics and/or vaccination; however, their effect is limited due to emergence of antibiotic resistant strains and infections with heterologous serotypes. Therefore, novel approaches are needed. Here, using the bioluminescent quorum sensing (QS) autoinducer 2 (AI-2) indicator Vibrio harveyi BB170, we screened the cell free culture supernatant of APEC O78 prepared from cultures grown in the presence of 4,182 small molecules (SMs; 100 μM). A total of 69 SMs inhibited > 75% of APEC O78 AI-2 activity in the indicator bacteria. Ten SMs that showed highest AI-2 inhibition were selected for further studies. Most of these SMs inhibited the AI-2 activity of other APEC serotypes and significantly reduced APEC O78 biofilm formation and motility. Most compounds showed minimal toxicity on human intestinal cells (Caco-2), chicken macrophage (HD-11), and chicken and sheep red blood cells, and reduced APEC survival in HD-11 and THP-1 macrophages. The SMs induced no or minimal toxicity and conferred protection against APEC in wax moth larval model. SMs affected the expression of APEC O78 QS, virulence, biofilm and motility associated genes providing insight on their potential mode(s) of action. Further testing in chickens will facilitate development of these SMs as novel therapeutics to control APEC in poultry and thereby also reduce zoonotic transmission.

Rotavirus (RV) is a primary cause of malabsorptive diarrhea in children and is associated with significant morbidity and mortality, especially in developing countries. The use of antibiotics exacerbates intestinal microbial imbalance and results in the persistence of RV-induced diarrhea.

Livestock farming provides a possible mechanism by which smallholder farmers can meet their household need for animal source foods (ASF), which may reduce the risk of stunting. However, direct/indirect contacts with domestic animals may increase colonization by Campylobacter spp., which has been associated with Environmental Enteric Dysfunction (EED) and stunting. A cross-sectional study involving 102 randomly selected children between 12 and 16 months of age was conducted in rural eastern Ethiopia to establish prevalence rates of Campylobacter colonization, EED, and stunting, and evaluate potential risk factors. Data were collected between September and December 2018. The prevalence of EED and stunting was 50% (95% CI: 40–60%) and 41% (95% CI: 32–51%), respectively. Among enrolled children, 56% had consumed some ASF in the previous 24 h; 47% had diarrhea and 50% had fever in the past 15 days. 54, 63, 71 or 43% of households owned at least one chicken, cow/bull, goat, or sheep; 54 (53%) households kept chickens indoors overnight and only half of these confined the animals. Sanitation was poor, with high levels of unimproved latrines and open defecation. Most households had access to an improved source of drinking water. The prevalence of Campylobacter colonization was 50% (95% CI: 41–60%) by PCR. In addition to the thermotolerant species Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis, non-thermotolerant species related to Campylobacter hyointestinalis and Campylobacter fetus were frequently detected by Meta-total RNA sequencing (MeTRS). Current breastfeeding and ASF consumption increased the odds of Campylobacter detection by PCR, while improved drinking water supply decreased the odds of EED. No risk factors were significantly associated with stunting. Further studies are necessary to better understand reservoirs and transmission pathways of Campylobacter spp. and their potential impact on child health.

Human rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model.

Campylobacter jejuni is among the leading causes of bacterial foodborne illness. Poultry is the major reservoir and source of human campylobacteriosis. Currently, there is no effective and practical method to decrease C. jejuni colonization in chickens or to reduce human infections. Additionally, antibiotic-resistant infections pose a serious public health concern; therefore, antibiotic-alternative approaches are needed to reduce transmission of C. jejuni including resistant bacteria from chickens to humans. Here, we evaluated the effect of E. coli Nissle 1917 (EcN) on innate responses of polarized HT-29 cells and consequently on C. jejuni 81176 infections in HT-29 cells. Pre-treatment of HT-29 cells with EcN for 4 h had a significant effect on the invasion of different C. jejuni strains (2 h post-infection) (P

Subtyping of bacterial isolates of the same genus and species is an important tool in epidemiological investigations. A number of phenotypic and genotypic subtyping methods are available; however, most of these methods are labor-intensive and time-consuming and require considerable operator skill and a wealth of reagents. Matrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF), an alternative to conventional subtyping methods, offers a rapid, reproducible method for bacterial identification with a high sensitivity and specificity and at minimal cost. The purpose of this study was to determine the feasibility of using MALDI-TOF to differentiate between six Salmonella serovars recovered from experimental microcosms inoculated with known strains of Salmonella. Following the establishment of a MALDI-TOF reference library for this project, the identity of 843 Salmonella isolates recovered from these microcosms was assessed using both MALDI-TOF and conventional methods (serotyping/PCR). All 843 isolates were identified as being Salmonella species. Overall, 803/843 (95%) of these isolates were identified similarly using the two different methods. Positive percent agreement at the serovar level ranged from 79 to 100%, and negative percent agreement for all serovars was greater than 98%. Cohen’s kappa ranged from 0.85 to 0.98 for the different serovars. This study demonstrates that MALDI-TOF is a viable alternative for the rapid identification and differentiation of Salmonella serovars.

