Risk-based critical concentrations of enteric pathogens for recreational water criteria and recommended minimum sample volumes for routine water monitoring.

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni., Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection.

Application of a microbial and pathogen source tracking toolbox to identify infrastructure problems in stormwater drainage networks: a case study.

Water scarcity and increasing urbanization are forcing municipalities to consider alternative water sources, such as stormwater, to fill in water supply gaps or address hydromodification of receiving urban streams. Mounting evidence suggests that stormwater is often contaminated with human feces, even in stormwater drainage systems separate from sanitary sewers. Pinpointing sources of human contamination in drainage networks is challenging given the diverse sources of fecal pollution that can impact these systems and the non-specificity of traditional fecal indicator bacteria (FIB) for identifying these host sources. As such, we used a toolbox approach that encompassed microbial source tracking (MST), FIB monitoring, and bacterial pathogen monitoring to investigate microbial contamination of stormwater in an urban municipality. We demonstrate that human sewage frequently contaminated stormwater (in >50% of routine samples), based on the presence of the human fecal marker HF183, and often exceeded microbial water quality criteria. Arcobacter butzleri, a pathogen of emerging concern, was also detected in >50% of routine samples, with 75% of these pathogen-positive samples also being positive for the human fecal marker HF183, suggesting human municipal sewage as the likely source for this pathogen. MST and FIB were used to track human fecal pollution in the drainage network to the most likely point source of contamination, for which a sewage cross-connection was identified and confirmed using tracer dyes. These results point to the ubiquitous presence of human sewage in stormwater and also provide municipalities with the tools to identify sources of anthropogenic contamination in storm drainage networks.IMPORTANCEWater scarcity, increased urbanization, and population growth are driving municipalities worldwide to consider stormwater as an alternative water source in urban environments. However, many studies suggest that stormwater is relatively poor in terms of microbial water quality, is frequently contaminated with human sewage, and therefore could represent a potential health risk depending on the type of exposure (e.g., irrigation of community gardens). Traditional monitoring of water quality based on fecal bacteria does not provide any information about the sources of fecal pollution contaminating stormwater (i.e., animals/human feces). Herein, we present a case study that uses fecal bacterial monitoring, microbial source tracking, and bacterial pathogen analysis to identify a cross-connection that contributed to human fecal intrusion into an urban stormwater network. This microbial toolbox approach can be useful for municipalities in identifying infrastructure problems in stormwater drainage networks to reduce risks associated with water reuse.

Salmonella Typhimurium and Vibrio cholerae can be transferred from plastic mulch to basil and spinach salad leaves.

Plastic pollution is increasingly found in agricultural environments, where it contaminates soil and crops. Microbial biofilms rapidly colonise environmental plastics, such as the plastic mulches used in agricultural systems, which provide a unique environment for microbial plastisphere communities. Human pathogens can also persist in the plastisphere, and enter agricultural environments via flooding or irrigation with contaminated water. In this study we examined whether Salmonella Typhimurium and Vibrio cholerae can be transferred from the plastisphere on plastic mulch to the surface of ready-to-eat crop plants, and subsequently persist on the leaf surface. Both S. Typhimurium and V. cholerae were able to persist for 14 days on fragments of plastic mulch adhering to the surface of leaves of both basil and spinach. Importantly, within 24 h both pathogens were capable of dissociating from the surface of the plastic and were transferred onto the surface of both basil and spinach leaves. This poses a further risk to food safety and human health, as even removal of adhering plastics and washing of these ready-to-eat crops would not completely remove these pathogens. As the need for more intensive food production increases, so too does the use of plastic mulches in agronomic systems. Therefore, there is now an urgent need to understand the unquantified co-pollutant pathogen risk of contaminating agricultural and food production systems with plastic pollution.

The use of oral fluids and sock samples for monitoring key pathogens in pig populations for surveillance purposes.

