Silvia Schmoger

Salmonella enterica subsp. enterica producing VIM-1 carbapenemase isolated from livestock farms

Sir, Thirdand fourth-generation cephalosporins and carbapenems are ‘critically important’ antimicrobials as classified by the WHO (www.who.int). In fact, carbapenems are last-line clinical antibiotics against infections caused by multidrug-resistant Gram-negative bacteria. In contrast to cephalosporins, carbapenems are not hydrolysed by most b-lactamases, including AmpC b-lactamases and extended-spectrum b-lactamases (ESBLs). However, during the last decade the prevalence of carbapenem resistance in Enterobacteriaceae has increased worldwide. Whereas the increase in the prevalence of ESBL-producing Enterobacteriaceae isolated from livestock is becoming an important public health problem, the increasing prevalence of carbapenemases has only affected hospitals and the community. Recently, however, the occurrence of carbapenemase-carrying commensal Escherichia coli isolated from livestock and their environment has been reported, and this could be the beginning of a new era in the antibiotic resistance field. Within the national RESET project (www.reset-verbund.de) several longitudinal and cross-sectional studies, collecting potential ESBL-carrier organisms from German farms, have been performed (using MacConkey agar with 1 mg/L cefotaxime as the selective medium). From the 221 isolates collected during 2011, 3 of them were ascribed to Salmonella enterica subsp. enterica (Table 1). The three Salmonella isolates (R3, R25 and R27) were obtained from two pig-fattening farms (R25 was collected outside the farm) and one broiler farm (Table 1). The three farms were distributed in different locations in the same German federal region, and although there was no apparent link between them, a common source cannot be excluded. The three isolates were tested for their susceptibility to 35 antimicrobials, including b-lactams/b-lactamase inhibitors (Table 1), phenicols, aminoglycosides, quinolones/fluoroquinolones, tetracycline, folate pathway antagonists, lipopeptides and fosfomycin, as previously described. For the present study, tigecycline (15 mg) and nitrofurantoin (300 mg) were included as well. The presence of ESBLs, AmpC b-lactamases and/or carbapenemase-encoding genes, class 1 and 2 integrons and other resistance genes was screened by PCR/sequencing, as previously described (Table S1, available as Supplementary data at JAC Online). The MIC values for some carbapenemase producers can be lower than the currently recommended breakpoints, and the results of the carbapenem susceptibility tests can be influenced by the genetic background. The Salmonella isolates R3, R25 and R27 showed decreased susceptibility to these antimicrobials [non-wild-type by the EUCAST epidemiological cut-off (ECOFF), but susceptible or intermediate according to the CLSI clinical breakpoint; Table 1]. This ‘decreased susceptibility’ could be transformed to a competent E. coli recipient, but conjugation or mobilization under the conditions used was unsuccessful. The three isolates carried both the AmpC-encoding gene blaACC-1 and the carbapenemase gene blaVIM-1, like in the previously reported E. coli isolates R178 and R29. When Salmonella R3 and the control strain E. coli R178 were grown in liquid medium with carbapenems (Luria-Bertani broth with 16 mg/L imipenem or 8 mg/L ertapenem inoculated with 1:1000 overnight culture), both isolates grew well, showing full carbapenem resistance (clinical breakpoints, CLSI versus EUCAST: imipenem ≥4 versus .8 mg/L; and ertapenem ≥1 versus .1 mg/L). Several class 1 integrons (In31, In70, In71, In110 and In450), transposons (Tn3, Tn402 and Tn21) and plasmids (incompatibility groups IncHI2, IncN, IncI1 and IncW) carrying blaVIM genes have been described. Like in E. coli R178 and R29, in the three Salmonella isolates the blaVIM-1 gene was located on a class 1 integron (variable region with blaVIM-1-aacA4-aadA1 gene cassettes) harboured by an 300 kb IncHI2 plasmid (determined by S1-nuclease PFGE analysis, PCR-based replicon typing and Southern blot hybridization, as previously described; HI2 double plasmid sequence typing failed). The plasmid also carried blaACC-1, strA/B, catA1 and a trimethoprim resistance gene (not identified with the primers used; see Table S1, available as Supplementary data at JAC Online). The sequence of the complete integron of Salmonella R3 (5436 bp, including complete sul1 and orf5, obtained using as template the pRHR3 plasmid of this isolate; see Table S1, available as Supplementary data at JAC Online) was identical to the one from E. coli R178 (accession number HE663536) and was related to Tn402 (like in GQ422826). Salmonella R27 was isolated from the same farm as both E. coli R178 and R29 (Table 1). However, the IncHI2 plasmids harboured by these Salmonella and E. coli isolates were different in size and gene content ( 300 versus 220 kb and presence versus absence of chloramphenicoland trimethoprimresistance genes), suggesting different plasmid evolutions. The three isolates were classified as S. enterica group C, antigenic formula ‘6,7:–:– ’ (www.pasteur.fr) at the National Salmonella Reference Laboratory (NRL-Salm, BfR). The sequence type ST32 (http://mlst.ucc.ie/mlst/dbs/Senterica) and the PFGE patterns found (Figure S1, available as Supplementary data at JAC Online) are typical of Salmonella Infantis (6,7:r:1,5) and have also been detected in German isolates from humans, poultry/ poultry meat and pig/pork meat. Salmonella Infantis is among the top 10 Salmonella serovars implicated in human salmonellosis worldwide (ranking in third place in 2011 in Europe; www.ecdc. europa.eu). Isolates from this serovar also caused disease with Research letters

Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm

and the source from which the isolate was obtained. Further target gene mutations were detected in single isolates: (i) a mutation that resulted in the Glu471Asp exchange in GrlB was present in an avian ST5 MRSA; (ii) a mutation at codon 517 in the gyrB gene (resulting in an Arg to Lys exchange) was found in the ST1791 MRSA of turkey meat origin; and (iii) the ST2269 isolate had an additional grlA mutation at codon 84 that caused a Glu to Asp exchange, and a gyrA mutation that resulted in a Glu88Asp exchange. The GrlA alterations Ser80Leu and Glu84Asp and the GyrA exchange Glu88Asp have not been identified so far in S. aureus. The role in fluoroquinolone resistance of the Glu422Asp exchange in GrlB needs further investigation as the corresponding mutation was present along with other mutations in staphylococci that varied in their enrofloxacin MICs between 1 and 8 mg/L, but it was also the only mutation detected in two porcine MRSA isolates with an enrofloxacin MIC of 1 mg/L. All but one of the MRSA and MSSA isolates investigated in this study showed one of two types of point mutations (A and B in Figure S1, available as Supplementary data at JAC Online) in the norA promoter region; these, however, affected neither the 235 and 210 positions, nor the norA-associated ribosome binding site. For one avian MSSA isolate, no norA-specific PCR product could be obtained in repeated attempts. The results of this study show that increased MICs of enrofloxacin among MRSA and MSSA isolates from diseased foodproducing animals or food of animal origin is mainly mediated by grlA and/or gyrA mutations, which—aside from the four novel mutations detected during this study—correspond to those reported previously in S. aureus isolates of different origin. 2

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

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

Spread and persistence of VIM-1 Carbapenemase-producing Enterobacteriaceae in three German swine farms in 2011 and 2012