High Campylobacter prevalence during early childhood has been associated with stunting and environmental enteric dysfunction (EED), especially in low resource settings. This study assessed the prevalence, diversity, abundance, and co-occurrence of Campylobacter spp. in stools from children in a rural area of eastern Ethiopia and their association with microbiome, diarrhea, and EED in children. Stool samples (n = 100) were collected from randomly selected children (age range: 360–498 days) in five kebeles in Haramaya District, Ethiopia. Diarrhea, compromised gut permeability, and gut inflammation were observed in 48, 45, and 57% of children, respectively. Campylobacter prevalence and species diversity were assessed using PCR and meta-total RNA sequencing (MeTRS). The prevalence of Campylobacter spp. in the children's stools was 50% (41–60%) by PCR and 88% (80–93.6%) by MeTRS (P < 0.01). Further, seven Campylobacter species (Campylobacter jejuni, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter coli, Campylobacter sp. RM6137, uncultured Campylobacter sp., and Campylobacter sp. RM12175) were detected by MeTRS in at least 40% of children stools in high abundance (>1.76-log read per million per positive stool sample). Four clusters of Campylobacter species (5–12 species per cluster) co-occurred in the stool samples, suggesting that Campylobacter colonization of children may have occurred through multiple reservoirs or from a reservoir in which several Campylobacter species may co-inhabit. No associations between Campylobacter spp., EED, and diarrhea were detected in this cross-sectional study; however, characteristic microbiome profiles were identified based on the prevalence of Campylobacter spp., EED severity, and diarrhea. Forty-seven bacterial species were correlated with Campylobacter, and 13 of them also correlated with gut permeability, gut inflammation and/or EED severity. Forty-nine species not correlated with Campylobacter were correlated with gut permeability, gut inflammation, EED severity and/or diarrhea. This study demonstrated that (1) in addition to C. jejuni and C. coli, multiple non-thermophilic Campylobacter spp. (i.e., Campylobacter hyointestinalis, Campylobacter fetus, and Campylobacter concisus) were frequently detected in the children's stools and (2) the Campylobacter, gut permeability, gut inflammation, EED severity, and diarrhea were associated with characteristic microbiome composition. Additional spatial and longitudinal studies are needed to identify environmental reservoirs and sources of infection of children with disparate Campylobacter species and to better define their associations with EED in low-income countries.

Human rotavirus (HRV) is a leading cause of morbidity and mortality in children, especially in developing countries. Malnutrition is prevalent in these countries, which may contribute to the decreased oral vaccine efficacy, posing a concern for global health. Neonatal gnotobiotic (Gn) pigs closely resemble human infants in their anatomy, physiology, and outbred status and are a unique model to investigate malnutrition, oral live attenuated HRV (AttHRV) vaccination, and subsequent virulent HRV (VirHRV) challenge. We evaluated the impact of malnutrition on AttHRV vaccine efficacy and B cell immune responses in neonatal germfree (GF) or Gn pigs transplanted with human infant fecal microbiota (HIFM). Pigs were fed either deficient or sufficient bovine milk diets. Malnutrition did not significantly affect the serum and intestinal contents total or HRV-specific IgG and IgA antibody titers pre VirHRV challenge. However, HRV-specific IgG and IgA antibody secreting cells (ASCs) were reduced in blood or intestinal tissues following AttHRV vaccination and pre VirHRV challenge in deficient HIFM transplanted pigs. Furthermore, post-VirHRV challenge, deficient HIFM pigs had decreased total Ig and HRV-specific IgG and IgA antibody titers in serum or intestinal contents, in addition to decreased HRV-specific IgG and IgA ASCs in blood and ileum, compared with sufficient HIFM pigs. Our results indicate that deficient diet impairs B cell mucosal, and systemic immune responses following HRV vaccination, and challenge. The impaired immune responses contributed to the decreased protective efficacy of the AttHRV vaccine, suggesting that malnutrition may significantly reduce the effectiveness of oral HRV vaccines in children in developing countries.

