for a one-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Wholegenome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. for E. faecium from cattle, resistance to β-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all β-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum. Public concern for antimicrobial use (AMU) and resistance (AMR) in livestock is increasing, as is continuing pressure for industries and governments to address these concerns. Science-based and epidemiologically sound research is critical to drive policy, communication, legislation, and inform consumer choices. To effectively investigate the current state of antimicrobial resistance, holistic One Health approaches are required to determine correlation between AMU and AMR across the human-agriculture-environment continuum. The genus Enterococcus is ubiquitous in nature and member species can be found in a range of habitats including soils, sediments, freshwater, marine water, beach sand, and a variety of plants 1,2. Enterococcus spp. are also common members of the normal gastrointestinal (GI) flora of both livestock and humans 3 , with their concentrations in human and animal feces typically ranging from 10 3-10 7 cells per gram 4-6. Enterococcus spp. are also commonly isolated from water contaminated by sewage or fecal wastes, and are widely used as bacteriological

Structures of free-living and protozoa-associated methanogen communities in the bovine rumen differ according to comparative analysis of 16S rRNA and mcrA genes Structures of free-living and protozoa-associated methanogen (PAM) communities from foragefed cattle were investigated by comparative sequence analysis of 16S rRNA and methyl coenzyme M reductase (mcrA) gene clone libraries. The free-living and protozoa-associated communities were composed of the same three genera [namely Methanobrevibacter, Methanomicrobium and rumen cluster C (RCC), which is distantly related to Thermoplasma]; however, the distribution of the methanogen genera differed between the two communities. Despite previous reports of potential bias for the degenerate mcrA primer set, the 16S rRNA (n5100 clones) and mcrA (n592 clones) gene libraries exhibited a similar distribution pattern for the three methanogenic genera. RCC was more abundant in the free-living community and represented 72 and 42 % of the 16S rRNA and mrcA gene sequences, respectively, versus 54 and 13 % of the 16S rRNA and mrcA gene sequences from the PAM community, respectively. The majority of RCC sequences from the free-living and protozoa-associated communities belonged to different species-level operational taxonomic units. Methanobrevibacter species were more abundant in the PAM community and represented 42 and 79 % of clones for the 16S rRNA and mrcA gene libraries, respectively, versus 9 and 27 % of 16S rRNA and mrcA gene clones from the free-living community, respectively. Methanomicrobium species were predominantly free-living. Primers for quantitative PCR were designed to target specific methanogen groups and used to assess the effect of a high-grain diet on methanogen species composition. Switching the ruminant diet from forage to high-grain resulted in reduced protozoal diversity, along with a profound overall reduction in the relative abundance of RCC and an increase in the relative abundance of free-living Methanobrevibacter spp. It was unclear whether the reduced abundance of RCC in grain-fed animals was due to the loss of symbiotic protozoa species or due to broader changes in the rumen environment that affected both RCC and protozoa. Importantly, results from this study emphasize the need to consider the different methanogen communities when developing strategies for mitigating methane emissions in ruminants. INTRODUCTIONRuminants are recognized as a major contributor to methane (CH 4 ) emissions within the agricultural sector. Methane, which is an end product of fermentation, is of concern because of its detrimental environmental effects (IPCC, 2007) and it represents a substantial loss of dietary energy (up to 12 %) to the animal (Johnson & Johnson, 1995). Practical and efficacious methods for decreasing ruminant enteric CH 4 emissions have yet to be developed, but are critical to ensuring environmentally and economically sustainable ruminant production.Methane is produced by methanogenic archaea ('methanogens'), which typically comprise ,3 % of the rumen...

The diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets. Methanobrevibacter spp. and rumen cluster C (distantly related to Thermoplasma spp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification. Within the rumen, methanogens can exist in a free-living form or in association with protozoa through extracellular attachment (ectosymbionts) or intracellular colonization (endosymbionts) (5). Protozoan-associated methanogens (PAMs) account for up to 37% of methane emitted from ruminants (4), and defaunation generally reduces ruminant methane emissions (7). Despite the fact that a thorough understanding of PAMs is essential for developing strategies to mitigate methane emissions from ruminants, species composition of this community has not been well characterized, particularly in cattle (10). According to a recent review, only four studies have characterized methanogens from isolated protozoa of ruminants, including sheep (three studies), goats (one study), and cattle (one study) (2,9,12,21).Given the inherent difficulty of culturing methanogens, most studies rely on the use of small-subunit (SSU) rRNA gene sequences for elucidating community structures. Numerous universal archaeal primer sets have been used by different groups to characterize ruminant methanogen communities (3,9,16,22,24,25). Sequence libraries created using these primers are subject to bias in which certain taxa are preferentially amplified due to factors such as the number and location of nucleotide mismatches between the primer and the target sequence. Furthermore, many of these primers were derived from outdated sequence databases and are therefore not comprehensive in their coverage. While these potential issues have been acknowledged (1,10,16,17), the impact of primer selection on the diversity of rumen methanogens has yet to be empirically assessed. In this study, we examined the effect of primer bias on the PAM community of cattle by using several primer sets that have been previously used to characterize rumen methanogens. Primers that amplified the same region of the SSU rRNA gene (i.e., V2 to V5) were selected to allow for direct phylogenetic comparison of sequences.Protozoan sampling and DNA extraction. Rumen fluid was obtained from four 10-month-old rumen-cannulated Black Angus heifers that were fed grass hay. Samples (250 ml from each of four sites, including the reticulum, ventral and caudal sacs, and the dorsoventral midline) were pooled and strained twice through two layers of PTEX mesh (355-m pore size; Sefar Inc., Kansas City, MO). To allow for separation, samples were incubated at 39°C for 30 min in a sealed container that was occasionally allowed to vent positive pressure. The top plant debris layer was discarded, and protozoa were separated by filtration through NITEX mesh (11-m pore size; Sefar Inc.). Prot...

Accessory gene fingerprinting may have important practical implications for improving the specificity of methods that are widely used for quantifying and identifying the sources of faecal contamination in surface water.

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