Antimicrobial-Resistant Bacterial Populations and Antimicrobial Resistance Genes Obtained from Environments Impacted by Livestock and Municipal Waste

Abstract

This study compared the populations of antimicrobial-resistant bacteria and the repertoire of antimicrobial resistance genes in four environments: effluent of three municipal wastewater treatment facilities, three cattle feedlot runoff catchment ponds, three swine waste lagoons, and two “low impact” environments (an urban lake and a relict prairie). Multiple liquid and solid samples were collected from each environment. The prevalences and concentrations of antimicrobial-resistant (AMR) Gram-negative (Escherichia coli and Salmonella enterica) and Gram-positive (enterococci) bacteria were determined from individual samples (n = 174). The prevalences of 84 antimicrobial resistance genes in metagenomic DNA isolated from samples pooled (n = 44) by collection date, location, and sample type were determined. The prevalences and concentrations of AMR E. coli and Salmonella were similar among the livestock and municipal sample sources. The levels of erythromycin-resistant enterococci were significantly higher in liquid samples from cattle catchment ponds and swine waste lagoons than in liquid samples from municipal wastewater treatment facilities, but solid samples from these environments did not differ significantly. Similarly, trimethoprim/sulfamethoxazole-resistant E. coli concentrations were significantly higher in swine liquid than in municipal liquid samples, but there was no difference in solid samples. Multivariate analysis of the distribution of antimicrobial resistance genes using principal coordinate analysis showed distinct clustering of samples with livestock (cattle and swine), low impact environment and municipal samples forming three separate clusters. The numbers of class A beta-lactamase, class C beta-lactamase, and fluoroquinolone resistance genes detected were significantly higher (P < 0.05) in municipal samples than in cattle runoff or swine lagoon samples. In conclusion, we report that AMR is a very widespread phenomenon and that similar prevalences and concentrations of antimicrobial-resistant bacteria and antimicrobial resistance genes exist in cattle, human, and swine waste streams, but a higher diversity of antimicrobial resistance genes are present in treated human waste discharged from municipal wastewater treatment plants than in livestock environments.

Effects of In-Feed Chlortetracycline Prophylaxis in Beef Cattle on Animal Health and Antimicrobial-Resistant Escherichia coli

“…Generic (isolated on media that did not contain antimicrobials of interest and were not isolated based on any specific resistance), 3GC r , and TET r E. coli bacteria were each enumerated and detected as previously described (23,24). Briefly, 50 l of the enumeration aliquot or an appropriate dilution of the enumeration aliquot was spirally plated using Autoplate 4000 (Spiral Biotech, Norwood, MA) onto CHROMagar E. coli (DRG International, Mountainside, NJ) with no supplemental antimicrobials (CEC), CEC supplemented with 2 mg/liter cefotaxime (CEC ϩ CTX), or CEC supplemented with 32 mg/liter tetracycline (CEC ϩ TET).…”

“…Briefly, 50 l of the enumeration aliquot or an appropriate dilution of the enumeration aliquot was spirally plated using Autoplate 4000 (Spiral Biotech, Norwood, MA) onto CHROMagar E. coli (DRG International, Mountainside, NJ) with no supplemental antimicrobials (CEC), CEC supplemented with 2 mg/liter cefotaxime (CEC ϩ CTX), or CEC supplemented with 32 mg/liter tetracycline (CEC ϩ TET). Antimicrobials were obtained from Sigma-Aldrich Corp., St. Louis, MO, and the antimicrobial concentrations were based on previous reports (19,23,24). Tetracycline was used in agar medium at 32 mg/liter, rather than the 16 mg/liter resistance breakpoint used by the National Antimicrobial Resistance Monitoring System (25), as in our experience, 16 mg/liter tetracycline in agar medium results in large numbers of false-positive colonies.…”

“…Tetracycline was used in agar medium at 32 mg/liter, rather than the 16 mg/liter resistance breakpoint used by the National Antimicrobial Resistance Monitoring System (25), as in our experience, 16 mg/liter tetracycline in agar medium results in large numbers of false-positive colonies. Agar plates were incubated at 37°C for 24 h. Blue colonies were considered presumptive E. coli colonies and were enumerated with an automated colony counter (ProtoCOL 3; Synbiosis, Frederick, MD) (19,23,24).…”

“…Secondary enrichments were prepared by inoculating 0.5 ml of the primary enrichment cultures into 2.5 ml of MacConkey broth (Becton Dickinson, Sparks, MD) with no supplemental antimicrobials (MCB), 2.5 ml of MCB supplemented with 2.4 mg/liter cefotaxime (MCB ϩ CTX), or 2.5 ml of MCB supplemented with 38.4 mg/liter tetracycline (MCB ϩ TET), as previously described (19,23,24). After incubation at 42°C for 18 h, MCB, MCB ϩ CTX, and MCB ϩ TET secondary enrichment cultures were streaked on CEC, CEC ϩ CTX, and CEC ϩ TET agar plates, respectively, and incubated at 37°C for 18 h to detect generic, 3GC r , and TET r E. coli, respectively.…”

Antimicrobial-Resistant Fecal Bacteria from Ceftiofur-Treated and Nonantimicrobial-Treated Comingled Beef Cows at a Cow–Calf Operation

“…16,17 ARB are ubiquitous in soils and other environments 16,17 and their numbers can be increased by supplying nutrients for growth through fecal deposition. 18,19 Beef cows present an interesting opportunity to study this paradigm. These animals receive many of the same treatments as animals in confined animal feeding operations, but the available nutrients in the excreted manure are spread over a larger land area and may not provide much potential for growth to ARB intrinsic to pasture soils.…”