Human rotavirus (HRV) infection is a major cause of viral gastroenteritis in young children worldwide. Current oral vaccines perform poorly in developing countries where efficacious vaccines are needed the most. Therefore, an alternative affordable strategy to enhance efficacy of the current RV vaccines is necessary. This study evaluated the effects of colonization of neonatal gnotobiotic (Gn) pigs with Escherichia coli Nissle (EcN) 1917 and Lacticaseibacillus rhamnosus GG (LGG) probiotics on immunogenicity and protective efficacy of oral attenuated (Att) HRV vaccine. EcN-colonized pigs had reduced virulent HRV (VirHRV) shedding and decreased diarrhea severity compared with the LGG-colonized group. They also had enhanced HRV-specific IgA antibody titers in serum and antibody secreting cell numbers in tissues pre/post VirHRV challenge, HRV-specific IgA antibody titers in intestinal contents, and B-cell subpopulations in tissues post VirHRV challenge. EcN colonization also enhanced T-cell immune response, promoted dendritic cells and NK cell function, reduced production of proinflammatory cytokines/Toll like receptor (TLR), and increased production of immunoregulatory cytokines/TLR expression in various tissues pre/post VirHRV challenge. Thus, EcN probiotic adjuvant with AttHRV vaccine enhances the immunogenicity and protective efficacy of AttHRV to a greater extent than LGG and it can be used as a safe and economical oral vaccine adjuvant.

Abstract The control of Salmonella from farm to fork is challenging due to the emergence of antimicrobial-resistant isolates and the limited effects of current control methods. Advanced chemical technologies have made accessible a wide range of uncharacterized small molecules (SMs) with encouraging chemical properties for antimicrobial treatment. Of the 4,182 SMs screened in vitro, four cidal SMs were effective at 10 µM and higher against several serotypes, antibiotic-resistant, and biofilm embedded Salmonella enterica subsp. enterica serotype Typhimurium by altering cell membrane integrity. The four SMs displayed synergistic effects with ciprofloxacin, meropenem and cefeprime against Salmonella. Further, the SMs were not pernicious to most eukaryotic cells at 200 μM and cleared internalized Salmonella in infected Caco-2, HD11, and THP-1 cells at 6.25 µM and higher. The SMs also increased the longevity of Salmonella-infected Galleria mellonella larvae and reduced the population of internalized S almonella Typhimurium. Two of the SMs (SM4 and SM5) also reduced S. Typhimurium load in infected chicken ceca as well as its systemic translocation into other tissues, with minimal impact on the cecal microbiota. This study demonstrated that SMs are a viable source of potential antimicrobials applicable in food animal production against Salmonella.

Abstract Background Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants; particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microbiota leading to sub-optimal functioning of the immune system and further predisposing infants to enteric infections. Therefore, we hypothesized that malnutrition exacerbates rotavirus disease severity in infants. Methods In the present study, we used a neonatal germ free (GF) piglets transplanted with a two-month-old human infant’s fecal microbiota (HIFM) on protein deficient and sufficient diets. We report the effects of malnourishment on the HRV infection and the HIFM pig microbiota in feces, intestinal and systemic tissues, using MiSeq 16S gene sequencing (V4-V5 region). Results Microbiota analysis indicated that the HIFM transplantation resulted in a microbial composition in pigs similar to that of the original infant feces. This model was then used to understand the interconnections between microbiota diversity, diet, and HRV infection. Post HRV infection, HIFM pigs on the deficient diet had lower body weights, developed more severe diarrhea and increased virus shedding compared to HIFM pigs on sufficient diet. However, HRV induced diarrhea and shedding was more pronounced in non-colonized GF pigs compared to HIFM pigs on either sufficient or deficient diet, suggesting that the microbiota alone moderated HRV infection. HRV infected pigs on sufficient diet showed increased microbiota diversity in intestinal tissues; whereas, greater diversity was observed in systemic tissues of HRV infected pigs fed with deficient diet. Conclusions These results suggest that proper nourishment improves the microbiota quality in the intestines, alleviates HRV disease and lower probability of systemic translocation of potential opportunistic pathogens/pathobionts. In conclusion, our findings further support the role for microbiota and proper nutrition in limiting enteric diseases.

