Effect of a glyphosate-containing herbicide on Escherichia coli and Salmonella Ser. Typhimurium in an in vitro rumen simulation system

Glyphosate (N-(phosphonomethyl)glycine) is the most-used herbicide worldwide. Many studies in the past have shown that residues of the herbicide can be found in many cultivated plants, including those used as livestock feed. Sensitivity to glyphosate varies with bacteria, particularly those residing in the intestine, where microbiota is exposed to glyphosate residues. Therefore, less susceptible pathogenic isolates could have a distinct advantage compared to more sensitive commensal isolates, probably leading to dysbiosis. To determine whether the ruminal growth and survival of pathogenic Escherichia coli or Salmonella serovar Typhimurium are higher when glyphosate residues are present in the feed, an in vitro fermentation trial with a “Rumen Simulation System” (RUSITEC) and a glyphosate-containing commercial formulation was performed. Colony forming units of E. coli and Salmonella ser. Typhimurium decreased steadily in all fermenters, regardless of the herbicide application. Minimum inhibitory concentrations of the studied Salmonella and E. coli strains did not change, and antibiotic susceptibility varied only slightly but independent of the glyphosate application. Overall, application of the glyphosate-containing formulation in a worst-case concentration of 10 mg/L neither increased the abundance for the tested E. coli and Salmonella strain in the in vitro fermentation system, nor promoted resistance to glyphosate or antibiotics

Minimum Inhibitory Concentration of Glyphosate and a Glyphosate-Containing Herbicide in Salmonella enterica Isolates Originating from Different Time Periods, Hosts, and Serovars

Glyphosate, the active compound of Roundup, is one of the most used pesticides in the world. Its residues are often detected in animal feed, but the impact on the animal gut microbiota and on pathogens of the intestine has not intensively been investigated. In this study, we analyzed the minimum inhibitory concentration (MIC) of glyphosate isopropylamine salt and a common glyphosate-containing herbicide formulation in 225 Salmonella enterica isolates by broth microdilution. A bacteriostatic effect of glyphosate on Salmonella growth was detected at the concentration range of 10 to 80 mg/mL for both the active ingredient and the ready-to-use formulation. Time/year of isolation, host species, and serovars revealed a statistically significant influence on MIC values. Recently collected Salmonella isolates had significantly higher MIC values for glyphosate and the glyphosate-containing product compared with isolates collected between 1981 and 1990. Isolates from pigs showed significantly higher MIC values compared with isolates from poultry, and isolates of the Salmonella serovar Typhimurium had significantly higher MIC values than Salmonella Enteritidis and Infantis isolates

WHO's latest rabies recommendations and guidance save lives and reduce the cost of treatment

Rabies vaccination is a crucial part of rabies post-exposure prophylaxis (PEP), but it tends to consist of long and costly regimens of intramuscular (IM) injections. Most human rabies deaths are caused by delayed access, unaffordability or ineffective delivery of PEP. Reducing these barriers is crucial to ensure that this incurable yet preventable disease does not cost lives. In 2022, WHO published new guidance towards the introduction or expansion of rabies vaccination into national immunization programmes to systematically drive down human rabies deaths effectively and cost-efficiently. Such guidance grounds on the latest scientific recommendation provided by WHO’s Strategic Advisory Group of Experts in 2018. WHO recommends a shortened 1-week rabies vaccination schedule, with visits on days 0, 3 and 7. On each visit, a 2-site intradermal (ID) injection (using only 0.1 ml of vaccine in each site) is administered. ID administration allows for vials to be shared among several patients within a 6-8 hours timeline. Compared to IM administration, ID is cost- and dose-sparing, even in low-throughput clinics. Additionally, this regimen requires only 3 visits to the healthcare facility, improving patient compliance. However, the uptake of this shortened ID regimen remains limited. It should now be a matter of urgency for Health Ministries in rabies-endemic settings to adopt the WHO-recommended shortened ID vaccination schedule and ensure appropriate medical training to improve PEP delivery. This will enable countries to improve PEP delivery and allow underserved populations to access affordable, life-saving rabies vaccines

Minimum Inhibitory Concentration of Glyphosate and of a Glyphosate-Containing Herbicide Formulation for Escherichia coli Isolates – Differences Between Pathogenicand Non-pathogenic Isolates and Between Host Species

