Studies on the prevalence, distribution and organization of extended-spectrum β-lactamase genes and transferable (fluoro)quinolone resistance genes among Enterobacteriaceae from defined disease conditions of companion and farm animals

Extended-spectrum β-lactamase (ESBL)- and Qnr-protein-producing Escherichia coli isolates have gained considerable public attention during recent years. However, information about such isolates from diseased animals in Germany is scarce. Thus, the aims of this study were (i) to determine how often and which subtypes of ESBL and qnr genes are present in E. coli from defined disease conditions of companion and farm animals and (ii) to gain insight into the location and organization of the resistance genes. For this, the E. coli isolates collected all over Germany in the BfT-GermVet study were used. In the BfT-GermVet study, 417 E. coli isolates from diseased dogs/cats (n = 228), horses (n = 102), and swine (n = 87) were tested for their susceptibility to 24 antimicrobial agents by broth microdilution. To identify potential ESBL-producers, all 100 ampicillin-resistant E. coli isolates from this collection were subjected to an initial screening for cefotaxime resistance and subsequent phenotypic confirmatory tests. In a second part of the project, all E. coli isolates were screened for qnr genes. The ESBL and qnr genes were detected by specific PCR assays and the complete resistance genes including their flanking regions were cloned and sequenced. Plasmids were transferred by conjugation and transformation into E. coli recipients and subjected to PCR-based replicon typing. One qnrB19-carrying plasmid was sequenced completely. Multilocus sequence typing (MLST) was performed for the ESBL-producing and for the qnr-positive E. coli isolates. Solely three E. coli isolates showed an ESBL phenotype. The canine isolate 913 from a urinary tract infection harboured a blaCTX-M-15 gene and the E. coli isolates from canine pneumonia (isolate 168) and porcine metritis-mastitis-agalactia syndrome (isolate 246) blaCTX-M-1 genes. The gene qnrB19 was detected in two E. coli isolates (2078 and 2086) from mares suffering from genital tract infections. MLST showed that isolate 913 had the sequence type ST410, while isolates 168 and 246 belonged to the novel sequence types ST1576 and ST1153, respectively. Isolates 2078 and 2086 were assigned to ST86. Isolate 913 harboured the ca. 50 kb IncF plasmid pCTX913. This plasmid carried the blaCTX-M-15 gene and an aac(6’)-Ib-cr gene, which confers resistance to kanamycin and reduced susceptibility to ciprofloxacin. Furthermore, pCTX913 conferred resistance to gentamicin and tetracycline. Upstream of blaCTX-M-15 the insertion sequence ISEcp1 was present and downstream a truncated orf477 and a fragment of a transposase gene tnpA. The two blaCTX-M-1 ESBL genes were located on structurally related plasmids of ca. 50 kb which did not confer any other resistance properties. PCR-based replicon typing identified both plasmids designated pCTX168 and pCTX246, to belong to IncN. Plasmid pCTX168 was conjugative. The blaCTX-M-1 upstream and the immediate downstream regions of pCTX168 and pCTX246 were similar whereas the sequences further downstream of blaCTX-M-1 differed. In the upstream region, a fragment of the insertion sequence ISEcp1, truncated by an IS26 element, was detected. In the downstream region, a fragment of orf477 and a truncated mrx gene were identified on both plasmids. On plasmid pCTX246, a complete mph(A) gene and another IS26 element were seen further downstream of the Δmrx gene, while on plasmid pCTX168 the mph(A) gene was truncated. The qnrB19-carrying conjugative plasmids pQNR2078 and pQNR2086 belonged also to IncN, were indistinguishable by restriction analysis and did not carry other resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence IS26 located in the same orientation. The completely sequenced plasmid, pQNR2078, had a size of 42,379 bp and exhibited 47 open reading frames. Except for the resistance gene region, the remaining part of pQNR2078 showed 99% nucleotide sequence identity to the blaCTX-M-65-carrying plasmid pKC396 from human E. coli. The blaCTX-M-15 gene region as identified on the IncF plasmid pCTX193 has been detected in diverse plasmid backgrounds in different members of the Enterobacteriaceae from human and animal sources isolated in several countries. In addition, the gene blaCTX-M-1 gene in combination with the IS26-ΔISEcp1-structure, the truncated mph(A)-mrx-mphR(A) gene cluster which is followed by a second IS26, linked to IncN plasmids has been described in a German human clinical E. coli ST131 isolate, but not in isolates of animal origin in Germany so far. The gene qnrB19 gene was detected in a new genetic environment on conjugative IncN plasmids and for the first time in an E. coli isolate of equine origin in Germany. Despite the fact, that the number of blaCTX-M and qnr genes among E. coli from the BfT-GermVet study was low, the results of this study showed that the integration of insertion sequences and interplasmid recombination events accounted for the structural variability of the blaCTX-M gene regions and the formation of the genetic environment of qnrB19 on pQNR2078. Furthermore, IncN plasmids, which carry blaCTX-M-1 or qnrB19 genes, might be involved in the dissemination of these resistance genes among Enterobacteriaceae of animal and human origin.