Foodborne pathogens significantly impact public health globally. Excessive antimicrobial use plays a significant role in the development of the public health crisis of antibiotic resistance. Here, we determined the prevalence and antimicrobial resistance profiles of E. coli O157, Salmonella, L. monocytogenes, and Campylobacter isolated between 2016 and 2020 from small scale agricultural settings that were amended with dairy cattle or poultry manure in Northeastern Ohio. The total prevalence of the foodborne pathogens was 19.3%: Campylobacter 8%, Listeria monocytogenes 7.9%, Escherichia coli O157 1.8%, and Salmonella 1.5%. The prevalence was significantly higher in dairy cattle (87.7%) compared to poultry (12.2%) manure amended farms. Furthermore, the prevalence was higher in manure samples (84%) compared to soil samples (15.9%; p < 0.05). Multiple drug resistance was observed in 73%, 77%, 100%, and 57.3% of E. coli O157, Salmonella, L. monocytogenes, and Campylobacter isolates recovered, respectively. The most frequently observed resistance genes were mphA, aadA, and aphA1 in E. coli O157; blaTEM, tet(B), and strA in Salmonella; penA, ampC, lde, ermB, tet(O), and aadB in L. monocytogenes and blaOXA-61, tet(O), and aadE in Campylobacter. Our results highlight the critical need to address the dissemination of foodborne pathogens and antibiotic resistance in agricultural settings.

The development of informatic tools to improve the identification of novel antimicrobials would significantly reduce the cost and time of drug discovery. We previously screened several plant (Xanthomonas sp., Clavibacter sp., Acidovorax sp., and Erwinia sp.), animal (Avian pathogenic Escherichia coli and Mycoplasma sp.), and human (Salmonella sp. and Campylobacter sp.) pathogens against a pre-selected small molecule library (n = 4182 SM) to identify novel SM (hits) that completely inhibited the bacterial growth or attenuated at least 75% of the virulence (quorum sensing or biofilm). Our meta-analysis of the primary screens (n = 11) using the pre-selected library (approx. 10.2 ± 9.3% hit rate per screen) demonstrated that the antimicrobial activity and spectrum of activity, and type of inhibition (growth versus virulence inhibitors) correlated with several physico-chemical properties (PCP; e.g., molecular weight, molar refraction, Zagreb group indexes, Kiers shape, lipophilicity, and hydrogen bond donors and acceptors). Based on these correlations, we build an in silico model that accurately classified 80.8% of the hits (n = 1676/2073). Therefore, the pre-selected SM library of 4182 SM was narrowed down to 1676 active SM with predictable PCP. Further, 926 hits affected only one species and 1254 hits were active against specific type of pathogens; however, no correlation was detected between PCP and the type of pathogen (29%, 34%, and 46% were specific for animal, human foodborne and plant pathogens, respectively). In conclusion, our in silico model allowed rational identification of SM with potential antimicrobial activity against bacterial pathogens. Therefore, the model developed in this study may facilitate future drug discovery efforts by accelerating the identification of uncharacterized antimicrobial molecules and predict their spectrum of activity.

Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducer-like proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The Δtlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (Ptlp2) was induced by Pi and iron, both in the ferrous (Fe2+) and ferric form (Fe3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The Δtlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avian species, and recent reports have suggested APEC as a potential foodborne zoonotic pathogen. Herein, we discuss the virulence and pathogenesis factors of APEC, review the zoonotic potential, provide the current status of antibiotic resistance and progress in vaccine development, and summarize the alternative control measures being investigated. In addition to the known virulence factors, several other factors including quorum sensing system, secretion systems, two-component systems, transcriptional regulators, and genes associated with metabolism also contribute to APEC pathogenesis. The clear understanding of these factors will help in developing new effective treatments. The APEC isolates (particularly belonging to ST95 and ST131 or O1, O2, and O18) have genetic similarities and commonalities in virulence genes with human uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) and abilities to cause urinary tract infections and meningitis in humans. Therefore, the zoonotic potential of APEC cannot be undervalued. APEC resistance to almost all classes of antibiotics, including carbapenems, has been already reported. There is a need for an effective APEC vaccine that can provide protection against diverse APEC serotypes. Alternative therapies, especially the virulence inhibitors, can provide a novel solution with less likelihood of developing resistance.

