Anne-Kathrin Schink

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla CTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla CTX-M-group-1 and bla OXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, bla OXA-1-like and bla CTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Analysis of extended-spectrum-β-lactamase-producing Escherichia coli isolates collected in the GERM-Vet monitoring programme

OBJECTIVES The aims of this study were (i) to detect extended-spectrum β-lactamase (ESBL) genes among 1378 Escherichia coli isolates from defined disease conditions of companion and farm animals and (ii) to determine the localization and organization of ESBL genes. METHODS E. coli isolates from the German resistance monitoring programme GERM-Vet were included in the study. Plasmids were transferred by conjugation or transformation and typed by PCR-based replicon typing. ESBL genes were detected by PCR; the complete ESBL genes and their flanking regions were sequenced by primer walking. Phylogenetic grouping and multilocus sequence typing (MLST) were performed for all ESBL-producing E. coli isolates. RESULTS Of the 27 ESBL-producing E. coli isolates detected, 22 carried blaCTX-M-1 genes on IncN (n = 16), IncF (n = 3), IncI1 (n = 2) or multireplicon (n = 1) plasmids. A blaCTX-M-3 gene was located on an IncN plasmid and a blaCTX-M-15 gene was located on an IncF plasmid. A multireplicon plasmid and an IncHI1 plasmid harboured blaCTX-M-2. A blaTEM-52c gene was identified within Tn2 on an IncI1 plasmid. The blaCTX-M genes located within the same or related genetic contexts showed differences due to the integration of insertion sequences. Various MLST types were detected, with ST10 (n = 7), ST167 (n = 4) and ST100 (n = 3) being the most common. CONCLUSIONS This study showed that the blaCTX-M-1 gene is the predominant ESBL gene among E. coli isolates from diseased animals in Germany and a considerable structural heterogeneity was found in the regions flanking the blaCTX-M-1 gene. Insertion sequences, transposons and recombination events are likely to be involved in alterations of the ESBL gene regions.

Analysis of blaCTX-M-Carrying Plasmids from Escherichia coli Isolates Collected in the BfT-GermVet Study

ABSTRACT In this study, 417 Escherichia coli isolates from defined disease conditions of companion and farm animals collected in the BfT-GermVet study were investigated for the presence of extended-spectrum β-lactamase (ESBL) genes. Three ESBL-producing E. coli isolates were identified among the 100 ampicillin-resistant isolates. The E. coli isolates 168 and 246, of canine and porcine origins, respectively, harbored bla CTX-M-1, and the canine isolate 913 harbored bla CTX-M-15, as confirmed by PCR and sequence analysis. The isolates 168 and 246 belonged to the novel multilocus sequence typing (MLST) types ST1576 and ST1153, respectively, while isolate 913 had the MLST type ST410. The ESBL genes were located on structurally related IncN plasmids in isolates 168 and 246 and on an IncF plasmid in isolate 913. The bla CTX-M-1 upstream regions of plasmids pCTX168 and pCTX246 were similar, whereas the downstream regions showed structural differences. The genetic environment of the bla CTX-M-15 gene on plasmid pCTX913 differed distinctly from that of both bla CTX-M-1 genes. Detailed sequence analysis showed that the integration of insertion sequences, as well as interplasmid recombination events, accounted for the structural variability in the bla CTX-M gene regions.

Susceptibility of canine and feline bacterial pathogens to pradofloxacin and comparison with other fluoroquinolones approved for companion animals

