Detection of pandemic B2-O25-ST131 Escherichia coli harbouring the CTX-M-9 extended-spectrum -lactamase type in a feral urban brown rat (Rattus norvegicus)

Sir, Apart from the increasing prevalence of extended-spectrum β-lactamases (ESBLs) in Escherichia coli isolates from human patients and diseased livestock1 their co-emergence in wildlife faecal samples has also been documented.2 As wildlife animals are known to disseminate bacteria of human and animal health concern and may carry, in particular, ESBL-producing E. coli, it is highly important to determine how widely these bacteria have spread into rural and urban ecosystems. Recently, broad geographical dissemination of an E. coli clone (B2-O25:H4-ST131) carrying the CTX-M-15 ESBL has been described for clinical and non-clinical settings in human medicine3 while only one very recent publication reports a case in one animal, namely a dog.4 The aim of this study was to determine the prevalence of E. coli harbouring genes of the CTX-M-ESBL type in a wildlife animal species with close contact with human settlements—the brown rat (Rattus norvegicus). Knowledge of the prevalence of antimicrobial-resistant E. coli in the faeces of feral rat species remains scarce,5 although rats have been known as carriers of various pathogens for centuries. Within the network ‘Rodent-borne pathogens’, faecal samples from 66 feral urban brown rats were collected in the city area of Berlin (Germany) for our study. Rats were captured and euthanized by pest control technicians and brought to the laboratory on the same day. After taking cloacal swabs the animals were passed to the researchers, who determined species affiliation. At least four E. coli isolates were obtained from each faecal sample by using CHROM agar orientation (Chromagar, Paris, France). Duplicate clones were excluded by macrorestriction analysis. The resulting 211 unique isolates were further processed by agar disc diffusion testing as a preliminary screen for antimicrobial resistance. Of these, 69 (32.7%) exhibited resistance phenotypes against multiple antibiotics and were additionally tested by the broth microdilution method (Micronaut breakpoint plate ‘Kleintier’; Genzyme Diagnostics, Rüsselsheim, Germany) against 17 antimicrobials including β-lactams, aminoglycosides, tetracyclines, sulphonamides, chloramphenicol and fluoroquinolones according to CLSI standards.6 One (0.5%) of the isolates sampled in the city area of Berlin with a dense human population (13 239 inhabitants/km2) showed a positive confirmatory test for ESBL production, as determined by the CLSI method and was therefore intensively characterized via a multifocus approach including phenotypic and genotypic methods. Multilocus sequence typing (http://mlst.ucc.ie/mlst/mlst/dbs/Ecoli/) and molecular determination of the rfb25b locus by PCR7 revealed sequence type ST131 and O antigen subtype O25b. Phenotypic antimicrobial resistance was detected for cefotaxime, cefalotin, cefovecin,a ampicillin, oxacillin, ticarcillin, doxycycline, gentamicin, enrofloxacin, difloxacin,a orbifloxacina and marbofloxacina (acommon veterinary antimicrobials for companion animals). Isoelectric focusing (IEF)8 and PCR-based screening9 and sequencing of genetic determinants for resistance led to the detection of the β-lactamases type CTX-M-9 (pI = 8.5) and TEM-1 (pI = 5.4) and the non-β-lactamase genes tet(A), sul2, strA, blaTEM-1, aac(3)IV and aac(6′)-Ib-cr. Southern blotting and subsequent PCR-based replicon typing10 for characterization of the resistance-providing plasmids resulted in the identification of the respective gene on a plasmid (>100 kb) of the FIA/FIB replicon type. Self-transferability of the plasmid was confirmed by mating experiments, in liquid and filter, at 37°C and 22°C, using an azide-resistant E. coli strain as receptor. Finally, PFGE revealed a close genetic similarity to other O25b-ST131 E. coli strains of the human pandemic clonal group3 as depicted in Figure 1. Dendrogram (created by software BioNumerics, Version 4.6; Applied Maths, Belgium) of the CHEF–PFGE electropherogram of XbaI-restricted genomic DNA of the CTX-M-9-type ESBL-producing E. coli (IMT19205) strain of sequence type ST131 and human control strain DSM22664 (RL102/05; O25b-CTX-M-15-ST131) representing the human pandemic virulent group of clonally related strains. In conclusion, the ESBL-producing E. coli isolate found in a brown rat from an urban region with a high human population density belongs to the currently spreading B2-O25b-ST131 clone. Although a direct clonal relatedness of this novel strain to human isolates was demonstrated via PFGE, its ESBL type was determined to be the more unusual CTX-M-9. This ESBL type has been recently found in a single Spanish clinical O25b-ST131 isolate.11 To our knowledge this is the first description of the B2-O25b-ST131 pandemic ESBL-producing E. coli strain in the brown rat. In addition to the long known threat of urban rats as a potential risk factor for human health, our finding provides the first evidence of a possible role of these urban rodents in the spread of CTX-M-type ESBL-producing E. coli. This work was supported by grants from the Federal Ministry of Education and Research Network Zoonosis (FBI-Zoo, grant no. 01KI07120), the Federal Institute for Risk Assessment (BfR-46-001) and the Med-Ved-Net (EU-funded Network of Excellence for the Prevention and Control of Zoonoses). None to declare. We would like to thank M. Stange, M. Heising and B. Barownick for providing their excellent assistance with rat trapping, and F. Aarestrup, H. Hasman, L. Martínez and A. Carattoli for providing control strains.