Glyphosate is the most extensively used herbicide in the world. However, concerns regarding its safety, side effects, and impact on other organisms have increased in recent years. This is the first study to analyze a large set of recent and historical Escherichia coli isolates varying in pathogenicity and beta-lactam resistance from different host species for their susceptibility to glyphosate isopropylamine salt (IPA), the active ingredient of the herbicide, and to a complete glyphosate-containing formulation (Roundup LB Plus). For this, minimum inhibitory concentrations (MIC) were determined for 238 E. coli isolates by broth microdilution in Mueller Hinton I media followed by the statistical analyses using Mann-Whitney-U test, multivariable analysis of variance (ANOVA) and a multivariable proportional-odds ordinal regression model. While the overall MIC distribution was narrow and lacked a highly resistant sub-population for both substances, statistical analyses revealed small but significant associations between glyphosate resistance levels and different factors tested. Mean MIC values for the entire dataset showed a higher level of resistance to the complete glyphosate-containing formulation (40 mg/ml IPA) than to pure glyphosate (10 mg/ml IPA) in E. coli. Isolates that originated from poultry had significantly higher MIC values for both pure glyphosate and the complete formulation. Pathogenic and non-extended-spectrum beta-lactamase (non-ESBL) E. coli isolates each showed significantly higher MIC values compared to commensals and ESBL-producing E. coli in pure glyphosate, but not in the complete formulation. Recently sampled isolates showed statistically higher MICs than the isolates of the historic standard E. coli collection of reference in pure glyphosate, when tested by nonparametric Mann-Whitney-U test, but not in the multivariable model. Further investigations are necessary to confirm whether these associations have a casual relationship with the glyphosate use or are the consequence of co-selection due to the increased application rates of antibiotics, heavy metals or other biocides. A possible accumulation of pathogenic bacteria in livestock animals fed with glyphosate-containing feed should also be considered

Selection for Resistance to a Glyphosate-Containing Herbicide in Salmonella enterica Does Not Result in a Sustained Activation of the Tolerance Response or Increased Cross-Tolerance and Cross-Resistance to Clinically Important Antibiotics

Evolution of bacterial tolerance to antimicrobials precedes evolution of resistance and may result in cross-tolerance, cross-resistance, or collateral sensitivity to other antibiotics. Transient exposure of gut bacteria to glyphosate, the world’s most widely used herbicide, has been linked to the activation of the stress response and changes in susceptibility to antibiotics. In this study, we investigated whether chronic exposure to a glyphosate-based herbicide (GBH) results in resistance, a constitutive activation of the tolerance and stress responses, and cross-tolerance or cross-resistance to antibiotics. Of the 10 farm animal-derived clinical isolates of Salmonella enterica subjected to experimental evolution in increasing concentrations of GBH, three isolates showed stable resistance with mutations associated with the glyphosate target gene aroA and no fitness costs. Global quantitative proteomics analysis demonstrated activation of the cellular tolerance and stress response during the transient exposure to GBH but not constitutively in the resistant mutants. Resistant mutants displayed no cross-resistance or cross-tolerance to antibiotics. These results suggest that while transient exposure to GBH triggers cellular tolerance response in Salmonella enterica, this response does not become genetically fixed after selection for resistance to GBH and does not result in increased cross-tolerance or cross-resistance to clinically important antibiotics under our experimental conditions

The glyphosate formulation Roundup® LB plus influences the global metabolome of pig gut microbiota in vitro

Glyphosate is the world's most widely used herbicide, and its potential side effects on the intestinal microbiota of various animals, from honeybees to livestock and humans, are currently under discussion. Pigs are among the most abundant livestock animals worldwide and an impact of glyphosate on their intestinal microbiota function can have serious consequences on their health, not to mention the economic effects. Recent studies that addressed microbiota-disrupting effects focused on microbial taxonomy but lacked functional information. Therefore, we chose an experimental design with a short incubation time in which effects on the community structure are not expected, but functional effects can be detected. We cultivated intestinal microbiota derived from pig colon in chemostats and investigated the acute effect of 228 mg/d glyphosate acid equivalents from Roundup® LB plus, a frequently applied glyphosate formulation. The applied glyphosate concentration resembles a worst-case scenario for an 8–9 week-old pig and relates to the maximum residue levels of glyphosate on animal fodder. The effects were determined on the functional level by metaproteomics, targeted and untargeted meta-metabolomics, while variations in community structure were analyzed by 16S rRNA gene profiling and on the single cell level by microbiota flow cytometry. Roundup® LB plus did not affect the community taxonomy or the enzymatic repertoire of the cultivated microbiota in general or on the expression of the glyphosate target enzyme 5-enolpyruvylshikimate-3-phosphate synthase in detail. On the functional level, targeted metabolite analysis of short chain fatty acids (SCFAs), free amino acids and bile acids did not reveal significant changes, whereas untargeted meta-metabolomics did identify some effects on the functional level. This multi-omics approach provides evidence for subtle metabolic effects of Roundup® LB plus under the conditions applied

