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

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

Quantification of glyphosate and aminomethylphosphonic acid from microbiome reactor fluids

Rationale: Glyphosate is one of the most widely used herbicides and it is suspected to affect the intestinal microbiota through inhibition of aromatic amino acid synthesis via the shikimate pathway.In vitromicrobiome bioreactors are increasingly used as model systems to investigate effects on intestinal microbiota and consequently methods for the quantitation of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in microbiome model systems are required. Methods: An optimized protocol enables the analysis of both glyphosate and AMPA by simple extraction with methanol:acetonitrile:water (2:3:1) without further enrichment steps. Glyphosate and AMPA are separated by liquid chromatography on an amide column and identified and quantified with a targeted tandem mass spectrometry method using a QTRAP 5500 system (AB Sciex). Results: Our method has a limit of detection (LOD) in extracted water samples of <2 ng/mL for both glyphosate and AMPA. In complex intestinal medium, the LOD is 2 and 5 ng/mL for glyphosate and AMPA, respectively. These LODs allow for measurement at exposure-relevant concentrations. Glyphosate levels in a bioreactor model of porcine colon were determined and consequently it was verified whether AMPA was produced by porcine gut microbiota. Conclusions: The method presented here allows quantitation of glyphosate and AMPA in complex bioreactor fluids and thus enables studies of the impact of glyphosate and its metabolism on intestinal microbiota. In addition, the extraction protocol is compatible with an untargeted metabolomics analysis, thus allowing one to look for other perturbations caused by glyphosate in the same sample

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

CO I Barcoding Reveals New Clades and Radiation Patterns of Indo-Pacific Sponges of the Family Irciniidae (Demospongiae: Dictyoceratida)

DNA barcoding is a promising tool to facilitate a rapid and unambiguous identification of sponge species. Demosponges of the order Dictyoceratida are particularly challenging to identify, but are of ecological as well as biochemical importance.Here we apply DNA barcoding with the standard CO1-barcoding marker on selected Indo-Pacific specimens of two genera, Ircinia and Psammocinia of the family Irciniidae. We show that the CO1 marker identifies several species new to science, reveals separate radiation patterns of deep-sea Ircinia sponges and indicates dispersal patterns of Psammocinia species. However, some species cannot be unambiguously barcoded by solely this marker due to low evolutionary rates.We support previous suggestions for a combination of the standard CO1 fragment with an additional fragment for sponge DNA barcoding

Einfluss von Glyphosat und Glyphosat-haltigen Herbiziden auf Salmonella enterica von Nutztieren

In this study, we investigated the question of the impact of glyphosate formulation on Salmonella enterica isolates in context of animal health and environmental hygiene. Within a screening of 225 S. enterica isolates from farm animals, a change in susceptibility within the last three decades became visible. Different susceptibility against glyphosate was found between different serovars and host animals. In general, the screening revealed higher MIC in isolates that were isolated in the last years. Also, higher MICs were found in pigs compared to poultry and cattle and the serovar Typhimurium had a higher MIC compared to Enteritidis and Infantis. This was the case not only for glyphosate as a pure substance but also for the formulation Roundup LB plus. In an in vitro fermenter experiment, we investigated whether this generally higher glyphosate tolerance in S. enterica lead to a better survival within cattle intestine e.g. rumen. Therefore, we performed an experiment with rumen simulation technic and infected fermenter vessels with S. enterica. Afterwards we challenged half of the vessels with RU, re-isolated S. enterica, counted them and determined the MIC to RU of the re-isolates. It turned out, that RU had no impact on survival time of S. enterica in fermenter, that no change of MIC within experiment took place and that no cross-resistance to antibiotics occurred. In laboratory experiments we finally investigated if it is possible to generate glyphosate resistance in S. enterica via a long-term evolution experiment. We challenged the pathogen with sub-lethal concentrations of RU. It was not easy to decrease sensitivity in S. enterica isolates and many days were needed. In the end, three isolates were selected due to a two to four times higher MIC after the experiment. Of these isolates, the whole genome was sequenced. Comparisons with sequenced ancestral strains revealed mutations in the aroA gene and close regions, but also revealed mutations in genes for stress response. In a fitness essay, no fitness costs were measurable. Also, no cross-resistance or cross-tolerance to antibiotics could be detected. In comparison of the proteome of ancestor and mutant not only the increase of EPSPS translation was visible, but also a variety of different proteins were up- and down-regulated, linked with stress response, iron metabolism and reproductions. In an in vivo animal trial, we finally wanted to investigate the impact of glyphosate and GBH on the shedding of S. enterica. Therefore, three groups of weaning piglets were fed with RU or glyphosate or functioned as control group. In qualitative analysis a significantly higher number of S. enterica was only found in colon samples of the control group compared to the RU group. All other comparisons, qualitatively and quantitatively, showed no differences between the control group, the group fed with glyphosate or the group fed with RU. The findings show a relatively low impact of glyphosate and glyphosate based herbicides in complex environments and with worst-case but still realistic concentrations on the survival, selection and shedding of S. enterica. In laboratory surroundings, it was possible to generate glyphosate resistant S. enterica isolates. These isolates showed mutations in the aroA gene. Furthermore, there was an inconsistent picture of the changes in adaptation to RU which can be brought back to other effects of glyphosate, apart from target enzyme and the effects of the undeclared ingredients of glyphosate formulation