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Objectives The characterization of CTX-M-₁₅ β-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. Methods E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-₁₅ genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-₁₅ were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. Results Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-₁₅ producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-₁₅ genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. Conclusions The prevalence of CTX-M-₁₅-producing isolates from non-human sources in Germany is still low. The blaCTX-M-₁₅ gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-₁₅ gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-₁₅-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.

Objectives: To discern the relevance of ST648 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli as a putative new group of multiresistant and extraintestinal pathogenic strains in animals, its frequency, ESBL types, antimicrobial resistance patterns and virulence gene (VG) profiles should be determined and compared with ST131 strains from the same collection of strains. Methods: ESBL-producing E. coli isolates (n=1152), consecutively sampled from predominantly dogs, cats and horses between 2008 and 2011, were assigned to a phylogenetic group by PCR. Partial multilocus sequence typingwas performed for group D and B2 strains and strains presumed to be D-ST648 and B2-ST131 were fully typed. ESBL genes and extraintestinal pathogenic E. coli (ExPEC)-like VGs were characterized by PCR and sequence analysis and antimicrobial resistance was determined by broth dilution. Clonal analysis was done by PFGE. Results: Forty (3.5%) ESBL-producing E. coli were determined as D-ST648, whereas B2-ST131 isolates occurred less frequently (2.8%). Although the predominant ESBL type in both groups was CTX-M-15 (72.5% versus 46.9%), ST648 strains from companion animals and horses displayed a lower variety of ESBL types (CTX-M-1,-3,-14,-15 and-61 versus CTX-M-1,-2,-14,-15,-27 and-55 and SHV-12). In contrast to ST131 strains, a higher proportion of ST648 strains showed resistance to most non-b-lactam antibiotics. Overall, VGs were less abundant in ST648 strains, although some strains had VG profiles comparable to those of ST131 strains. ExPEC-associated serotype O1:H6 was predominant (46.8%) among the ST648 strains. Some PFGE clusters comprised ST648 isolates from pets, horses and wild birds and humans included from previous studies. Conclusions: Our findings demonstrate that certain subgroups of E. coli D-ST648-CTX-M may represent a novel genotype that combines multiresistance, extraintestinal virulence and zoonotic potential. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Acquired carbapenemases pose one of the most pressing public health threats relating to antibiotic resistance. In most countries, the number of carbapenemase-producing bacteria from human clinical specimens is rising, and the epidemiological status of these multiresistant bacteria is progressively worsening. Furthermore, there is a growing number of reports of carbapenemases found either in bacteria isolated from non-human sources or in Salmonella enterica subsp. enterica, a zoonotic species. However, carbapenemases are not yet systematically sought in bacteria from non-human sources, reports of them are largely observational, and there is limited investigation of carbapenemase-positive bacteria in animals and possible links with people who may have acted as potential sources. Active surveillance and monitoring for carbapenem-resistant bacteria in the food chain and other non-human sources is urgently needed, with an enhanced and rigorous follow-up of all positive results. The carbapenems are currently our last good defence against multiresistant Gram-negative bacteria. Our ability to limit the rise and spread of carbapenemase producers, which occur only at basal levels in many countries at present, should serve as a key performance indicator for the success or failure of the efforts that have been called for by international organizations and governments to reduce the impact of antibiotic resistance.

To characterize a 5.9 kb aac(6')-Ib-cr-harbouring plasmid that was detected in a clinical Salmonella Typhimurium DT104B strain. Methods: Extraction and purification of plasmid DNA and electrotransformation assays were carried out in order to obtain kanamycin-resistant transformants. MICs of several fluoroquinolones and aminoglycosides were determined. DNA sequencing was performed by primer walking on purified plasmid preparations. The new plasmid nucleotide sequence was analysed and compared with available sequences using bioinformatic tools. Results: pMdT1 is a 5.9 kb mobilizable ColE1-like plasmid that harbours aac(6')-Ib-cr4, a gene encoding a new variant of the AAC(6')-Ib-cr protein (225 amino acids). This active protein conferred resistance to tobramycin and kanamycin, and also decreased susceptibility to ciprofloxacin and norfloxacin in the transformant strain, as MICs demonstrated. The mobilization region, necessary for horizontal transfer and composed of the mobA, mobB, mobC and mobD genes, displayed a high degree of identity with those from representative ColE1-like plasmids. The basis of mobility (bom), oriT and origin of replication regions were also detected. Apart from the acetylase-encoding gene, three other open reading frames (ORFs) were determined. No similarities were found when the ORF1 sequence was compared with the sequences included in GenBank. The deduced ORF2 protein predicted a CopG-like structure characteristic of transcriptional regulators, and the deduced ORF3 protein was identical to macrophage stimulating factors. Conclusions: The pMdT1 is the smallest mobilizable ColE1-like plasmid containing an aac(6')-Ib-cr gene that has been described so far. ©The Author 2013.