In this study, 908 bacterial pathogens from defined infections of dogs and cats were tested for their susceptibility to the novel fluoroquinolone pradofloxacin, which was approved in 2011 for use in cats and dogs. Most of the bacteria tested (Staphylococcus aureus, Staphylococcus pseudintermedius, Escherichia coli, β-haemolytic streptococci, Pasteurella multocida and Bordetella bronchiseptica) exhibited low pradofloxacin MIC(90) values of ≤ 0.25 μg/ml. Solely Proteus spp. and Pseudomonas aeruginosa had higher MIC(90) values of ≥ 4 μg/ml. Only six (3.4%) of 177 S. pseudintermedius and 12 (5.3%) of 227 E. coli isolates showed pradofloxacin MICs of ≥ 2 μg/ml. Analysis of the quinolone resistance determining regions of the target genes identified double mutations in GyrA that resulted in amino acid exchanges S83L+D87N or S83L+D87Y and single or double mutations in ParC that resulted in amino acid exchanges S80I or S80I+E84G in all 12 E. coli isolates. The six S. pseudintermedius isolates exhibited amino acid exchanges S84L or E88K in GyrA and S80I in GrlA. Comparative analysis of the MICs of pradofloxacin and the MICs determined for enrofloxacin and its main metabolite ciprofloxacin, but also marbofloxacin, orbifloxacin, difloxacin and ibafloxacin was conducted for the target pathogens S. pseudintermedius, E. coli and P. multocida. This comparison confirmed that pradofloxacin MICs were significantly lower than those of the other tested fluoroquinolones.

Detection of qnr genes among Escherichia coli isolates of animal origin and complete sequence of the conjugative qnrB19-carrying plasmid pQNR2078

OBJECTIVES The aims of this study were to identify qnr genes among quinolone-resistant Escherichia coli isolates from defined disease conditions of companion and farm animals obtained in the BfT-GermVet study, and to gain insight into their localization and the organization of the qnr gene regions. METHODS The qnr genes were detected by PCR and confirmed by sequencing. qnr-positive isolates were checked for mutations in DNA gyrase and topoisomerase IV genes by PCR and sequencing of the quinolone resistance-determining regions. Multilocus sequence typing (MLST) was performed for the qnr-positive E. coli isolates. Plasmids harbouring qnr genes were transferred by conjugation into E. coli recipients, subjected to PCR-based replicon typing and plasmid-MLST, and one qnrB19-carrying plasmid was sequenced completely. RESULTS Only 2 of 417 E. coli isolates investigated carried qnr genes. Both isolates originated from horses and showed MLST type ST86. They harboured conjugative qnrB19-carrying plasmids, which proved to be indistinguishable by restriction analysis, belonged to incompatibility group IncN, showed plasmid-MLST type ST8 and did not carry other resistance genes. The qnrB19 gene was flanked by copies of the insertion sequence IS26. One of these plasmids, pQNR2078, was sequenced completely and had a size of 42 379 bp. Except for the resistance gene region, plasmid pQNR2078 closely resembled the bla(CTX-M-65)-carrying plasmid pKC396 from E. coli. CONCLUSIONS qnr genes were rarely detected among E. coli from animals in the BfT-GermVet study. The qnrB19 gene was detected on conjugative plasmids, with IS26 being likely involved in the mobility of qnrB19.

Antimicrobial Resistance in Escherichia coli

Multidrug resistance in Escherichia coli has become a worrying issue that is increasingly observed in human but also in veterinary medicine worldwide. E. coli is intrinsically susceptible to almost all clinically relevant antimicrobial agents, but this bacterial species has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer. The most problematic mechanisms in E. coli correspond to the acquisition of genes coding for extended-spectrum β-lactamases (conferring resistance to broad-spectrum cephalosporins), carbapenemases (conferring resistance to carbapenems), 16S rRNA methylases (conferring pan-resistance to aminoglycosides), plasmid-mediated quinolone resistance (PMQR) genes (conferring resistance to [fluoro]quinolones), and mcr genes (conferring resistance to polymyxins). Although the spread of carbapenemase genes has been mainly recognized in the human sector but poorly recognized in animals, colistin resistance in E. coli seems rather to be related to the use of colistin in veterinary medicine on a global scale. For the other resistance traits, their cross-transfer between the human and animal sectors still remains controversial even though genomic investigations indicate that extended-spectrum β-lactamase producers encountered in animals are distinct from those affecting humans. In addition, E. coli of animal origin often also show resistances to other-mostly older-antimicrobial agents, including tetracyclines, phenicols, sulfonamides, trimethoprim, and fosfomycin. Plasmids, especially multiresistance plasmids, but also other mobile genetic elements, such as transposons and gene cassettes in class 1 and class 2 integrons, seem to play a major role in the dissemination of resistance genes. Of note, coselection and persistence of resistances to critically important antimicrobial agents in human medicine also occurs through the massive use of antimicrobial agents in veterinary medicine, such as tetracyclines or sulfonamides, as long as all those determinants are located on the same genetic elements.

