Stefan S. Olsen

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins

OBJECTIVES To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. METHODS Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. RESULTS ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. CONCLUSIONS The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers.

Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark.

During 2001–2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans.

Emergence of ampicillin-resistant Enterococcus faecium in Danish hospitals

BACKGROUND Ampicillin-resistant Enterococcus faecium isolates are reported in increasing numbers in many European hospitals. The clonal complex 17 (CC17) characterized by ampicillin resistance has been associated with nosocomial E. faecium outbreaks and infections in five continents. The aim was to investigate how prevalent ampicillin resistance is in clinical E. faecium isolates from Denmark and to investigate their clonal affiliation, especially to CC17. METHODS Microbiology data from 2002 through 2006 on E. faecium and Enterococcus faecalis blood isolates was received from Departments of Clinical Microbiology in 11 Danish counties. From January 2004 through December 2004, we collected 275 clinical enterococci from four of these departments. Multilocus sequence typing (MLST) and PFGE were performed on the 84 ampicillin-resistant E. faecium isolates from this collection. RESULTS A 68% increase in the number of infections caused by enterococci was observed from 2002 through 2006. The increase was mainly caused by E. faecium isolates, which tripled, whereas the number of E. faecalis isolates increased by only 23% during the same period. There was also a significant increase in the number of ampicillin-resistant E. faecium isolates. MLST showed that 98% of the tested ampicillin-resistant E. faecium isolates belonged to CC17. PFGE showed eight different clusters and we found indications of clonal spread within the hospitals. CONCLUSIONS Ampicillin-resistant E. faecium isolates have increased in frequency in Denmark during 2002-2006. Most of the ampicillin-resistant E. faecium isolates belong to complex CC17.

Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat

Recently, it has been suggested that the Escherichia coli causing urinary tract infection (UTI) may come from meat and animals. The purpose was to investigate if a clonal link existed between E. coli from animals, meat and UTI patients. Twenty-two geographically and temporally matched B2 E. coli from UTI patients, community-dwelling humans, broiler chicken meat, pork, and broiler chicken, previously identified to exhibit eight virulence genotypes by microarray-detection of approximately 300 genes, were investigated for clonal relatedness by PFGE. Nine isolates were selected and tested for in vivo virulence in the mouse model of ascending UTI. UTI and community-dwelling human strains were closely clonally related to meat strains. Several human derived strains were also clonally interrelated. All nine isolates regardless of origin were virulent in the UTI model with positive urine, bladder and kidney cultures. Further, isolates with the same gene profile also yielded similar bacterial counts in urine, bladder and kidneys. This study showed a clonal link between E. coli from meat and humans, providing solid evidence that UTI is zoonosis. The close relationship between community-dwelling human and UTI isolates may indicate a point source spread, e.g. through contaminated meat.

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012–13, investigated by WGS, MLST and PFGE

OBJECTIVES In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm. METHODS A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE. RESULTS One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. CONCLUSIONS This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Trends in occurrence of antimicrobial resistance in Campylobacter jejuni isolates from broiler chickens, broiler chicken meat, and human domestically acquired cases and travel associated cases in Denmark.

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis. Often it causes self-limiting disease but severe or prolonged cases may require antimicrobial treatment. The agricultural use of antimicrobial agents selects for resistance among C. jejuni which is transmitted to humans via food. In Denmark, the use of fluoroquinolones in animal husbandry has been restricted since 2003. The purpose of the present study was to look at trends in occurrence of resistance among C. jejuni from broiler chickens, broiler chicken meat and human domestically acquired or travel associated cases. From 1997 through 2007, C. jejuni isolates were obtained from The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) and susceptibility tested for ciprofloxacin, erythromycin, nalidixic acid, and tetracycline. Erythromycin resistance was at a low level in all the reservoirs during the study period. Resistance to ciprofloxacin, nalidixic acid and tetracycline was significantly higher in C. jejuni from imported broiler chicken meat compared to Danish broiler chicken meat. In domestically acquired human C. jejuni isolates, resistance to ciprofloxacin and nalidixic acid was for most years significantly higher compared to the level found in isolates from Danish broiler chicken meat, whereas the resistance level was similar to the level found in isolates from imported broiler chicken meat. Imported broiler chicken meat may therefore contribute to the high level of ciprofloxacin and nalidixic acid resistance in C. jejuni isolates from domestically acquired human infections. In 2006 and 2007, the occurrence of resistance to ciprofloxacin, nalidixic acid and tetracycline was significantly higher in travel associated C. jejuni isolates compared to isolates acquired domestically. Even though the use of fluoroquinolones is restricted for animal use in Denmark, Danes are still often infected by fluoroquinolone resistant C. jejuni from imported chicken meat or by travelling.