United against rabies forum:The one health concept at work

Human deaths from rabies are preventable and can be eliminated by applying a systematic One Health approach. However, this ancient disease still threatens the lives of millions of people in up to 150 countries and kills an estimated 59, 000 people every year. Rabies today is largely a disease of poverty, almost always linked to dog bites, with most deaths occurring in neglected communities in Africa and Asia. The disease places an immense economic burden on its victims, a cost that far outweighs the investment needed to control it. A global framework for rabies elimination in humans is set out in Zero by 30: The Global Strategic Plan to end human deaths from dog-mediated rabies by 2030. Despite the existence of proven control strategies and agreement on the path to eliminating human rabies deaths, mortality numbers from rabies remain high, and COVID-19 has set back efforts even further. But COVID-19 has also highlighted the value of a One Health approach to zoonotic disease and pandemic prevention. Rabies control programs offer a practical route to building One Health capacities that can also address other zoonotic threats, including those with pandemic potential. The United Against Rabies Forum aims to accelerate progress on rabies elimination while applying a One Health approach. The Forum promotes cross-sector collaboration among stakeholders and supports countries in their rabies elimination efforts. Increased political engagement and resource mobilization, both internationally and nationally, will be needed to achieve global rabies goals and can also make One Health implementation a reality

Auswirkungen von Glyphosat auf Escherichia coli und Bakteriengemeinschaften in vitro und in vivo