Despite the prevalence of co-infections and the association of over 50 viral and 46 bacterial pathogens with pig diseases, little is known about their simultaneous occurrence, particularly in commercial pig farming environments where health programs are in place. To address this knowledge gap, this study aimed to evaluate the pathogen threshold of respiratory and enteric pathogens in pig herds using the Pork MultiPath™ (PMP1 and PMP2, respiratory and enteric respectively) technology, which detects multiple pathogens simultaneously in a single reaction with high sensitivity and specificity. In this study the most prevalent respiratory pathogens, Mycoplasma hyrohinis, Pasteurella multocida, and Haemophilus parasuis detected by PMP1 were effectively controlled during the nursery stage through strategic treatment with tiamulin. Even though the major respiratory incidences were reduced, the recorded coughing and sneezing rates were associated with the levels of H. parasuis and M. hyrohinis, which were set at 1356 and 1275 copies/reaction, respectively. In addition, one of the identified co-infection patterns indicated a strong relationship between the occurrence of H. parasuis and M. hyorhinis at the sample and pen levels, highlighting the high likelihood of detecting these two pathogens together. Testing with enteric panel PMP2 revealed that the most frequently detected virulence factors during the early nursery stage were Escherichia coli genes for toxins - ST1, ST2, and fimbriae - F4 and F18. Moreover, a co-infection with Rotavirus B and C was often observed during the nursery stage, and a strong positive correlation between these two markers has been identified. Additionally, the levels of several markers, namely E. coli F4, F5, F18, LT, ST1, and ST2, have been associated with a higher likelihood of sickness in pig populations. In addition, the onset of Brachyspira pilosicoli during the nursery and grower stages was found to be associated with an increased risk of diarrhoea, with a set threshold at around 500 copies/reaction. Although simultaneous detection of multiple pathogens is not yet widely used in the pig industry, it offers a significant advantage in capturing the diversity and interactions of co-infections. Testing pooled samples with Pork MultiPath™ is cost-effective and practical to regularly monitor the health status of pig populations.

Beneficial effect of probiotics supplementation on quality of edible fresh table eggs during storage.

Background: This study discussed the effect of probiotic supplementation on laying hens' diets and the enhancement of egg quality during the cold storage period. Aim: To study the efficacy of the addition of probiotics to hen diets in terms of improving the egg's quality during the cold storage period and protection against enteric pathogens. Methods: 100 table eggs were collected from farms of laying hens on a battery system, 46 weeks old HylineW36 white in Sharkia Government. The collected eggs were separated into 2 groups (50 each); the control group from hens fed on diets without probiotics, and the probiotic group from hens fed on diets with (100 g/ton) of supplemented probiotics preparation. All groups were separated into 5 sub-groups for the examinations; on the fresh day, 7th, 14th, 21st, and 28th days on cold storage at 4°C. Chemical, physical, and microbiological examinations were done for internal egg contents and eggshells. Results: Our results showed that probiotics supplements have advantageous effects on the quality of eggs during cold storage periods. Also, microbiological examination proved that eggshells from hens fed on diets with probiotics supplemented (100 g/ton) have decreased the level of bacterial contamination with Salmonella and Escherichia coli than hens fed on a regular diet. Conclusion: It could be shown that the probiotics supplementation may decrease and reduce the effect of the storage period on the quality of shell, albumen, and yolk.

Rain splash-mediated dispersal of Escherichia coli from fecal deposits to field-grown lettuce in the mid- and south Atlantic U.S. regions is affected by mulch type.