Characterization of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

The aim of the study was to investigate the molecular epidemiology of 87 third-generation cephalosporin-resistant Escherichia coli (3GC-R Ec) from bloodstream infections in Denmark from 2009. Sixty-eight of the 87 isolates were extended-spectrum beta-lactamase (ESBL) producers, whereas 17 isolates featured AmpC mutations only (without a coexpressed ESBL enzyme) and 2 isolates were producing CMY-22. The majority (82%) of the ESBL-producing isolates in our study were CTX-M-15 producers and primarily belonged to phylogroup B2 (54.4%) or D (23.5%). Further, one of the two CMY-22-producing isolates belonged to B2, whereas only few of the other AmpCs isolates belonged to B2 and D. Pulsed-field gel electrophoresis revealed that both clonal and nonclonal spread of 3GC-R Ec occurred. ST131 was detected in 50% of ESBL-producing isolates. The remaining ESBL-producing isolates belonged to 17 other sequence types (STs), including several other internationally spreading STs (e.g., ST10, ST69, and ST405). The majority (93%) of the ESBL-producing isolates and one of the CMY-22-producing isolates were multiresistant. In conclusion, 3GC-R in bacteriaemic E. coli in Denmark was mostly due to ESBL production, overexpression of AmpC, and to a lesser extent to plasmid-mediated AmpC. The worldwide disseminated CTX-M-15-ST131 was strongly represented in this collection of Danish, bacteriaemic E. coli isolates.

Concurrent emergence of multidrug resistance and heat resistance by CTX-M-15-encoding conjugative plasmids in Klebsiella pneumoniae.

A plasmid‐encoded ClpK protein was recently identified as a predictor of a heat‐resistant phenotype in the opportunistic pathogen Klebsiella pneumoniae. This study was undertaken to evaluate the presence of the clpK gene in extended‐spectrum β‐lactamase (ESBL)‐producing K. pneumoniae and to assess the probable co‐transfer of multi‐resistance with the heat resistance phenotype. A Danish collection of 80 ESBL‐producing K. pneumoniae bloodstream infection isolates was screened for clpK by colony hybridization. Nineteen isolates (24%) were positive for clpK; some of them representing major clones identified in Denmark. Among these, nine isolates belonged to a single K. pneumoniae CTX‐M‐15 clone with sequence type (ST)16 exhibiting a heat‐resistant phenotype. This clone has a multi‐hospital occurrence and has also been detected outside Denmark. Horizontal co‐transfer of multiple antibiotic resistances, including the CTX‐M‐15 resistance determinant, and the heat resistance phenotype was observed. Thus, the clpK gene is harbored by different ESBL‐producing K. pneumoniae isolates including a clone of ST16 internationally spread. The co‐localization of clpK on transferable ESBL‐encoding plasmids allowing co‐dissemination of multiple drug resistance with bacterial heat resistance is a highly interesting phenomenon that may further complicate the prevention of spreading of certain successful clones of multi‐resistant K. pneumoniae.

