Decay of Fecal Indicator Bacterial Populations and Bovine-Associated Source-Tracking Markers in Freshly Deposited Cow Pats

Understanding the survival of fecal indicator bacteria (FIB) and microbial source-tracking (MST) markers is critical to developing pathogen fate and transport models. Although pathogen survival in water microcosms and manure-amended soils is well documented, little is known about their survival in intact cow pats deposited on pastures. We conducted a study to determine decay rates of fecal indicator bacteria (Escherichia coli and enterococci) and bovine-associated MST markers (CowM3, Rum-2-bac, and GenBac) in 18 freshly deposited cattle feces from three farms in northern Georgia. Samples were randomly assigned to shaded or unshaded treatment in order to determine the effects of sunlight, moisture, and temperature on decay rates. A general linear model (GLM) framework was used to determine decay rates. Shading significantly decreased the decay rate of the E. coli population (P < 0.0001), with a rate of −0.176 day⁻¹ for the shaded treatment and −0.297 day⁻¹ for the unshaded treatment. Shading had no significant effect on decay rates of enterococci, CowM3, Rum-2-bac, and GenBac (P > 0.05). In addition, E. coli populations showed a significant growth rate (0.881 day⁻¹) in the unshaded samples during the first 5 days after deposition. UV-B was the most important parameter explaining the decay rate of E. coli populations. A comparison of the decay behaviors among all markers indicated that enterococcus concentrations exhibit a better correlation with the MST markers than E. coli concentrations. Our results indicate that bovine-associated MST markers can survive in cow pats for at least 1 month after excretion, and although their decay dynamic differs from the decay dynamic of E. coli populations, they seem to be reliable markers to use in combination with enterococci to monitor fecal pollution from pasture lands.     

Co-occurrence of antibiotic,biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site

Contaminants such as heavy metals may contribute to the dissemination of antimicrobial resistance (AMR) by enriching resistance gene determinants via co-selection mechanisms. In the present study, a survey was performed on soils collected from four areas at the Savannah River Site (SRS), South Carolina, USA, with varying contaminant profiles: relatively pristine (Upper Three Runs), heavy metals (Ash Basins), radionuclides (Pond B) and heavy metal and radionuclides (Tim’s Branch). Using 16S rRNA gene amplicon sequencing, we explored the structure and diversity of soil bacterial communities. Sites with legacies of metal and/or radionuclide contamination displayed significantly lower bacterial diversity compared to the reference site. Metagenomic analysis indicated that multidrug and vancomycin antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) including those associated with copper, arsenic, iron, nickel and zinc were prominent in all soils including the reference site. However, significant differences were found in the relative abundance and diversity of certain ARGs and MRGs in soils with metal/radionuclide contaminated soils compared to the reference site. Co-occurrence patterns revealed significant ARG/MRG subtypes in predominant soil taxa including Acidobacteriaceae, Bradyrhizobium, Mycobacterium, Streptomyces, Verrumicrobium, Actinomadura and Solirubacterales. Overall, the study emphasizes the potential risk of human activities on the dissemination of AMR in the environment.     

Escaping the fate of Sisyphus: assessing resistome hybridization baits for antimicrobial resistance gene capture

Finding, characterizing and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19 933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement.