Antimicrobial Resistance Mechanisms in Canine Multidrug-Resistant Escherichia coli and Enterobacter spp.

Sidjabat, Hanna (2007). Antimicrobial Resistance Mechanisms in Canine Multidrug-Resistant Escherichia coli and Enterobacter spp. PhD Thesis, School of Veterinary Science, University of Queensland.

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Author Sidjabat, Hanna
Thesis Title Antimicrobial Resistance Mechanisms in Canine Multidrug-Resistant Escherichia coli and Enterobacter spp.
School, Centre or Institute School of Veterinary Science
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Abstract/Summary Multidrug-resistant (MDR) Enterobacteriaceae exhibiting resistance to expanded-spectrum cephalosporins have recently emerged as a cause of opportunistic infections in hospitalised dogs. At the University of Queensland Veterinary Teaching Hospital (UQVTH) and other referral veterinary hospitals in Brisbane Australia, MDR E. coli (MDREC) and MDR Enterobacter spp were the most commonly isolated strains. Plasmid-mediated resistance genes and clonality were determined for 11 MDREC and 10 MDR Enterobacter spp. isolates associated with opportunistic infections in hospitalised dogs. Phenotypic (broth microdilution and disc susceptibility testing, modified disc diffusion testing and IEF for ~Iactamases) and genotypic tests (pCR and sequencing, plasmid analysis and Southern blot hybridisation, bacterial conjugation and transformation) were used to identify and localise specific antimicrobial resistance genes to plasmids. The clonality of the isolates was investigated by PFGE and plasmid analysis. MDREC isolates could be divided into two clonal groups (CG 1 and 2) with distinct PFGE and plasmid profiles. In all MDREC stmins, resistance to expanded-spectrum cephalosporins was imparted by an AmpC ~Iactamase gene, blacMY_7 which was located on a nonconjugative ~93 kb plasmid that did not contain any other known resistance genes. There was a marked difference in the distribution of plasmid-mediated resistance genes between CG 1 and CG 2. The identified resistance genes located on ~170 kb plasmid in MOREC CG 1 strains were blaTEM, catAl and class I integron-associated genes (sull and dfrA17-aadA5). In MDREC CG 2 strains, a second -93 kb plasmid contained biaTEM and co-transferred resistance to sulfonamide/trimethoprim, tetracycline and streptomycin. This plasmid also contained a class I integron, but the gene cassette could not be identified, except for the presence of dfrV in a single isolate. Canine MDR Enterobacter spp. clinical isolates were genotypically diverse based on PFGE; and belonged to either Enterobacter cloacae or Enterobacter hormaechei based on 16S rRNA gene sequence analysis. Of the ten canine Enterobacter spp., nine isolates carried plasmid-mediated blasHv.12 on -140 kb plasmid and the remaining one isolate possessed blacMY_2 on a ~93 kb plasmid. The blasHV_12-carrying plasmid also co-transferred resistance to chloramphenicol (cauV), streptomycin/spectinomycin (aadA2), gentamicin, tetracycline, sulfonamide/trimethoprim; whereas, the blacMY.2-carrying plasmid did not contain any known resistance genes to other antimicrobials. Both blaSHV.12 and blaCM'{.2 carrying plasmids were both readily transmissible. This is the first report of plasmid-mediated blaCM'{.7 in MDREC and blaSHV'12 and blaCM¥.2 in Enterobacter spp. isolated from animals. The fact that the blaSHV.12 carrying plasmid can transfer readily and could be co-selected by several traditional antimicrobials provides an exceptional threat of rapid spread of this plasmid between bacteria causing extraintestinal infections in both humans and animals. In contrast, the blaCM'{'2 or blaCM'{.7 carrying plasmids will only be spread when there is selection pressure created by use of expandedspectrum cephalosporins or broad spectrum ~-Iactam/clavulanic acid combinations. A I-year infection control study was conducted to limit further occurrence of extraintestinal infections and determine the molecular epidemiology of MDREC, and, to a lesser extent, MDR Enterobacter spp. emergence and spread within UQVTH. Selective media were used to