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.

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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 a chronic exposure to a glyphosate-containing herbicide (GBH) results in resistance, a constitutive activation of the tolerance and stress response and cross-tolerance or cross-resistance to antibiotics. Of the ten farm animals-derived clinical isolates of Salmonella enterica subjected to experimental evolution in increasing concentrations of GBH, three isolates showed a stable resistance with mutations associated with the glyphosate target gene aroA and no fitness costs. The 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. Importance Glyphosate-based herbicides (GBH) are among the world's most popular, with traces commonly found in food, feed and the environment. Such high ubiquity means that the herbicide may come into contact with various microorganisms, on which it acts as an antimicrobial and may select for resistance and cross-resistance to clinically important antibiotics. It is therefore important to estimate whether the widespread use of pesticides may be an underappreciated source of antibiotic resistant microorganisms that may compromise efficiency of antibiotic treatments in humans and animals.

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.

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 vitro microbiome 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.