bla _CTX-M-32 on an IncN Plasmid in Escherichia coli from Beef Cattle in the United States

Abstract

d C TX-M-type enzymes are the most extensively distributed extended-spectrum ␤-lactamases (ESBLs) conferring resistance to advanced-generation cephalosporins in Enterobacteriaceae worldwide (1). While these enzymes have also become the most common ESBLs detected in U.S. human infections (2, 3), data on bla CTX-M genes in bacteria from U.S. livestock still remain scarce (4,5).In this report, we describe the detection of bla CTX-M-32 , carried in Escherichia coli strains collected from a cohort of steers housed on a U.S. cattle research-dedicated feedlot in 2009. bla CTX-M -positive isolates were discovered during investigation of antimicrobial resistance in commingling steers receiving injectable long-acting ceftiofur and/or chlortetracycline in feed regimens. Fecal samples were collected from 88 steers in a 26-day period, and E. coli was isolated on MacConkey agar without antibiotics. bla CTX-M was detected in E. coli isolates from 29/88 steers over the course of the study using the PCR primers CTX-M-F-CCGCTGCCGGTYT TATC and CTX-M-R-ATGTGCAGYACCAGTAA. Of these 29 steers, 14 were treated with ceftiofur, four received chlortetracycline, and 11 received both treatments. Sequencing showed that E. coli from each animal carried bla CTX-M-32 (GenBank accession number AJ557142), a group 1 bla CTX-M gene not previously described in U.S. animals but seen throughout Europe in both human and animal isolates (6, 7). A subset of 12 bla CTX-M-32 -positive E. coli strains from different steers was selected for further investigation. Pulsed-field gel electrophoresis (8) revealed six distinct E. coli strains and seven of the 12 E. coli strains with identical banding patterns, indicating that dissemination of bla CTX-M-32 may therefore be the result of both a clonal expansion and horizontal gene transfer. To investigate plasmid vectors, plasmid DNA from each of the 12 isolates was extracted using a Qiagen plasmid midi kit and transformed into naïve DH10B cells, and bla CTX-M-32 -carrying plasmids were successfully selected on agar containing 2 g/ml cefotaxime. Plasmids were shown to be self-transmissible to E. coli recipient 711 (nalidixic acid resistant [NAL r ]) and conferred no additional antibiotic resistance to the bacterial host. PCR-based replicon typing (9) and plasmid multilocus sequence typing (pMLST) (10) revealed plasmids to be IncN sequence type 1 (ST1), a plasmid group responsible for the extensive spread of group 1 bla CTX-M variants in humans and livestock in Europe (10-12).To our knowledge, this is the first report of bla CTX-M-32 in isolates from animals in the United States. Furthermore, bla CTX-Mpositive bacteria were found in feces in greater abundance than previously reported (4, 5), even without use of selective antibiotic enrichment. While steers may have acquired bla CTX-M-32 -carrying E. coli before entering the feedlot, our data strongly suggest that both spread of an E. coli clone between animals and horizontal transfer of an IncN plasmid contributed to wide dissemination of bla CTX-M-32 among this coho...

Impact of treatment strategies on cephalosporin and tetracycline resistance gene quantities in the bovine fecal metagenome

“…The bla CTX-M gene was detected in E. coli 2526 as reported earlier, and was also quantified from community DNA samples derived from feces in this study. For both of the targeted ceftiofur-resistance genes, bla CMY-2 and bla CTX-M gene copies per gram feces significantly increased in treatment groups where all animals were administered CCFA compared with the pens in which only one animal received CCFA treatment.…”

“…The full study was conducted in two replicates of 88 steers each. Fecal samples were evaluated both by non-type-specific (NTS) E. coli culture-based242526 as well as metagenome-based assays. This manuscript focuses on results obtained from an additional analysis of resistance genes copies present in the entire fecal metagenome.…”

“…If results from our previous work23 proved to be robust across the entire metagenome, chlortetracycline would be expected to minimize the proliferation and accumulation of ceftiofur-resistant bacteria and their resistance elements in production-animal agriculture settings. Overall, in the present study, samples were evaluated both at phenotypic24 and genotypic levels, the latter involving both analysis of fecal bacterial isolates242526 as well as the broader metagenome. This manuscript focuses on the results obtained from the analysis of resistance genes present in fecal community DNA and will compare and contrast those findings with the isolate-based work.…”

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

“…23 3GC r E. coli were tested for the presence of bla CMY-2 32 and bla CTX-M . 33 COT r E. coli were tested for trimethoprim (dfr1, dfr5, dfr7/17, and dfr12) and sulfonamide (sul1, sul2, and sul3) resistance genes. 34 DNA lysates from the PCR-confirmed NTS enterococci isolates (n = 1,007) and ERY r enterococci colonies (n = 507) were PCR screened for the presence of tetM and ermB genes.…”

Antimicrobial usage and resistance in beef production

“…Cottell et al [128] evaluated E. coli originating from 88 steers that were treated with ceftiofur and/or chlortetracycline in an experimental US feedlot. The ESBL bla CTX-M-32 , was detected in cefoxatime-resistant E. coli in 29 animals, and was found to be present on a self-transmissible IncN-family plasmid (reviewed in [129]).…”