Comparative diversity of microbiomes and Resistomes in beef feedlots, downstream environments and urban sewage influent.

BACKGROUND: Comparative knowledge of microbiomes and resistomes across environmental interfaces between animal production systems and urban settings is lacking. In this study, we executed a comparative analysis of the microbiota and resistomes of metagenomes from cattle feces, catch basin water, manured agricultural soil and urban sewage. RESULTS: Metagenomic DNA from composite fecal samples (FC; n = 12) collected from penned cattle at four feedlots in Alberta, Canada, along with water from adjacent catchment basins (CB; n = 13), soil (n = 4) from fields in the vicinity of one of the feedlots and urban sewage influent (SI; n = 6) from two municipalities were subjected to Illumina HiSeq2000 sequencing. Firmicutes exhibited the highest prevalence (40%) in FC, whereas Proteobacteria were most abundant in CB (64%), soil (60%) and SI (83%). Among sample types, SI had the highest diversity of antimicrobial resistance (AMR), and metal and biocide resistance (MBR) classes (13 & 15) followed by FC (10 & 8), CB (8 & 4), and soil (6 & 1). The highest antimicrobial resistant (AMR) gene (ARG) abundance was harboured by FC, whereas soil samples had a very small, but unique resistome which did not overlap with FC & CB resistomes. In the beef production system, tetracycline resistance predominated followed by macrolide resistance. The SI resistome harboured ß-lactam, macrolide, tetracycline, aminoglycoside, fluoroquinolone and fosfomycin resistance determinants. Metal and biocide resistance accounted for 26% of the SI resistome with a predominance of mercury resistance. CONCLUSIONS: This study demonstrates an increasing divergence in the nature of the microbiome and resistome as the distance from the feedlot increases. Consistent with antimicrobial use, tetracycline and macrolide resistance genes were predominant in the beef production system. One of the feedlots contributed both conventional (raised with antibiotics) and natural (raised without antibiotics) pens samples. Although natural pen samples exhibited a microbiota composition that was similar to samples from conventional pens, their resistome was less complex. Similarly, the SI resistome was indicative of drug classes used in humans and the greater abundance of mercury resistance may be associated with contamination of municipal water with household and industrial products.

A One Health Comparative Assessment of Antimicrobial Resistance in Generic and Extended-Spectrum Cephalosporin-Resistant Escherichia coli from Beef Production, Sewage and Clinical Settings.

This study aimed to compare antimicrobial resistance (AMR) in extended-spectrum cephalosporin-resistant and generic Escherichia coli from a One Health continuum of the beef production system in Alberta, Canada. A total of 705 extended-spectrum cephalosporin-resistant E. coli (ESCr) were obtained from: cattle feces (CFeces, n = 382), catch basins (CBasins, n = 137), surrounding streams (SStreams, n = 59), beef processing plants (BProcessing, n = 4), municipal sewage (MSewage; n = 98) and human clinical specimens (CHumans, n = 25). Generic isolates (663) included: CFeces (n = 142), CBasins (n = 185), SStreams (n = 81), BProcessing (n = 159) and MSewage (n = 96). All isolates were screened for antimicrobial susceptibility to 9 antimicrobials and two clavulanic acid combinations. In ESCr, oxytetracycline (87.7%), ampicillin (84.4%) and streptomycin (73.8%) resistance phenotypes were the most common, with source influencing AMR prevalence (p < 0.001). In generic E. coli, oxytetracycline (51.1%), streptomycin (22.6%), ampicillin (22.5%) and sulfisoxazole (14.3%) resistance were most common. Overall, 88.8% of ESCr, and 26.7% of generic isolates exhibited multi-drug resistance (MDR). MDR in ESCr was high from all sources: CFeces (97.1%), MSewage (96.9%), CHumans (96%), BProcessing (100%), CBasins (70.5%) and SStreams (61.4%). MDR in generic E. coli was lower with CFeces (45.1%), CBasins (34.6%), SStreams (23.5%), MSewage (13.6%) and BProcessing (10.7%). ESBL phenotypes were confirmed in 24.7% (n = 174) ESCr and 0.6% of generic E. coli. Prevalence of bla genes in ESCr were blaCTXM (30.1%), blaCTXM-1 (21.6%), blaTEM (20%), blaCTXM-9 (7.9%), blaOXA (3.0%), blaCTXM-2 (6.4%), blaSHV (1.4%) and AmpC ß-lactamase blaCMY (81.3%). The lower AMR in ESCr from SStreams and BProcessing and higher AMR in CHumans and CFeces likely reflects antimicrobial use in these environments. Although MDR levels were higher in ESCr as compared to generic E. coli, AMR to the same antimicrobials ranked high in both ESCr and generic E. coli sub-populations. This suggests that both sub-populations reflect similar AMR trends and are equally useful for AMR surveillance. Considering that MDR ESCr MSewage isolates were obtained without enrichment, while those from CFeces were obtained with enrichment, MSewage may serve as a hot spot for MDR emergence and dissemination.