Mechanisms of bacterial resistance to antimicrobial agents.

During the past decades resistance to virtually all antimicrobial agents has been observed in bacteria of animal origin. This chapter describes in detail the mechanisms so far encountered for the various classes of antimicrobial agents. The main mechanisms include enzymatic inactivation by either disintegration or chemical modification of antimicrobial agents, reduced intracellular accumulation by either decreased influx or increased efflux of antimicrobial agents, and modifications at the cellular target sites (i.e., mutational changes, chemical modification, protection, or even replacement of the target sites). Often several mechanisms interact to enhance bacterial resistance to antimicrobial agents. This is a completely revised version of the corresponding chapter in the book Antimicrobial Resistance in Bacteria of Animal Origin published in 2006. New sections have been added for oxazolidinones, polypeptides, mupirocin, ansamycins, fosfomycin, fusidic acid, and streptomycins, and the chapters for the remaining classes of antimicrobial agents have been completely updated to cover the advances in knowledge gained since 2006.

Detection of extended-spectrum β-lactamase (ESBL)-carrying plasmids in Escherichia coli isolates collected in the GERM-Vet program

Introduction: The association between the major gastric pathogen Helicobacter pylori and humans dates back at least 100,000 years. Since that time H. pylori evolved in parallel with its human host, accompanying anatomically modern humans ‘out of Africa’ and differentiating into seven known biogeographic populations. Three of these are indigenous to Africa: hpNEAfrica, hpAfrica1, and hpAfrica2. The most divergent H. pylori population, hpAfrica2, is associated with the ancient San hunter-gatherers of southern Africa. This striking parallel between the oldest human and bacterial lineages led us to investigate the prevalence and genetic structure of H. pylori in another ancient hunter-gatherer population, the Cameroonian Baka Pygmies. Methods: Gastric biopsies were obtained for a total of 178 individuals, 77 of them belonging to the Baka Pygmy population and the remainder to neighboring agricultural Bantu communities. H. pylori was cultured from both antrum and corpus biopsies from each individual. The isolates were analyzed using MLST (Multilocus sequence typing). The population structure of distinct haplotypes was determined by Bayesian clustering using the program STRUCTURE. Phylogenetic analyses were perfomed using ClonalFrame. Results: The H. pylori prevalence among Baka pygmies (20%) was significantly lower compared to the non-Baka individuals (80%). MLST analysis of all isolates identified 113 unique haplotypes, ten of which were shared by more than one person. Subsequent STRUCTURE analyses assigned these isolates to either hpNEAfrica (67), hpAfrica1 (44), or hpEurope (2) populations. Among infected Baka and non-Baka individuals, the infection rates with hpNEAfrica and hpAfrica1 isolates were similar. In phylogenetic analyses using ClonalFrame we observed an intermediate position of the Cameroonian hpAfrica1 isolates between the already described hspSAfrica and hspWAfrica subpopulations corresponding to the geographic location of Cameroon between South and West Africa. Further STRUCTURE analyses of the hpNEAfrica population revealed a possible subdivision into an East African and a Central African subpopulation. Discussion: The results suggest that the Baka pygmies, which belong to one of the oldest branches of the human population tree and still live as hunter-gatherers, were initially H. pylori free and acquired H. pylori more recently from their neighboring Bantu communities.