Introduction: Wildlife feces can contaminate vegetables when enteric bacteria are released by rain and splashed onto crops. Regulations require growers to identify and not harvest produce that is likely contaminated, but U.S. federal standards do not define dimensions for no-harvest zones. Moreover, mulching, used to retain soil moisture and maximize crop yield may impact rain-mediated bacterial dispersal from feces. Methods: To assess Escherichia coli dissemination from a fecal point source to lettuce grown on various mulches, lettuce cv. 'Magenta' was transplanted into raised beds with plastic, biodegradable plastic, straw, or left uncovered at field sites in Maryland and Georgia. Eleven days post-transplant, 10 g of rabbit manure spiked with ~8 log CFU g-1 E. coli were deposited in each bed. One day following natural or simulated rain events, lettuce was sampled along 1.5 m transects on either side of fecal deposits. Lettuce-associated E. coli was semi-quantified with an MPN assay and dependence on fecal age (stale or fresh), lettuce age (baby leaf or mature head), distance from point source, mulch and post-rain days were statistically evaluated. Results: Distance (p<0.001), fecal age (p<0.001) and mulch (p<0.01) were factors for E. coli transfer from point source to lettuce. The highest and lowest E. coli estimates were measured from lettuce grown on biodegradable plastic and straw, respectively, with a 2-log MPN difference (p<0.001). Mulch and distance were also significant factors in E. coli recovery 3 days post-rain (both p<0.001), where plastic mulches differed from bare ground and straw (p<0.01). For all treatments, fewer E. coli were retrieved from lettuce at 0.3 m, 3 days post-rain compared to 1 day (p<0.001). Fitting the data to a Weibull Model predicated that a 7-log reduction in E. coli from fecal levels would be achieved at 1.2-1.4 m from the point source on plastic mulches, 0.75 m on bare soil (p<0.05) and 0.43 m on straw (p<0.01). Discussion: Straw and bare ground limited rain-mediated E. coli dispersal from feces to lettuce compared to plastic mulches. Fecal age was negatively associated with E. coli dispersal. These findings can inform harvesting recommendations for measures related to animal intrusion in vegetable production areas.

The Effect of Colostrum Supplementation during the First 5 Days of Life on Calf Health, Enteric Pathogen Shedding, and Immunological Response.

The objective of this dairy farm study was to investigate the preweaning health, performance, immunity, and enteric pathogen shedding in calves supplemented with colostrum during five days after birth compared to calves not supplemented with colostrum. The colostrum supplementation was the previously frozen colostrum added to the milk replacer from day 2-5 given to 39 calves, and 36 Control calves received a milk-derived supplement. There was no significant difference in preweaning weight gain between the treatment groups. Serum samples collected on days 2, 7, 14, and 21 indicated that total and antigen-specific IgG levels against rotavirus, coronavirus, and E. coli F5 were not significantly different between the treatment groups. Fecal samples taken on days 7, 14, and 21 were all negative for coronavirus and E. coli F5, whereas there were low levels of Cryptosporidia and a trend for low levels of rotavirus on day 14 in colostrum-supplemented compared to Control calves. Respiratory clinical signs, depressed attitude and body temperature tended to be reduced in colostrum-supplemented compared to Control calves. This study shows that, even in calves with good colostrum status and high plane of nutrition, there can be benefits of post-closure colostrum supplementation including reduced Cryptosporidia and rotavirus shedding and reduced respiratory disease.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

Risk factors and provider awareness of sexually transmitted enteric pathogens among men who have sex with men.