Presence and Diversity of Extended-Spectrum Cephalosporin Resistance Among Escherichia coli from Urban Wastewater and Feedlot Cattle in Alberta, Canada.

A recent preliminary study from our group found that extended-spectrum cephalosporin-resistance determinants can be detected in the majority of composite fecal samples collected from Alberta feedlot cattle. Most notably, blaCTX-M genes were detected in 46.5% of samples. Further isolate characterization identified blaCTX-M-15 and blaCTX-M-27, which are widespread in bacteria from humans. We hypothesized that Escherichia coli of human and beef cattle origins share the same pool of blaCTX-M genes. In this study, we aimed to assess and compare the genomic profiles of a larger collection of blaCTX-M-positive E. coli recovered from fecal composite samples from Canadian beef feedlot cattle and human wastewater through whole-genome sequencing. The variants blaCTX-M-55, blaCTX-M-32, blaCTX-M-27, blaCTX-M-15, and blaCTX-M-14 were found in both urban wastewater and cattle fecal isolates. Core genome multilocus sequence typing showed little similarity between the fecal and wastewater isolates. Thus, if the dissemination of genes between urban wastewater and feedlot cattle occurs, it does not appear to be related to the expansion of specific clonal lineages. Further investigations are warranted to assemble and compare plasmids carrying these genes to better understand the modalities and directionality of transfer.

Author Correction: Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a One-Health continuum.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Surveillance of Enterococcus spp. reveals distinct species and antimicrobial resistance diversity across a One-Health continuum.

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. Whole-genome 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 ß-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 ß-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Characterization of tetracycline- and ampicillin-resistant Escherichia coli isolated from the feces of feedlot cattle over the feeding period.

The objective of this study was to investigate tetracycline and ampicillin resistance in Escherichia coli isolated from the feces of 50 crossbred steers housed in 5 feedlot pens. The steers were not administered antibiotics over a 246-day feeding period. A total of 216 isolates were selected for further characterization. The E. coli isolates were selected on MacConkey agar or on MacConkey agar amended with ampicillin (50 microg/mL) or tetracycline (4 microg/mL). Pulsed-field gel electrophoresis (PFGE) typing (XbaI digestion), screening against 11 antibiotics, and multiplex PCR for 14 tet and 3 beta-lactamase genes were conducted. Prevalence of antimicrobial resistance in E. coli at each sampling day was related both temporally and by pen. Multiplex PCR revealed that tet(B) was most prevalent among tetracycline-resistant isolates, whereas beta-lactamase tem1-like was detected mainly in ampicillin-resistant isolates. Our results suggest that antimicrobial resistance in E. coli populations persists over the duration of the feeding period, even in the absence of in-feed antibiotics. Many of the isolates with the same antibiograms had indistinguishable PFGE patterns. Characterization of the factors that influence the nature of this nonselective resistance could provide important information for consideration in the regulation of in-feed antimicrobials for feedlot cattle.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.

Comparison of antimicrobial resistance genes in feedlots and urban wastewater.

The use of antibiotics in livestock production in North America and possible association with elevated abundance of detectable antimicrobial resistance genes (ARG) is a growing concern. Real-time, quantitative PCR (RT-qPCR) was used to determine the relative abundance and diversity of ARG in fecal composite and catch basin samples from 4 beef feedlots in Alberta. Samples from a surrounding waterway and municipal wastewater treatment plants were also included to compare the ARG profile of urban environments and fresh water with that of feedlots. The relative abundance of 18 resistance genes across 5 antibiotic families including sulfonamides, tetracyclines, macrolides, fluoroquinolones, and ß-lactams was examined. Sulfonamide, fluoroquinolone, and ß-lactam resistance genes predominated in wastewater treatment samples, while tetracycline resistance genes predominated in cattle fecal composite samples. These results reflect the types of antibiotic that are used in cattle versus humans, but other factors such as co-selection of ARG and variation in the composition of bacterial communities associated with these samples may also play a role.