Salmonella is one of the leading causes of human foodborne gastroenteritis in the United States. In addition, Salmonella contributes to morbidity and mortality in livestock. The control of Salmonella is an increasing problematic issue in livestock production due to lack of effective control methods and the constant adaptation of Salmonella to new management practices, which is often related to horizontal acquisition of virulence or antibiotic resistance genes. Salmonella enterica serotype Heidelberg is one of the most commonly isolated serotypes in all poultry production systems in North America. Emergence and persistence of multi-drug resistant Salmonella Heidelberg isolates further impact the poultry production and public health. We hypothesized that distinct poultry production environments affect Salmonella genomic content, and by consequence its survival and virulence abilities. This study compared the genomic composition of S. Heidelberg isolated from environmental samples (19 chicken and 12 turkey isolates) of different breeder farms (16 chicken and 8 turkey farms) in the Midwest, United States. Whole genome comparison of 31 genomes using RAST and SEED identified differences in specific sub-systems in isolates between the chicken- and turkey-associated farm environmental samples. Genes associated with the type IV secretion system (n = 12) and conjugative transfer (n = 3) were absent in turkey farm isolates compared to the chicken ones (p-value < 0.01); Further, turkey farm isolates were enriched in prophage proteins (n = 53; p-value < 0.01). Complementary studies using PHASTER showed that prophages were all Caudovirales phages and were more represented in turkey environmental isolates than the chicken isolates. This study corroborates that isolates from distinct farm environment show differences in S. Heidelberg genome content related to horizontal transfer between bacteria or through viral infections. Complementary microbiome studies of these samples would provide critical insights on sources of these variations. Overall, our findings enhance the understanding of Salmonella genome plasticity and may aid in the development of future effective management practices to control Salmonella.

Campylobacter jejuni is a leading cause of bacterial food poisoning in humans. Due to the rise in antibiotic-resistant Campylobacter, there exists a need to develop antibiotic-independent interventions to control infections in humans. Here, we evaluated the impact of Escherichia coli Nissle 1917 (EcN), a probiotic strain, on C. jejuni’s invasion and intracellular survival in polarized human colonic cells (HT-29). To further understand how EcN mediates its impact, the expression of 84 genes associated with tight junctions and cell adhesion was profiled in HT-29 cells after treatment with EcN and challenge with C. jejuni. The pre-treatment of polarized HT-29 cells with EcN for 4 h showed a significant effect on C. jejuni’s invasion (∼2 log reduction) of the colonic cells. Furthermore, no intracellular C. jejuni were recovered from EcN pre-treated HT-29 cells at 24 h post-infection. Other probiotic strains tested had no significant impact on C. jejuni invasion and intracellular survival. C. jejuni decreased the expression of genes associated with epithelial cells permeability and barrier function in untreated HT-29 cells. However, EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity. The results suggest that EcN impedes C. jejuni invasion and subsequent intracellular survival by affecting HT-29 cells barrier function and tight junction integrity. We conclude that EcN might be a viable alternative for controlling C. jejuni infections.

ABSTRACT Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103+ and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing the rates of death from infectious diseases. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. We have established the first human infant microbiota-transplanted neonatal pig model of childhood malnutrition that reproduced the impaired immune, intestinal, and other physiological functions seen in malnourished children. This model can be used to evaluate relevant dietary and other health-promoting interventions. Our findings provide an explanation of why adequate nutrition alone may lack efficacy in malnourished children.

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P

Campylobacter jejuni is a common gastrointestinal pathogen that colonizes food animals; it is transmitted via fecal contamination of food, and infections in immune-compromised people are more likely to result in serious long-term illness. Environmental phosphate is likely an important sensor of environmental fitness and the ability to obtain extracellular phosphate is central to the bacteria's core metabolic responses. PhoX is the sole alkaline phosphatase in C. jejuni, a substrate of the TAT transport system. Alkaline phosphatases mediate the hydrolytic removal of inorganic phosphate (Pi) from phospho-organic compounds and thereby contribute significantly to the polyphosphate kinase 1 (ppk1) mediated formation of poly P, a molecule that regulates bacterial response to stresses and virulence. Similarly, deletion of the tatC gene, a key component of the TAT system, results in diverse phenotypes in C. jejuni including reduced stress tolerance and in vivo colonization. Therefore, here we investigated the contribution of phoX in poly P synthesis and in TAT-system mediated responses. The phoX deletion mutant showed significant decrease (P

BACKGROUND: Inorganic polyphosphate (poly P) plays an important role in stress tolerance and virulence in many bacteria. PPK1 is the principal enzyme involved in poly P synthesis, while PPK2 uses poly P to generate GTP, a signaling molecule that serves as an alternative energy source and a precursor for various physiological processes. Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans, possesses homologs of both ppk1 and ppk2. ppk1 has been previously shown to impact the pathobiology of C. jejuni. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The Deltappk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Deltappk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Deltappk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization. CONCLUSIONS/SIGNIFICANCE: Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. PPK2 is absent in humans and animals; therefore, can serve as a novel target for therapeutic intervention of C. jejuni infections.