Sexual transmission of enteric pathogens among men who have sex with men (MSM) is well documented, although whether providers are cognizant of this risk when MSM patients present with gastrointestinal symptoms has not been studied. Over 34 months at a major tertiary metropolitan medical system, this study retrospectively analyzed 436 BioFire FilmArray Gastrointestinal results from 361 patients documented as MSM. An extensive chart review was performed, including specific sexual behaviors, socioeconomic risk factors, and whether providers charted a sexual history when a patient presented for care. Overall BioFire positivity rate was 62% with no significant difference in positivity between persons living with HIV and those without. Patients charted as sexually active had a significantly increased odds ratio (OR) of a positive result compared to those who were not. Anilingus had the highest OR. Providers charted any type of sexual history in 40.6% of cases, and HIV/infectious disease providers were significantly more likely to do this compared to other subspecialties. Sexual transmission of enteric pathogens within MSM is ongoing, and patients are at risk regardless of living with HIV. Not all sexual behaviors have the same associated risk, highlighting opportunities to decrease transmission. Increased provider vigilance and better patient education on sexual transmission of enteric pathogens are needed to reduce the disease burden. IMPORTANCE: Our work adds several key findings to the growing body of literature describing the epidemiology of enteric pathogens as sexually transmitted infections among men who have sex with men (MSM). We analyzed clinical test results, housing status, provider awareness, sexual behaviors, and symptoms for 361 patients. We found that any sexual activity was associated with an increased risk of diarrheal pathogen detection, whereas being unhoused was not a risk factor. These findings suggest separate transmission networks between unhoused persons, who are also at risk of infectious diarrhea, and MSM. Moreover, our study suggested low awareness among patient-facing clinicians that diarrheal pathogens can be sexually transmitted. Together, our findings indicate an important opportunity to disrupt transmission cycles by educating clinicians on how to assess and counsel MSM patients.

Enhancing enteric pathogen detection: implementation and impact of multiplex PCR for improved diagnosis and surveillance.

BACKGROUND: Syndromic surveillance of acute gastroenteritis plays a significant role in the diagnosis and management of gastrointestinal infections that are responsible for a substantial number of deaths globally, especially in developing countries. In Lebanon, there is a lack of national surveillance for acute gastroenteritis, and limited data exists regarding the prevalence of pathogens causing diarrhea. The one-year study aims to investigate the epidemiology of common gastrointestinal pathogens and compare our findings with causative agents of diarrhea reported by our study collaborative centers. METHODS: A multicenter, cross-sectional study was conducted over a one-year period. A total of 271 samples were obtained from outpatients and inpatients presenting with symptoms of acute gastroenteritis at various healthcare facilities. The samples were then analyzed using Allplex gastrointestinal assay that identifies a panel of enteric pathogens. RESULTS: Overall, enteropathogens were detected in 71% of the enrolled cases, 46% of those were identified in patients as single and 54% as mixed infections. Bacteria were observed in 48%, parasites in 12% and viruses in 11%. Bacterial infections were the most prevalent in all age groups. Enteroaggregative E. coli (26.5%), Enterotoxigenic E. coli (23.2%) and Enteropathogenic E. coli (20.3%) were the most frequently identified followed by Blastocystis hominis (15.5%) and Rotavirus (7.7%). Highest hospitalization rate occurred with rotavirus (63%), Enterotoxigenic E. coli (50%), Blastocystis hominis (45%) and Enteropathogenic E. coli (43%). Enteric pathogens were prevalent during summer, fall and winter seasons. CONCLUSIONS: The adoption of multiplex real-time PCR assays in the diagnosis of gastrointestinal infections has identified gaps and improved the rates of detection for multiple pathogens. Our findings highlight the importance of conducting comprehensive surveillance to monitor enteric infections. The implementation of a syndromic testing panel can therefore provide healthcare professionals with timely and accurate information for more effective treatment and public health interventions.

Assessment of wastewater derived pollution using viral monitoring in two estuaries.

Human wastewater-derived pollution of the environment is an emerging health risk that increases the number of waterborne and foodborne illnesses globally. To better understand and mitigate such health risks, we investigated the prevalence of faecal indicator bacteria, Escherichia coli, and indicator virus (crAssphage) along with human and animal enteric viruses (adenoviruses, noroviruses, sapoviruses, hepatitis E virus) in shellfish and water samples collected from two shellfish harvesting areas in the UK. Human noroviruses were detected at higher detection rates in oyster and water samples compared to mussels with peaks during the autumn-winter seasons. Human enteric viruses were sporadically detected during the warmer months, suggesting potential introduction by tourists following the relaxation of COVID-19 lockdown measures. Our results suggest that viral indicators are more suitable for risk assessment and source tracking than E. coli. The detection of emerging hepatitis and sapoviruses, support the need for comprehensive viral monitoring in shellfish harvesting areas.