En Amérique du Nord, l'utilisation des antibiotiques dans la production du bétail et l'association possible avec une abondance élevée détectable de gènes de résistance aux antimicrobiens (GRA) est une préoccupation grandissante. Une épreuve d'amplification en chaîne par la polymérase quantitative en temps réel a été utilisée afin de déterminer l'abondance relative et la diversité des GRA dans des échantillons composites de fèces et de bassin de rétention de quatre parcs d'engraissement de bovins en Alberta. Des échantillons d'un cours d'eau avoisinant et de l'usine municipale de traitement des eaux usées ont également été inclus afin de comparer le profil des GRA provenant d'un milieu urbain et d'eau fraîche à celui des parcs d'engraissement. L'abondance relative de 18 gènes de résistance issus de cinq familles d'antibiotiques incluant les sulfonamides, les tétracyclines, les macrolides, les fluoroquinolones, et les ß-lactames fut examinée. Les gènes de résistance aux sulfonamides, aux fluoroqunolones, et aux ß-lactames prédominaient dans les échantillons d'eaux usées, alors que les gènes de résistance à la tétracycline étaient prédominants dans les échantillons composites de fèces des bovins. Ces résultats reflètent les types d'antibiotiques qui sont utilisés chez les bovins versus les humains, mais d'autres facteurs tels que la co-sélection de GRA et la variation dans la composition des communautés bactériennes associées à ces échantillons peuvent également jouer un rôle.(Traduit par Docteur Serge Messier).

Effect of subtherapeutic vs. therapeutic administration of macrolides on antimicrobial resistance in Mannheimia haemolytica and enterococci isolated from beef cattle.

Macrolides are the first-line treatment against bovine respiratory disease (BRD), and are also used to treat infections in humans. The macrolide, tylosin phosphate, is often included in the diet of cattle as a preventative for liver abscesses in many regions of the world outside of Europe. This study investigated the effects of administering macrolides to beef cattle either systemically through a single subcutaneous injection (therapeutic) or continuously in-feed (subtherapeutic), on the prevalence and antimicrobial resistance of Mannheimia haemolytica and Enterococcus spp. isolated from the nasopharynx and faeces, respectively. Nasopharyngeal and faecal samples were collected weekly over 28 days from untreated beef steers and from steers injected once with tilmicosin or tulathromycin or continuously fed tylosin phosphate at dosages recommended by manufacturers. Tilmicosin and tulathromycin were effective in lowering (P < 0.05) the prevalence of M. haemolytica, whereas subtherapeutic tylosin had no effect. M. haemolytica isolated from control- and macrolide-treated animals were susceptible to macrolides as well as to other antibiotics. Major bacteria co-isolated with M. haemolytica from the nasopharynx included Pasteurella multocida, Staphylococcus spp., Acinetobacter spp., Escherichia coli and Bacillus spp. With the exception of M. haemolytica and P. multocida, erythromycin resistance was frequently found in other isolated species. Both methods of macrolide administration increased (P < 0.05) the proportion of erythromycin resistant enterococci within the population, which was comprised almost exclusively of Enterococcus hirae. Injectable macrolides impacted both respiratory and enteric microbes, whereas orally administered macrolides only influenced enteric bacteria.

Comparison of CMY-2 plasmids isolated from human, animal, and environmental Escherichia coli and Salmonella spp. from Canada.

A total of 244 CMY-2 plasmids from 5 separate studies involving Escherichia coli and Salmonella human clinical cases as well as E. coli from feedlots and water sources were examined. Genetically similar CMY-2 plasmids isolated from either E. coli or Salmonella from human, animal, and environmental sources are widely distributed across Canada and cluster into replicon types I1, A/C, and K/B and an unidentified group.

Mapping the core: chlamydia and gonorrhea infections in Calgary, Alberta.

OBJECTIVES: To examine the spatial patterning of the individuals with gonorrhea or chlamydia infection in the Calgary Health Region (CHR) to target prevention and control activities. METHODS: A Geographic Information System was used to map the prevalence rates of gonorrhea and chlamydia infection in the CHR to 2001 Census Tracts in the CHR. Data from the 2001 Canadian Census were used to describe the socioeconomic status (SES) of these areas. RESULTS: Low SES indicators correlated with each other (low median household income, lower education, single mothers) as did high SES indicators (married, owning a dwelling, high median income, university education). A correlation was detected between areas of low SES and areas of high prevalence rates for gonorrhea and for chlamydia. These areas clustered primarily downtown and in the northeast part of the city. CONCLUSIONS: Nodes and corridors of activity in Calgary were detected in correlation studies of the 2001 Census variables used. The core (high prevalence) areas should be the areas targeted for sexually transmitted infection prevention and control. This can be done at the community level through measures such as more sexually transmitted infection clinics operating with longer hours in areas identified from this mapping.