obtain MDR coliform isolates from the hospital environment and rectal swabs of hospitalised dogs and hospital employees. Furthermore, a multiplex PCR was designed to identify the MDR coliform isolates as E. coli, and to determine their clonality. A subset of representative isolates was characterised by PFGE, plasmid analysis and serotyping. Similar to the MDREC clinical cases, only two COs were identified. The clinical infections were therefore likely to be endogenous and influenced by prolonged hospitalisation periods and concurrent antimicrobial therapy. Direct, dog-to-dog, faecal-oral transmission was likely to have occurred as well as exposure to a contaminated environment for MDREC CG I and Enterobacter spp. strains. More importantly, the isolation of MDREC CG 2 from two hospital employees showed the possibility ofMDREC transfer between dogs and humans via the faecal-oral route. The clinical cases caused by MDREC and MDREC spread within hospitalised dogs and the hospital environment were therefore resultant from clonal dissemination oftwo distinct clonal lineages. A human clinical MDREC strain isolated from a Brisbane patient with a urmary tract infection Was shown to be genotypically related to MDREC CG I and possessed the same blaCMY.7 plasmid, confirming that dog-to-human, or indeed, human-to-dog MDREC transmission, can also be hypothesised for MDREC CG 1 strains. The level of blaCMY.7 RNA expression and plasmid copy number were determined for representative canine and human MDREC CG 1 and CG 2 strains. It was compared with relative expression in Salmonella enterica serotype Typhimurium (S. typhimurium) strain 100 and laboratory E. coli strains containing the native blaCMY.7 plasmid or the cloned blaCM'{.7 gene plus the transcriptional start site. The constitutive expression level per gene copy of blaCM'{.7 in canine MDREC CG 1 (-1 copy per cell) and CG 2 (-2 copies per cell) Was 100-fold and ISO-fold greater than chromosomal ampC expression in the C. freundii wild-type, respectively. The constitutive expression per copy number of cloned blaCMY.7 insert (-15 copies) was IS-fold lower than corresponding expression in both the E. coli laboratory strain containing the blacMY.7 native plasmid (-2 copies) and the original clinical donor strain. The constitutive expression data correlated with the expanded-spectrum cephalosporin MICs and high-level AmpC expression did not compromise the growth rate of E. coli. Typical ExPEC virulence genes were identified in the three human, 25 canme and 6 environmental MDREC isolates. Regardless of sample source, ExPEC VFs were distributed according to clonality and phylotype. MDREC CG 1 strains belonged to phylogenetic group A (typical commensal E. coli phylotype) and possessed only iutA and traT; whereas MDREC CG 2 were all phylogenetic group D (typical ExPEC phylotype) and possessed iutA, ibeA, fimH, and kpsAfT K5, which are important virulence genes in extraintestinal infections. The fact that MDREC CG 2 possessed type 1 :fimbriae and flagella in comparison to CG 1 isolates may contribute to colonisation ability and virulence, both in the gastrointestinal tract and extraintestinal sites, such as bladder. This is confirmed in three different mouse models (gastrointestinal colonisation, extraintestinal virulence and urinary tract infection) in which CG 1 and CG 2 index strains were compared. Both CGs of MDREC strains isolated from extraintestinal infections in dogs were not only important sources of plasmid-mediated antimicrobial resistance genes, but strong evidence was obtained supporting their ability to colonise and cause extraintestinal infection in humans. Dogs therefore represent an important reservoir of MDR coliforms that cause opportunistic infections and the transfer of strains and/or their plasmid-mediated resistance genes, particularly AmpC j3-lactamase and ESBL genes commonly occurs between humans and dogs. This finding has important public health implications and must be considered in treatment and prevention strategies in human and veterinary settings.

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Created: Fri, 21 Nov 2008, 16:26:18 EST