Combatting infectious diarrhea: innovations in treatment and vaccination strategies.

INTRODUCTION: The escalating prevalence of infectious diseases is an important cause of concern in society. Particularly in several developing countries, infectious diarrhea poses a major problem, with a high fatality rate, especially among young children. The condition is divided into four classes, namely, acute diarrhea, invasive diarrhea, acute bloody diarrhea, and chronic diarrhea. Various pathogenic agents, such as bacteria, viruses, protozoans, and helminths, contribute to the onset of this condition. AREAS COVERED: The review discusses the scenario of infectious diarrhea, the prevalent types, as well as approaches to management including preventive, therapeutic, and vaccination strategies. The vaccination techniques are extensively discussed including the available vaccines, their advantages as well as limitations. EXPERT OPINION: There are several approaches available to develop new-improved vaccines. In addition, route of immunization is important and aerosols/nasal sprays, oral route, skin patches, powders, and liquid jets to minimize needles can be used. Plant-based vaccines, such as rice, might save packing and refrigeration costs by being long-lasting, non-refrigerable, and immunogenic. Future research should utilize predetermined PCR testing intervals and symptom monitoring to identify persistent pathogens after therapy and symptom remission.

Antimicrobial properties of tomato juice and peptides against typhoidal Salmonella.

Tomatoes are readily available and affordable vegetables that offer a range of health benefits due to their bioactive molecules, such as antioxidants and antimicrobials. In contrast to the widely recognized antioxidant properties of tomatoes, their antimicrobial properties remain largely unexplored. Here, we present our findings on the antimicrobial properties of tomato juice and peptides, namely, tomato-derived antimicrobial peptides (tdAMPs), in relation to their effectiveness against typhoidal Salmonella. Our research has revealed that tomato juice demonstrates significant antimicrobial properties against Salmonella Typhi, a pathogen that specifically affects humans and is responsible for causing typhoid fever. By employing computational analysis of the tomato genome sequence, conducting molecular dynamics simulation, and performing functional analyses, we have successfully identified two tdAMPs, namely, tdAMP-1 and tdAMP-2. These tdAMPs have demonstrated potent antimicrobial properties by effectively disrupting bacterial membranes. The efficacy of tdAMP-2 is shown to be more effective than tdAMP-1. The efficacy of tdAMP-1 and tdAMP-2 has been demonstrated against drug-resistant S. Typhi, as well as hyper-capsular S. Typhi variants that possess hypervirulent characteristics, which are presently circulating in countries with endemicity. Tomato juice, along with the two tdAMPs, has demonstrated effectiveness against uropathogenic Escherichia coli as well. This underscores their potential as viable agents in combating certain Gram-negative pathogens. This study provides valuable insights into the development of effective and sustainable public health strategies that utilize tomato and its derivatives as lifestyle interventions.IMPORTANCEIn this study, we investigate the antimicrobial properties of tomato juice, the most widely consumed affordable vegetables, as well as tomato-derived antimicrobial peptides, in relation to their effectiveness against foodborne pathogens with an emphasis on Salmonella Typhi, a deadly human-specific pathogen.

Exogenous butyrate inhibits butyrogenic metabolism and alters virulence phenotypes in Clostridioides difficile.

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile-inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile, which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

Ligilactobacillus animalis 506 Protects the Intestinal Barrier from the Damaging Effects of Enteric Pathogens and Deoxynivalenol.

This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.

Systemic inflammation, enteropathogenic E. Coli, and micronutrient insufficiencies in the first trimester as possible predictors of preterm birth in rural Bangladesh: a prospective study.