The occurrence of carbapenemase-producing Enterobacteriaceae in livestock is considered as a threat for public health. In Germany, VIM-1-producing Escherichia (E.) coli sequence type (ST) 88 and Salmonella Infantis isolates harbouring blaVIM-1IncHI2 plasmids have been isolated from swine and poultry farms. A retrospective study was performed to determine if there was a broader distribution of VIM-1-positive isolates in any of the carbapenemase-positive swine farms. Selective incubation (carbapenem-containing broth) of 249 conserved cultures collected in three farms (2011-2012), allowed the detection of 40 blaVIM-1-positive isolates. Apart from the already known non-motile Salmonella Infantis isolate R25 (farm S1) and R27 (S2), a third isolate was recovered from farm S3. For E. coli, additional to isolates R29 and R178 (S2), 35 new isolates were identified in the same farm during all the sampling periods (three dates, 2011) and in samples from different animals, farm environment, manure and flies. The newly identified E. coli and Salmonella isolates showed similar genetic and phenotypic characteristics (XbaI-PFGE profiles, antimicrobial resistance patterns, plasmid content, phylogroups, antigenic formula) to those in the previously described strains, suggesting microevolution within the clonal lines within one fattening period. The study shows that persistence of carbapenemase-producing clonal lines in livestock farms is possible, and underlines the need for harmonised monitoring and surveillance studies to follow up the occurrence of such bacteria in European livestock.

CTX-M-15-Producing E. coli Isolates from Food Products in Germany Are Mainly Associated with an IncF-Type Plasmid and Belong to Two Predominant Clonal E. coli Lineages

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA−1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation.

Recurrent detection of VIM-1-producing Escherichia coli clone in German pig production.

this last case, the description here of blaNDM-1-carrying A. pittii in France is made in a patient with no recent history of travel. The diagnosis was made fortuitously from a rectal swab sample. The context of this diagnosis suggests that the circulation of ACB species carrying blaNDM may be underestimated in France. This case raises questions about the management of patients with carbapenemase-producing A. pittii carriage in hospitals. Here, all the patients hospitalized in the same ward and screened for carbapenemase carriage were negative. Dissemination of NDM1-producing A. pittii has been noted in an ICU. NDM-producing A. pittii had then been isolated in the air within the ICU, being suspected to contribute to the dissemination of the bacterium. Therefore, early detection of carbapenemases in Acinetobacter species seems critical to control the dissemination of carbapenemase-producing isolates. Here, this first French case of NDM-producing A. pittii in a patient with no history of travel enhances the problem of carbapenemaseproducing ACB species and their management in hospitals.

Diversity of CTX-M-1-producing E. coli from German food samples and genetic diversity of the blaCTX-M-1 region on IncI1 ST3 plasmids

Antimicrobial resistance to cephalosporins is commonly mediated by extended-spectrum β-lactamases (ESBL) or plasmidic AmpC β-lactamases (pAmpC). In livestock blaCTX-M-1 is the most frequently detected ESBL-encoding gene. As transmission to consumers through contaminated food is often proposed, this study characterized ESBL/pAmpC-producing E. coli collected from food samples. Therefore, samples from food products of animal origin and vegetables were screened for phenotypically resistant E. coli by selective cultivation. The ESBL genotype was confirmed for 404 isolates with the majority of them (n = 212) harboring the blaCTX-M-1 gene. PFGE and MLST analyses as well as plasmid characterization were carried out for 89 isolates, selected under epidemiological aspects. In addition, 44 isolates were investigated by whole genome sequencing and/or sequencing of their plasmids on an Illumina Miseq platform. MLST and PFGE indicated a diverse population of CTX-M-1-producing E. coli in German food samples with no spread of single clonal lineages. The majority of the isolates harbored the blaCTX-M-1 gene on IncI1 plasmids. Frequently, the gene was associated with the ISEcp1 element and located on a ∼100 kb IncI1 plasmid depicting the plasmid multilocus sequence type (ST) 3. The blaCTX-M-1 gene and its flanking sequences were located within the shufflon of the type IV pilus region in diverse orientations. In conclusion, dissemination of the CTX-M-1 β-lactamase within food samples of animal origin is driven by the transmission of a ∼100 kb large IncI1 ST3 plasmid. Apart from conjugal transfer of IncI1 ST3 plasmids the transmission of the blaCTX-M-1 gene might be further promoted through mobilization due to its location within a recombination hot-spot of IncI1 plasmids.