NDM-1 carbapenemase-producing Salmonella enterica subsp. enterica serovar Corvallis isolated from a wild bird in Germany

Abstract

Sir, Thirdand fourth-generation cephalosporins, as well as carbapenems, are b-lactam antimicrobial agents with a broad in vitro spectrum against many human pathogens. They are considered critically important antimicrobials for human treatment. During the last decade, the prevalence of antimicrobial-resistant carbapenemase-producing Enterobacteriaceae among humans has increased and is considered a public health concern worldwide. However, the isolation of carbapenemase-producing enteric bacteria from animals has rarely been reported. – 6 One of the most widespread carbapenemases is the New Delhi metallo-b-lactamase (NDM). The genes encoding this enzyme have been found in different bacterial species (mostly Acinetobacter spp., Escherichia coli and Klebsiella spp.) and are located on highly efficient mobile genetic elements. NDM-1-producing bacteria, mostly isolated from human patients, are frequent in some environmental niches (i.e. water and sewage in India) and have recently been isolated from food-producing animals in China. The Indian subcontinent and the Balkan states seem to be the main reservoirs for NDM-1-producing strains. Among Salmonella, the blaNDM-1 gene has rarely been reported, and in all cases only in patients who had travelled to India. The German Salmonella Reference Laboratory (NRL-Salm Federal Institute of Risk Assessment, BfR) collection contains about 67000 isolates gathered since 1997, mainly from foodproducing animals and foods, but also from non-food-producing animals, the environment, feed and humans. Among this collection, 184 Salmonella spp. isolates that showed clinical resistance to third-generation cephalosporins (MICs ≥4 mg/L for cefotaxime, collected since 2006) were further characterized for their susceptibility to a panel of b-lactams/b-lactamase inhibitors and the presence of extended-spectrum b-lactamase/AmpC genes as previously described. One Salmonella enterica subsp. enterica serovar Corvallis (NRL-Salm-12-1738), multilocus sequence type ST1541 (http:// mlst.ucc.ie/mlst/dbs/Senterica) isolated from a wild bird (black kite, Milvus migrans) showed carbapenem susceptibility values that suggested the presence of a carbapenemase (zone diameters of 24, 20 and 24 mm for, respectively, imipenem, ertapenem and meropenem 10 mg discs, Oxoid-Thermo Fisher Scientific, Wesel, Germany). When Salmonella Corvallis 12-1738 was tested for the MICs of these compounds (Etest, bioMerieux, Craponne, France), it showed reduced susceptibility to them all (0.25, 0.5 and 0.125 mg/L for imipenem, ertapenem and meropenem, respectively; non-wild-type by the EUCAST cut-off values, but susceptible or intermediate according to the CLSI clinical breakpoints). When the isolate was grown in liquid medium with imipenem (Luria-Bertani broth with 16 mg/L imipenem inoculated with 1:1000 overnight culture), it showed full resistance (clinical breakpoints CLSI versus EUCAST, imipenem≥4 versus.8 mg/L). This isolate also showed resistance to chloramphenicol (floR), kanamycin [accA4, also named aac(6)-Ib], tetracycline[tet(A)], trimethoprim(dfrA17), streptomycin (strA/B), sulphonamides (sul1, sul2) and fosfomycin, carried the plasmid-mediated quinolone resistance geneqnrS, andwas susceptible to tigecycline and nitrofurantoin. The resistance determinants (genes cited above, integrons and plasmids) responsible for these resistance phenotypes were analysed by PCR amplification/sequencing and plasmid typing as described before. In this way, the presence of a blaNDM-1 carbapenemase gene was determined (European Nucleotide Archive accession number HG007972; sequence of a 960 bp PCR fragment obtained using the ISAb125-F1/bleBML-B1: TTGAAACTGTCGCACC TCA/TCCAACTCGTCGCAAAGC primers designed for the present work), together with a blaCMY-16 AmpC gene. Both genes were co-localized on pRH-1738, an 180 kb IncA/C conjugative plasmid, which also harboured two class 1 integrons (carrying dfrA1-aadA5 or aacA4 in their variable regions) as well as all resistance genes detected except qnrS. The blaNDM-1 genes have been found located on plasmids of different sizes and incompatibility groups, with IncA/C and IncHI1 being the most frequent. The low number of Salmonella Corvallis in the NRL-Salm collection (62 isolates from different countries isolated from food, livestock, the environment and non-food animals) reflects the low prevalence of Salmonella Corvallis in human infection in Germany (579 cases of salmonellosis, 0.09%, since 2001; Robert Koch-Institut: SurvStat, http://www3.rki.de/SurvStat, data status: 21 May 2013) and Europe (http://www.efsa.europa.eu/en/ efsajournal/doc/3129.pdf). However, Salmonella Corvallis has been detected with noticeably higher prevalence in humans and food products in non-European countries, is endemic in SouthEast Asia (associated with pig and pork) and is emerging in North Africa and Nigeria, where it is widely found in different animal species and the environment (R. S. Hendriksen, Technical University of Denmark, personal communication). The intake of water polluted with faeces or human waste seems to be the most important route for wild birds to acquire antimicrobial-resistant bacteria. The black kite is a migratory bird of prey that lives close to water and spends the summer in Europe