Glyphosate (N-(phosphonomethyl)glycine) is the most-used herbicide worldwide. Many studies have found residues in feed and food. Naturally, concerns about its safety and side effects on other organisms have been raised. With only insufficient and contradictory data about the susceptibility for the widely used herbicide glyphosate, our study is the first to systematically analyse a large amount of E. coli from livestock isolated at different points in time. According to standards for antimicrobial susceptibility testing, we determined minimum inhibitory concentrations (MICs) by means of broth microdilution for the active ingredient (AI) isopropylamine salt (IPA) and the glyphosate-containing formulation Roundup® LB Plus (RU), commonly used in Germany. In total, 238 E. coli isolates, mainly isolated from poultry, pigs and cattle were investigated. Samples isolated between 2014 and 2015 were compared to historic samples of the standard E. coli collection of reference (ECOR) from 1984. For further statistical analysis, samples were divided into extended-spectrum beta-lactamase (ESBL) and non-ESBL producing E. coli as well as into commensal and pathogenic isolates. Mean and mode for all isolates showed a higher level of tolerance for RU (40 mg/ml IPA) compared to GLY (10 mg/ml IPA). In general, the distribution was narrow, and a clearly resistant subpopulation was lacking. To identify less susceptible isolates, a 95% cut-off was calculated (20 mg/ml for GLY and 40 mg/ml for RU). Isolates above the cut-off were sequenced and their aroA gene, coding for the glyphosate target enzyme, compared. No previously known resistance mechanisms were found, however, most differences occurred close to positions described in the literature. Isolates from poultry showed significantly higher MICs in RU and GLY, both in nonparametric Mann-Whitney U tests and statistical models (multivariable variance analysis for GLY and multivariable proportional-odds regression model for RU). In addition, both pathogenic and non-ESBL isolates showed significantly higher MICs in the GLY group, verified by both statistical methods. Solely in the nonparametric test for GLY, historic isolates were less tolerant than recently sampled isolates. However, with only very few isolates from livestock preserved in the ECOR collection, the sample size is a limiting factor of this model. Hence, future studies should include more equivalent historic isolates. To determine whether the growth and survival of a pathogenic E. coli and a S. Typhimurium isolate in an in vitro ruminal experiment is influenced by 10 mg/l RU as a worst-case concentration, a ’Rumen Simulation System’ (RUSITEC) was used. Fermenters were inoculated with 109 colony forming units (CFU) of each strain, leading to a starting concentration of 106 CFU/ml. Initially, the number of CFU of Salmonella doubled after 2 to 4h. Apart from this brief increase, the number of bacteria continuously declined in all fermenters, without being influenced by the RU application. E. coli was no longer detectable in quantitative tests from day 4 and in qualitative tests from day 5 onwards. S. Typhimurium remained detectable until the end of the experiment on day 7, although only a few CFU survived. MICs for RU did not change after the exposure, while antibiotic susceptibility did not vary significantly. In conclusion, the exposure to RU neither increased the abundance, nor promoted resistance. Considering the fermentation experiment focused only on two Enterobacteriaceae in an artificial environment, a more extensive in vivo experiment with pigs was conducted. Weaned piglets (naturally colonized with ESBL E. coli) were infected with 108 CFU of the same S. Typhimurium DT104 strain used in the RUSITEC experiment, at the age of five weeks. One week later, half of the animals per group (n=14/2) were sacrificed as an internal control. The other half was further exposed to nothing (CTRL), GLY or RU, in worst-case concentrations of 2.85 mg/kg bw/d, based on residue levels described in pig feed. The feeding experiment lasted for two weeks, during which faecal samples were checked twice weekly for Salmonella and weekly for ESBL E. coli. Finally, different organs were investigated and faeces and caecum contents were frozen and sent for 16S rRNA analysis via Illumina MiSeq. Neither the exposure to GLY nor to RU increased the shedding or accumulation in organs. As in the fermentation experiment, MICs of the isolates for RU or GLY did not change. The 16S rRNA analysis revealed some differences between the microbial compositions in the different study groups. In general, the RU group showed greater diversity in both faecal and caecum samples. For the GLY group, a tendency was only observed in faeces. Overall, more differences between the CTRL and the exposed groups were found in caecum samples than in faeces. In both caecum and faeces samples, Lactobacillaceae (genus Lactobacillus) increased in pigs from the RU and Enterobacteriaceae (genus Escherichia) increased in pigs from the GLY group. In contrast to previous reports, the number of Clostridia did not increase, but rather decreased in some samples. Future studies should focus on identifying reasons for inter- and intra-species susceptibility variation by taking a closer look at resistance mechanisms and target structures. Moreover, a possible link between antibiotic and glyphosate tolerance should be investigated. Effects of glyphosate on more vulnerable microbiota that are more sensitive for lack of aromatic amino acids (i.e. after birth or after weaning, infection, antibiotic treatment or immunosuppression) should be investigated.Glyphosat (N-(Phosphonomethyl)glycin) ist das am häufigsten genutzte Unkrautvernichtungsmittel