Carbapenamases are present in Enterobacteria from non-domestic animals...more frequent than we thought

Objective: To investigate the occurrence of carbapenemases in E. coli and Salmonella isolates collected from livestock animals in Germany within the National Project RESET. Methods: 36 frozen cultures were tested for the presence of carpapenemases. They were obtained from samples collected during vertical studies (2011- 2012) in two German swine and one German poultry farm which resulted positive for VIM-1 carbapenemases (Fischer et al., JAC 2012). The cultures originated from single animal faeces, pooled faeces samples, dust, mouse faeces, flies, manure, environmental samples in- and outside the farm, and were taken in pig fattening farms S1 (11 samples) and S2 (9 samples) and in a broiler fattening farm G1 (18 samples). Samples artificially contaminated with VIM-1, NDM, OXA-48, IMP and KPC-producing isolates were used as positive control. The cultures were incubated in pepton water during 24 h, and tested with the hyplex® SuperBug ID System (detection of VIM-1, IMP, NDM, KPC and OXA-48; Amplex Diagnostics GmbH). Positive samples were confirmed by multiplex-PCR. Producing bacteria were isolated using MacConkey Agar with 1 μg/ml Cefotaxime. Spezies were confirmed by biochemical methods. E. coli isolates were tested by phylogenetic grouping. Positive isolates are currently typed by XbaI-PFGE and plasmid analyses (S1-nuclease PFGE, and PCR-based replicon typing). Results: At least four of the 36 samples analysed resulted positive for VIM-1 carbapenemases. Three of them were E. coli-phylogroup A isolated from a fly, manure, and a swine rectal sample collected in farm S2. The fourth isolate, Salmonella serovar Infantis, was isolated from mouse faeces collected in farm S1. In these farms farms two E. coli (Phylogroup A; farm S2) and thee S. Infantis (G1, S1, S2) had been detected in a previous study. These isolates were clonally related and carried large IncHI2 plasmids (300 kb Salmonella and 240 kb E. coli) in which a class 1 integron carrying blaVIM-1, aacA4 and the aadA1 gene cassettes were present. The clonal or plasmid/integron dispersion of blaVIM-1 in the new isolates is being investigated by further molecular analyses. Conclusions: Carbapenamase occurrence had been primarily ascribed to human community and hospital settings. However carbapenemase producing isolates are also present in livestock animals, more frequently than we thought, and can be spread by different animal vectors like flies and mice, and to the environment by manure.