BACKGROUND: An incomplete understanding of preterm birth is especially concerning for low-middle income countries, where preterm birth has poorer prognoses. While systemic proinflammatory processes are a reportedly normal component of gestation, excessive inflammation has been demonstrated as a risk factor for preterm birth. There is minimal research on the impact of excessive maternal inflammation in the first trimester on the risk of preterm birth in low-middle income countries specifically. METHODS: Pregnant women were enrolled at the rural Bangladesh site of the National Institute of Child Health Global Network Maternal Newborn Health Registry. Serum samples were collected to measure concentrations of the inflammatory markers C-reactive protein (CRP) and Alpha-1-acid glycoprotein (AGP), and stool samples were collected and analyzed for enteropathogens. We examined associations of maternal markers in the first-trimester with preterm birth using logistic regression models. CRP and AGP were primarily modeled with a composite inflammation predictor. RESULTS: Out of 376 singleton births analyzed, 12.5% were preterm. First trimester inflammation was observed in 58.8% of all births, and was significantly associated with increased odds of preterm birth (adjusted odds ratio [aOR] = 2.23; 95% confidence interval [CI]: 1.03, 5.16), independent of anemia. Maternal vitamin B12 insufficiency (aOR = 3.33; 95% CI: 1.29, 8.21) and maternal anemia (aOR = 2.56; 95% CI: 1.26, 5.17) were also associated with higher odds of preterm birth. Atypical enteropathogenic E. coli detection showed a significant association with elevated AGP levels and was significantly associated with preterm birth (odds ratio [OR] = 2.36; 95% CI: 1.21, 4.57), but not associated with CRP. CONCLUSIONS: Inflammation, anemia, and vitamin B12 insufficiency in the first trimester were significantly associated with preterm birth in our cohort from rural Bangladesh. Inflammation and anemia were independent predictors of premature birth in this low-middle income setting where inflammation during gestation was widespread. Further research is needed to identify if infections such as enteropathogenic E. coli are a cause of inflammation in the first trimester, and if intervention for infection would decrease preterm birth.

Effectiveness of Ultra-High Irradiance Blue-Light-Emitting Diodes to Control Salmonella Contamination Adhered to Dry Stainless Steel Surfaces.

Controlling Salmonella contamination in dry food processing environments represents a significant challenge due to their tolerance to desiccation stress and enhanced thermal resistance. Blue light is emerging as a safer alternative to UV irradiation for surface decontamination. In the present study, the antimicrobial efficacy of ultra-high irradiance (UHI) blue light, generated by light-emitting diodes (LEDs) at wavelengths of 405 nm (841.6 mW/cm2) and 460 nm (614.9 mW/cm2), was evaluated against a five-serovar cocktail of Salmonella enterica dry cells on clean and soiled stainless steel (SS) surfaces. Inoculated coupons were subjected to blue light irradiation treatments at equivalent energy doses ranging from 221 to 1106 J/cm2. Wheat flour was used as a model food soil system. To determine the bactericidal mechanisms of blue light, the intracellular concentration of reactive oxygen species (ROS) in Salmonella cells and the temperature changes on SS surfaces were also measured. The treatment energy dose had a significant effect on Salmonella inactivation levels. On clean SS surfaces, the reduction in Salmonella counts ranged from 0.8 to 7.4 log CFU/cm2, while, on soiled coupons, the inactivation levels varied from 1.2 to 4.2 log CFU/cm2. Blue LED treatments triggered a significant generation of ROS within Salmonella cells, as well as a substantial temperature increase in SS surfaces. However, in the presence of organic matter, the oxidative stress in Salmonella cells declined significantly, and treatments with higher energy doses (>700 J/cm2) were required to uphold the antimicrobial effectiveness observed on clean SS. The mechanism of the bactericidal effect of UHI blue LED treatments is likely to be a combination of photothermal and photochemical effects. These results indicate that LEDs emitting UHI blue light could represent a novel cost- and time-effective alternative for controlling microbial contamination in dry food processing environments.

