Functional bla_KPC-2 Sequences Are Present in U.S. Beef Cattle Feces Regardless of Antibiotic Use

Carbapenems are classified as critically important antibiotics since they are employed when resistant Gram-negative bacterial infections fail to respond to other antibiotic therapies. Carbapenem-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 bla_KPC-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 internal fragment of the bla_KPC-2 gene was targeted by the qPCR detection method, we sought to determine if functional bla_KPC-2-like sequences are present in beef cattle feces. Full-length bla_KPC-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 bla_KPC-2 cloned in the same orientation as the P_lac promoter had carbapenemase activity and imipenem minimum inhibitory concentrations ≥32 μg/mL. We conclude that the bla_KPC-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.