A. van Essen-Zandbergen

Occurrence and characteristics of extended-spectrum-β-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses

OBJECTIVES To investigate the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. METHODS Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤ 25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. RESULTS Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were bla(CTX-M-1, -2, -9, -14, -15,) bla(TEM-52), bla(CMY-2) and bla(CMY-)(39). bla(CTX-M-1) was predominant (n = 17). bla(CTX-M-9) occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying bla(CTX-M-1) belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. CONCLUSIONS This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs.

Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry.

To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006 showing this phenotype were characterized by miniaturized micro-array, PCR and DNA-sequencing. Presence and size of plasmids were determined by S1-digest pulsed-field gel electrophoresis and further characterized by PCR-based replicon typing. Transfer of resistance plasmids was tested by conjugation and transformation experiments. To link resistance genes and plasmid type, Southern blot hybridization experiments were conducted. In 42 isolates, five (bla(CTX-M-1), bla(CTX-M-2), bla(TEM-20), bla(TEM-52), bla(SHV-2)) different extended spectrum beta-lactamase (ESBL)-genes and two (bla(ACC-1), bla(CMY-2)) AmpC-genes were present. Three of the detected ESBL-genes (bla(CTX-M-1), bla(TEM-52) and bla(CTX-M-2)) were located on similar types of plasmids (IncI1 and IncHI2/P) in both E. coli and Salmonella. Two other detected ESBL- and AmpC-genes bla(SHV-2) and bla(CMY-2) respectively (on IncK plasmids), were only found in E. coli, whereas the AmpC-gene bla(ACC-1) (on non-typable plasmids), and the ESBL-gene bla(TEM-20) (on IncI1 plasmids), were only detected in Salmonella. In two isolates, no ESBL- or AmpC-gene could be detected through these methods. The increase in the number of E. coli and S. enterica isolates from the gastro-intestinal tract of broilers exhibiting non-wild type MICs for cefotaxime is mainly due to an increase in IncI1 plasmids containing bla(CTX-M-1). The reason for the successful spread of this plasmid type in these species is not yet understood.

Extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers

OBJECTIVES The aim of this study was to establish the prevalence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase-producing Escherichia coli at Dutch broiler farms and in farmers and to compare ESBL/AmpC-producing isolates from farmers and their animals. METHODS Twenty-five to 41 cloacal swabs collected from broilers at each of 26 farms and 18 faecal samples from 18 broiler farmers were analysed for determination of the presence of ESBL/AmpC-producing E. coli. ESBL/AmpC genes were characterized by microarray, PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing or restriction fragment length polymorphism. E. coli genotypes were determined by multilocus sequence typing. RESULTS Birds from all farms were positive for ESBL/AmpC-producing E. coli, and on 22/26 farms the within-farm prevalence was ≥ 80%. Six of 18 farmers carried isolates containing ESBL/AmpC genes bla(CTX-M-1), bla(CMY-2) and/or bla(SHV-12), which were also present in the samples from their animals. In five of these isolates, the genes were located on identical plasmid families [IncI1 (n = 3), IncK (n = 1) or IncN (n = 1)], and in isolates from two farmers the genes were carried on identical plasmid subtypes (IncI1 ST12 and IncN ST1, where ST stands for sequence type) as in the isolates from their animals. CONCLUSIONS This study shows a high prevalence of birds carrying ESBL/AmpC-producing E. coli at Dutch broiler farms and a high prevalence of ESBL/AmpC-producing E. coli in farmers. This is undesirable due to the risk this poses to human health. Future research should focus on identification of the source of these isolates in the broiler production chain to make interventions resulting in reduction of these isolates possible.

Extended-spectrum and AmpC β-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: prevalence, risk factors and molecular characteristics

OBJECTIVES The objectives of this study were to: estimate the prevalence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.

Increasing prevalence and diversity of ESBL/AmpC-type β-lactamase genes in Escherichia coli isolated from veal calves from 1997 to 2010

OBJECTIVES Several studies on faecal carriage of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli have been performed in cattle, but little is known about faecal carriage in veal calves. This study describes the prevalence and molecular characteristics of ESBL/AmpC genes in E. coli isolated from faecal samples of veal calves from 1997 to 2010. METHODS Pooled faecal samples were inoculated using selective enrichment broth and subsequently selective MacConkey agar. All isolates with reduced susceptibility to cefotaxime were screened by PCR and sequencing analysis for the presence of ESBL/AmpC genes. RESULTS The prevalence of E. coli with reduced susceptibility to cefotaxime showed a discontinuous increasing trend, ranging from 4% in 1998 and 1999 to 39% in 2010. Promoter mutations of the chromosomal ampC gene were present in all years. In 2000, ESBL genes blaCTX-M-1, blaTEM-52 and blaTEM-20 were first observed. Before 2005 the majority of E. coli with reduced susceptibility to cefotaxime harboured ampC promoter mutations. From 2005 onwards the majority harboured blaCTX-M genes, of which blaCTX-M-1 was the most abundant, followed by blaCTX-M-14 and blaCTX-M-15. The diversity of blaCTX-M genes gradually increased from one variant in 2000 to six variants in 2010. The prevalence of blaTEM-52 was relatively low, but it was detected from 2000 onwards. blaCMY and blaSHV were found sporadically. CONCLUSIONS The prevalence and molecular diversity of genes encoding cefotaxime resistance in E. coli isolated from veal calves over a 14 year period showed an increasing trend. From 2005 onwards, blaCTX-M genes were most abundant, especially blaCTX-M-1.

Transmission of methicillin-resistant Staphylococcus aureus isolates on broiler farms

The aim of the present study was to investigate the resistance pheno- and genotypes and the molecular typing characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from broiler farms in order to explore transmission between the different reservoirs. Thirty-seven MRSA CC398 isolates (11 from broilers, 15 from the broiler houses, 5 from farm residences and 6 from humans living and/or working on the farms) cultured from samples at four different farms during a previous study, were included. In addition to the previously determined spa types, the isolates were characterized by dru typing, SCCmec typing, pulsed-field gel electrophoresis and DNA microarray. Resistance phenotypes were determined by broth microdilution. Resistance genes were detected by DNA microarray or specific PCR assays. Selected isolates from broilers and humans (n=7) were analysed by whole genome mapping. On the same farm, isolates from chickens, broiler houses, the farm residences and humans were often closely related or indistinguishable. On three of the four farms, however, MRSA isolates with different characteristics were present. On the one hand, the apparent similarity of MRSA isolates from the same farm indicates transmission between broilers, humans and their environment. On the other hand, different MRSA isolates were present on the same farm, indicating introduction from different sources or diversification over time. This study shows that different typing methods should be used to investigate epidemiological links between isolates and that whole genome mapping can be a useful tool to establish these links.

Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves.

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC ≥ 0.125 mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.

Complete Genome Sequences of IncI1 Plasmids Carrying Extended-Spectrum β-Lactamase Genes

ABSTRACT Extended spectrum beta-lactamases (ESBLs) confer resistance to clinically relevant antibiotics. Often, the resistance genes are carried by conjugative plasmids which are responsible for dissemination. Five IncI1 plasmids carrying ESBLs from commensal and clinical Escherichia coli isolates were completely sequenced and annotated along with a non-ESBL carrying IncI1 plasmid.

Analysis of resistance pheno- and genotypes in methicillin-resistant Staphylococcus aureus (MRSA) CC398 isolates from broiler farms

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.