Sugar-binding and split domain combinations in repeats-in-toxin adhesins from Vibrio cholerae and Aeromonas veronii mediate cell-surface recognition and hemolytic activities.

Many pathogenic Gram-negative bacteria use repeats-in-toxin adhesins for colonization and biofilm formation. In the cholera agent Vibrio cholerae, flagellar-regulated hemagglutinin A (FrhA) enables these functions. Using bioinformatic analysis, a sugar-binding domain was identified in FrhA adjacent to a domain of unknown function. AlphaFold2 indicated the boundaries of both domains to be slightly shorter than previously predicted and assisted in the recognition of the unknown domain as a split immunoglobulin-like fold that can assist in projecting the sugar-binding domain toward its target. The AlphaFold2-predicted structure is in excellent agreement with the molecular envelope obtained from small-angle X-ray scattering analysis of a recombinant construct spanning the sugar-binding and unknown domains. This two-domain construct was probed by glycan micro-array screening and showed binding to mammalian fucosylated glycans, some of which are characteristic erythrocyte markers and intestinal cell epitopes. Isothermal titration calorimetry further showed the construct-bound l-fucose with a Kd of 21 µM. Strikingly, this recombinant protein construct bound and lysed erythrocytes in a concentration-dependent manner, and its hemolytic activity was blocked by the addition of l-fucose. A protein ortholog construct from Aeromonas veronii was also produced and showed a similar glycan-binding pattern, binding affinity, erythrocyte-binding, and hemolytic activities. As demonstrated here with Hep-2 cells, fucose-based inhibitors of this sugar-binding domain can potentially be developed to block colonization by V. cholerae and other pathogenic bacteria that share this adhesin domain.IMPORTANCEThe bacterium, Vibrio cholerae, which causes cholera, uses an adhesion protein to stick to human cells and begin the infection process. One part of this adhesin protein binds to a particular sugar, fucose, on the surface of the target cells. This binding can lead to colonization and killing of the cells by the bacteria. Adding l-fucose to the bacteria before they bind to the human cells can prevent attachment and has promise as a preventative drug to protect against cholera.

The Planetary Child Health & Enterics Observatory (Plan-EO): a protocol for an interdisciplinary research initiative and web-based dashboard for mapping enteric infectious diseases and their risk factors and interventions in LMICs.

Background: Diarrhea remains a leading cause of childhood illness throughout the world that is increasing due to climate change and is caused by various species of ecologically sensitive pathogens. The emerging Planetary Health movement emphasizes the interdependence of human health with natural systems, and much of its focus has been on infectious diseases and their interactions with environmental and human processes. Meanwhile, the era of big data has engendered a public appetite for interactive web-based dashboards for infectious diseases. However, enteric infectious diseases have been largely overlooked by these developments. Methods: The Planetary Child Health and Enterics Observatory (Plan-EO) is a new initiative that builds on existing partnerships between epidemiologists, climatologists, bioinformaticians, and hydrologists as well as investigators in numerous low- and middle-income countries. Its objective is to provide the research and stakeholder community with an evidence base for the geographical targeting of enteropathogen-specific child health interventions such as novel vaccines. The initiative will produce, curate, and disseminate spatial data products relating to the distribution of enteric pathogens and their environmental and sociodemographic determinants. Discussion: As climate change accelerates there is an urgent need for etiology-specific estimates of diarrheal disease burden at high spatiotemporal resolution. Plan-EO aims to address key challenges and knowledge gaps by making rigorously obtained, generalizable disease burden estimates freely available and accessible to the research and stakeholder communities. Pre-processed environmental and EO-derived spatial data products will be housed, continually updated, and made publicly available to the research and stakeholder communities both within the webpage itself and for download. These inputs can then be used to identify and target priority populations living in transmission hotspots and for decision-making, scenario-planning, and disease burden projection. Study registration: PROSPERO protocol #CRD42023384709.