Objectives This study was initiated to collect retrospective information on the occurrence of plasmid-mediated quinolone resistance (PMQR) in Salmonella enterica and Escherichia coli isolates in Europe and to identify the responsible genes. Methods Databases of national reference laboratories containing MIC values for Salmonella and E. coli isolated between 1994 and 2009 in animals, humans, food and the environment from 13 European countries were screened for isolates exhibiting a defined quinolone resistance phenotype, i.e. reduced susceptibility to fluoroquinolones and nalidixic acid. PCR and sequence analysis were performed to identify the responsible PMQR genes. Results Screening of databases of 13 European countries resulted in a selection of 1215 Salmonella and 333 E. coli isolates. PMQR genes were identified in 59% of the Salmonella isolates and 15% of the E. coli isolates selected. In Salmonella, qnrS1 (n¿=¿125) and variants of qnrB (n¿=¿138) were frequently identified, whereas qnrA1 (n¿=¿3) and aac(6')-1b-cr (n¿=¿3) were rarely found. qnrD was detected in 22 Salmonella isolates obtained from humans and animals. In E. coli, qnrS1 was identified in 19 isolates and qnrB19 was found in one isolate. No qnrC or qepA genes were detected in either Salmonella or E. coli. Conclusions This study shows the occurrence and dissemination of PMQR genes in Salmonella and E. coli in Europe with a defined quinolone resistance phenotype. We also report the first detection of qnrD in Salmonella collected in Europe.

Objectives To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical isolates of Salmonella enterica serotype 4,5,12:i:- belonging to the Spanish clone. Methods The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization. Results The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in different combinations). Most of the responsible genes were provided by a conventional class 1 integron with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-psp-aadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21 with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or two (A/C and N) replicons, as well as virulence genes of pSLT. Conclusions IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant human and animal bacterial pathogens, and hence are regarded as an important threat to public health. Those found in the Spanish clone of Salmonella 4,5,12:i:- constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.

Objectives: In view of the intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15 extended-spectrum b-lactamase (ESBL) in human clinical settings it would be of great interest to explore itsexistence in animals to unravel a possible reservoir function and the origin and transmission of this group ofmultiresistant strains.Methods: A total of 177 clinical phenotypically ESBL-producing E. coli isolates, mainly obtained from companionanimals with urinary tract infections, wound infections and diarrhoea, were collected in a veterinary diagnosticlaboratory covering a European-wide service area. They were screened for molecular subtype O25b and multilocussequence type 131. O25b-ST131 isolates were subsequently tested for ESBL types, and phenotypic andgenotypic resistance determinants. Further characterization of the strains was performed by PFGE and virulencegene typing.Results: Ten (5.6%) of 177 phenotypically ESBL-producing E. coli isolates, nine strains from dogs and one strainfrom a horse, were allocated to the B2-O25b-ST131 lineage. Nine of these isolates harboured a CTX-M-15-typeb-lactamase enzyme while one strain possessed an SHV-12-type ESBL. Macrorestriction analysis revealed acluster formation of six of the animal CTX-M-15-type ESBL-producing strains from five different Europeancountries together with a human control strain constituting a group of clonally related strains at a similarityvalue of 87.0%.Conclusions: Our findings demonstrate that the group of clonally related human B2-O25:H4-ST131 CTX-M-15-type ESBL-producing E. coli strains is present in companion animals from various European countries. This highlightsthe possibility of inter-species transmission of these multiresistant strains from human to animal and viceversa.

To study the molecular characteristics of the quinolone and associated ampicillin resistance mechanisms present in Salmonella enterica serovar Virchow isolated from Turkish foods.Nine epidemiologically unrelated Salmonella Virchow strains isolated from foods (chicken and minced meat) sold in different markets in Ankara were analysed for their susceptibility to 17 antimicrobials. The strains were typed by PFGE and plasmid profiling and investigated by molecular methods (PCR/sequencing) for the presence of several resistance genes, class 1 integrons and mutations in the quinolone resistance-determining regions. Plasmids conferring quinolone resistance were analysed by restriction fragment length polymorphism (RFLP) analysis, DNA hybridization, sequencing, replicon-typing PCR and mating experiments.All strains showed nalidixic acid resistance (MICor= 128 mg/L) together with a decreased susceptibility to ciprofloxacin (three strains with an MIC of 1 mg/L and six with an MIC of 0.25 mg/L), associated with mutations within the gyrA gene (Asp-87 --Tyr-87). In three strains, qnrS1 genes were detected. Ampicillin resistance encoded by a bla(CTX-M3) gene and/or bla(TEM-1-like) gene was found in four strains. Three of these strains carried an approximately 45 kb conjugative plasmid, designated pRQ2006, harbouring qnrS1 and a Tn3-like transposon. Partial sequencing and RFLP of pRQ2006 indicated its similarity to the qnrS1 plasmid pAH03786 found in a Japanese Shigella flexneri 2b isolate.This is the first study describing the presence of qnrS1 genes in bacterial isolates from Turkey. The pRQ2006 plasmid seems to be more related to the S. flexneri 2b qnrS1 plasmid pAH0376 than to the Salmonella qnrS1-carrying plasmids pINF5 and TPqnrS-2.

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