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IntroductionEnteric pathogens are a leading causes of diarrheal deaths in low-and middle-income countries. The Exposure Assessment of Campylobacter Infections in Rural Ethiopia (EXCAM) project, aims to identify potential sources of bacteria in the genus Campylobacter and, more generally, fecal contamination of infants during the first 1.5 years of life using Escherichia coli as indicator.MethodsA total of 1,310 samples (i.e., hand rinses from the infant, sibling and mother, drinking and bathing water, food and fomite provided to or touched by the infants, areola swabs, breast milk and soil) were collected from 76 households between May 2021 and June 2022. Samples were assigned to two groups by infant age: TP1 (time point 1), infants between 4 and 8 months of age, and TP2, infants between 11 and 15 months of age. Fluorometric and semi-selective colorimetric approaches were used to quantify E. coli in the field samples.ResultsOverall, E. coli was ubiquitous within selected households (56.8% across the study). E. coli was more frequently detected than average (>53%) with high concentration (>2-log CFU) in soil (g) and per pair of hand, while the opposite trend (51%), but at low concentration ( 0.3).DiscussionUsing E. coli as surrogate, our study identified mother (hands and areola) as reservoirs likely to be involved in frequent transmission of fecal contaminants to infants within rural Ethiopian households.

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.

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.

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.

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.

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.

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.