Location: Meat Safety and QualityTitle: Functional blaKPC-2 sequences are present in U.S. beef cattle feces regardless of antibiotic use
Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2018
Publication Date: 7/1/2018
Citation: Vikram, A., Schmidt, J.W. 2018. Functional blaKPC-2 sequences are present in U.S. beef cattle feces regardless of antibiotic use. Foodborne Pathogens and Disease. 15(7):444-448. https://doi.org/10.1089/fpd.2017.2406.
Interpretive Summary: Recently, we detected a gene that encodes carbapenem resistance in the feces of 36 lots beef cattle "raised without antibiotics" and 36 lots raised "conventionally". Previous data indicated that carbapenem resistance was rare in food animals. Thus, this finding was unprecedented, but since the assay was designed to detect only a portion of the gene, it was possible that the full-length gene encoding functional carbapenem resistance was not present. The goal of this work was to determine if full-length functional gene sequences could be recovered from 18 (9 conventional and 9 raised without antibiotics) randomly selected samples from the original 72 lots. Full-length genes were recovered from all 18 samples, with functionality confirmed for 14 samples. We conclude that carbapenem resistant bacteria are likely present in the feces of more US beef cattle than previously believed. More research is critically needed to determine the levels of carbapenem resistance genes in human feces, feces from other food-animals, wildlife, companion animals, and the environment to accurately assess public health implications.
Technical Abstract: Carbapenems are classified as critically important antibiotics since they are employed when resistant Gramnegative bacterial infections fail to respond to other antibiotic therapies. arbapenem-resistant bacteria (CRB) were traditionally understood to be rare in the U.S. food-producing animals. Recently, using quantitative polymerase chain reaction (qPCR), our group detected blaKPC-2 in all 72 metagenomic DNA (mgDNA) samples prepared from the feces of 36 lots of beef cattle ‘‘raised without antibiotics’’ (RWA) and 36 lots raised ‘‘conventionally’’ (CONV). Since a small nternal fragment of the blaKPC-2 gene was targeted by the qPCR detection method, we sought to determine if functional blaKPC-2- ike sequences are present in beef cattle feces. Full-length blaKPC-2 sequences were amplified from 18 mgDNA samples (9 CONV and 9 RWA), cloned into pCR4Blunt-TOPO vectors, and transformed into Escherichia coli TOP10 cells. All 14 of the samples with blaKPC-2 cloned in the same orientation as the Plac promoter had carbapenemase activity and imipenem minimum inhibitory concentrations ‡32 lg/mL. We conclude that the blaKPC-2 genes detected in our previous study were functional, which indicates that CRB were present in those fecal samples. Identification of functional Klebsiella pneumoniae carbapenemases in fecal samples from both CONV and RWA cattle strongly suggests that CRB are more common in U.S. beef cattle feces than previously believed. Critically, more research using similar qPCR methods to determine the levels of carbapenem-resistant genes in human feces, feces from other food animal species, wildlife, companion animals, and the environment are required to accurately assess public health implications.