der Welt. In vielen Untersuchungen konnten Spuren davon in Lebens- und Futtermitteln gefunden werden, die dementsprechend die Frage nach der Sicherheit des Produktes und der potenziellen Effekte auf die Umwelt, auf Tiere und den Menschen aufwarfen. Aufgrund der sehr geringen und zum Teil widersprüchlichen Datenlage zur Empfindlichkeit von Nutztier-assoziierten E. coli gegenüber Glyphosat, wurden insgesamt 238 E. coli-Isolate, größtenteils aus Geflügel-, Schweine- und Rinderhaltungen, untersucht. Angelehnt an Standardverfahren zur antimikrobiellen Empfindlichkeitsprüfung, wurden minimale Hemmkonzentrationen (MHKs) mittels Mikrodilutionsverfahren bestimmt. Dabei wurde neben dem aktiven Wirkstoff (GLY) als Isopropylamin-Salz (IPA) auch die in Deutschland gängige Formulierung Roundup® LB Plus (RU) untersucht. Historische Isolate einer E. coli Referenzsammlung (ECOR) aus dem Jahr 1984 dienten dabei als Vergleich zu aktuellen Isolaten aus den Jahren 2014 und 2015. Weiterhin wurden die Isolate bezüglich ihres ESBL-Status sowie gemäß ihrer Isolierung als ‘Pathogen’ oder ‘Kommensale’ kategorisiert. Die getesteten Isolate wiesen allgemein eine höhere Toleranz gegenüber RU als für GLY auf (Median- und Modalwert der MHK in RU: 40 mg/ml IPA und in GLY: 10 mg/ml IPA). Da die MHK-Verteilung gering und ohne klar abgrenzbare resistente Subpopulation war, wurde ein 95 % Cutoff-Wert berechnet. Isolate mit einer MHK über diesem Wert wurden sequenziert um die aroA Gene, welche das Ziel-Enzym für Glyphosat codieren, mit weniger toleranten Isolaten zu vergleichen. Dabei wurden Veränderungen gehäuft an Positionen in der Nähe zu schon bekannten Veränderungen resistenter Enzyme gefunden. Isolate aus Geflügelbetrieben zeigten sowohl in nicht-parametrischen Mann-Whitney U Tests, als auch in statistischen Modellierungen (multivariable Varianzanalyse für GLY und multivariable Regressionsanalyse für RU) statistisch signifikant höhere MHK-Werte für GLY und RU. Dies war auch sowohl für nicht-ESBL produzierende als auch für als pathogen kategorisierte E. coli in GLY der Fall. Historische Isolate zeigten allein in nicht-parametrischen Tests in Glyphosat eine geringere Toleranz als aktuelle. Durch die nur geringe Anzahl an Nutztier-assoziierten Isolaten in der ECOR Sammlung ist die Aussage dieser Tests (v.a. für die Modellierungen) jedoch begrenzt und es sollten mehr historische Isolate aus dem Nutztierbereich untersucht werden. Zur Untersuchung der Wachstumskinetik von E. coli und S. Typhimurium DT 104 unter Glyphosatwirkung wurde ein in vitro Pansenfermentationsexperiment mit einer worst-case Konzentration von 10 mg/l RU durchgeführt. Die Fermenter wurden dabei mit jeweils 109 Kolonie-bildenden Einheiten (KbE) eines E. coli und S.Typhimurium DT 104 Isolates beimpft, was zu einer Startkonzentration von 106 KbE/ml in den Fermentern führte. Nach 2-4 h verdoppelte sich die Anzahl der Salmonellen, sank danach jedoch, wie auch die Anzahl der E coli-Isolate, in allen Fermentern, unabhängig der RU-Gabe, stetig ab. An Tag 4 post-inoculationem waren quantitativ und an Tag 5 qualitativ keine E. coli mehr nachzuweisen. Salmonellen waren am Ende des Versuches an Tag 7 nur noch qualititiv isolierbar. Insgesamt wurden durch die Gabe von RU weder die Erregerzahl noch deren Empfindlichkeit gegenüber RU oder Antibiotika beeinflusst. Zur weiteren Beurteilung der Auswirkung von Glyphosat auf ESBL E. coli und S. Typhimurium sowie auf ein enterales mikrobielles Ökosystem, wurde ein in vivo Expositions-Versuch mit Schweinen durchgeführt. Dabei wurden 5 Wochen alte, natürlich mit ESBL-E. coli kolonisierte Absatzferkel mit 108 KbE des auch schon in den Fermenterversuchen eingesetzten Salmonella-Stammes infiziert. Nach einer Woche wurde die Hälfte einer Gruppe (n=14/2) als interne Kontrolle euthanasiert. Die andere Hälfte wurde jeweils entweder 2,85 mg/kg KGW/d GLY oder RU in Anlehnung an entsprechende worst-case Konzentrationen in Schweinefutter oder keinem Zusatz (Kontrollgruppe, CTRL) ausgesetzt. Während dem 2-wöchigen Fütterungsexperiment wurden Kotproben auf Salmonellen (2 mal wöchentlich) und auf ESBL-E. coli (wöchentlich) hin untersucht. Final wurden verschiedene Organe auf ein Vorkommen von Salmonellen getestet und Kot- und Caecumproben mittels 16S rRNA-Ganzgenomsequenzierung untersucht. Weder GLY noch RU erhöhte die Ausscheidungsrate der untersuchten Isolate oder deren Vorkommen in den bei der Sektion gewonnenen Organen. In Übereinstimmung mit dem Fermentationsexperiment änderten sich die MHK-Werte durch den Kontakt zu GLY oder RU nicht. Die 16S rRNA-Analyse zeigte einige Unterschiede in der mikrobiellen Zusammensetzung zwischen den Gruppen. Kot- und Caecum-Proben der RU Gruppe wiesen allgemein eine höhere Diversität auf, für GLY zeigte sich solch eine Tendenz hingegen nur in Kot. Im Vergleich zur Kontrollgruppe, konnten sowohl in Caecum- als auch in Kotproben der RU-Gruppe vermehrt Lactobacillaceae (Genus Lactobacillus) und in Proben der GLY-Gruppe vermehrt Enterobacteriaceae (Genus Escherichia) nachgewiesen werden. Im Gegensatz zu Hinweisen aus der Literatur, waren nicht mehr Clostridia, sondern in einigen Proben sogar weniger zu finden. Die Gründe für unterschiedliche Empfindlichkeiten gegenüber GLY und RU innerhalb einer, aber auch zwischen verschiedenen Bakterienspezies, sollten in zukünftigen Studien genauer untersucht werden. Dabei sollte der Fokus auf Resistenzmechanismen und Zielstruktur sowie einem möglichen Zusammenhang zwischen Antibiotika-Resistenz und Glyphosat-Toleranz liegen. Weiterhin sollten die Auswirkungen von Glyphosat auf Mikrobiota, die für einen Mangel an aromatischen Aminosäuren empfindlich sind (z. B. nach der Geburt oder während des Absetzzeitraumes) untersucht werden