Sublethal exposure to commercial formulations of the herbicides dicamba, 2,4-dichlorophenoxyacetic acid, and glyphosate cause changes in antibiotic susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium.

UNLABELLED: Biocides, such as herbicides, are routinely tested for toxicity but not for sublethal effects on microbes. Many biocides are known to induce an adaptive multiple-antibiotic resistance phenotype. This can be due to either an increase in the expression of efflux pumps, a reduced synthesis of outer membrane porins, or both. Exposures of Escherichia coli and Salmonella enterica serovar Typhimurium to commercial formulations of three herbicides-dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup)-were found to induce a changed response to antibiotics. Killing curves in the presence and absence of sublethal herbicide concentrations showed that the directions and the magnitudes of responses varied by herbicide, antibiotic, and species. When induced, MICs of antibiotics of five different classes changed up to 6-fold. In some cases the MIC increased, and in others it decreased. Herbicide concentrations needed to invoke the maximal response were above current food maximum residue levels but within application levels for all herbicides. Compounds that could cause induction had additive effects in combination. The role of soxS, an inducer of the AcrAB efflux pump, was tested in ß-galactosidase assays with soxS-lacZ fusion strains of E. coli. Dicamba was a moderate inducer of the sox regulon. Growth assays with Phe-Arg ß-naphtylamide (PAßN), an efflux pump inhibitor, confirmed a significant role of efflux in the increased tolerance of E. coli to chloramphenicol in the presence of dicamba and to kanamycin in the presence of glyphosate. Pathways of exposure with relevance to the health of humans, domestic animals, and critical insects are discussed. IMPORTANCE: Increasingly common chemicals used in agriculture, domestic gardens, and public places can induce a multiple-antibiotic resistance phenotype in potential pathogens. The effect occurs upon simultaneous exposure to antibiotics and is faster than the lethal effect of antibiotics. The magnitude of the induced response may undermine antibiotic therapy and substantially increase the probability of spontaneous mutation to higher levels of resistance. The combination of high use of both herbicides and antibiotics in proximity to farm animals and important insects, such as honeybees, might also compromise their therapeutic effects and drive greater use of antibiotics. To address the crisis of antibiotic resistance requires broadening our view of environmental contributors to the evolution of resistance.

Size exclusion-based purification and PCR-based quantitation of MS2 bacteriophage particles for environmental applications.

MS2 bacteriophage is the most commonly used surrogate for pathogenic viruses in laboratory and field studies. In order to determine the number of infectious viral particles in samples, the use of accurate quantitation methods is essential. We have optimised a size exclusion chromatography-based method for MS2 purification and a SYBR Green-based single-step quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay for the quantitation of MS2. The qRT-PCR enabled accurate quantitation of viral RNA of the purified stock with a detection limit of 2 genome copy equivalents/µl. Detection inhibition, if any, was eliminated by reducing sample volume added to the qRT-PCR reaction mix when MS2 was detected in environmental water samples. The purification method eliminated the impurities and the purified stock yielded a high concentration of infectious MS2 particles. The qRT-PCR assay enabled the accurate quantitation of the viral particles thus providing an alternative to the traditional plaque assays. A combined use of purified MS2 stock and PCR-based quantitation gives the opportunity to explore virus characteristics, behaviour and interactions in the environment.