Characterization of CTX-M-14- and CTX-M-15-producing Escherichia coli of porcine origin

Sir, The emergence of extended-spectrum b-lactamase (ESBL)producing Escherichia coli among human patients is largely due to the spread of CTX-M, especially the clonal spread of sequence type (ST) 131 E. coli producing CTX-M-15. In recent years ESBLproducing E. coli have also been detected in food and animals and ST131 has been detected in E. coli of animal origin. To our knowledge, E. coli ST131 producing CTX-M-15 has been detected in companion animals, but not in other animals or in meat. Major questions include whether animals can be a reservoir of the STs that have been associated with ESBL-producing E. coli causing infections in humans, and whether animals and meat can be a reservoir of ESBL genes that can be transferred to some of the pathogenic E. coli STs (e.g. ST131) in the human intestine. Recently Agerso et al. reported on the prevalence of cephalosporinase-producing E. coli from Danish pig (n1⁄4786), Danish pork (n1⁄4153) and imported pork (n1⁄4173) samples taken in 2009. CTX-M-1-producing E. coli were most common among the ESBL-producing E. coli from pigs and pork, whereas a few isolates were producers of CTX-M-14 and CTX-M-15; whether these types are established in pigs is unknown. In the present study, one CTX-M-14-producing E. coli isolate from imported pork, six CTX-M-14-producing E. coli from Danish pigs and two CTX-M-15-producing E. coli from Danish pigs originating from the cephalosporinase prevalence study mentioned above were characterized for PFGE profile, multilocus sequence typing (MLST) ST, resistance profile, phylotype, clonal group A status, virulence genes and transfer of blaCTX-M genes (Table 1). PFGE with XbaI was performed on the nine E. coli isolates. MLST was performed using seven conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) (http://mlst. ucc.ie/mlst/dbs/Ecoli). The MICs of chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline and trimethoprim for the isolates were determined (Trek Diagnostic, East Grinstead, UK). Results were interpreted according to the EUCAST (www.EUCAST.org) clinical breakpoint except for results for nalidixic acid, tetracycline, streptomycin and sulfamethoxazole, which were interpreted according to CLSI. E. coli ATCC 25922 was used for quality control. The phylogenetic background (A, B1, B2 or D) of the isolates was determined by PCR. Results obtained allowed classification of the isolates into the four major E. coli phylogenetic lineages or non-typeable isolates. Isolates classified as phylogroup D were tested for the presence of single-nucleotide polymorphisms in fumB and gyrB, which has been associated with a clonal group of extraintestinal pathogenic E. coli (ExPEC), designated clonal group A. Furthermore, the isolates were investigated for the ExPEC-related virulence genes kpsM II, papA, papC, iutA, sfaS, focG, afa and dra. In vitro matings were performed with a rifampicin-resistant mutant of an ST131 B2 E. coli of Danish pork origin as recipient. Three selected transconjugants from each mating were examined for the presence of the transferred blaCTX-M gene by PCR. Furthermore, PFGE was performed on the three selected transconjugants, the donor and the recipient. The isolates had different PFGE types (≤80% identical) and were not clonally related to any of the 80 ESBL-producing E. coli blood isolates of human origin from 2009 obtained in our BioNumerics database (data not shown). The nine CTX-M-producing E. coli isolates in the present study had nine different STs, of which ST48, ST101, ST117 and ST167 had previously been detected in human patients (Table 1). – 8 Six of the nine isolates were resistant to other classes of antimicrobial agents beside the b-lactams (Table 1). Urinary tract infections are most often associated with phylogroup B2 and to some extent with phylogroup D. None of the nine isolates belonged to phylogroup B2 (Table 1). However, blaCTX-M-14and blaCTX-M-15-producing E. coli isolates from patients with infections belonging to phylogroup A, B1 and D have been detected. – 5 Two of the tested E. coli belonged to phylogroup D, but not to the clonal group A. A previous Danish study of phylogroup D E. coli of porcine origin likewise failed to identify any clonal group A isolates. Isolate 2700-1 (CTX-M-14) was positive for focG (F1C fimbriae) and iutA (aerobactin siderophore) and isolate 2469-1 (CTX-M-14) was positive for papA/papC (P fimbriae) and iutA, which defined both isolates as ExPEC isolates according to the molecular definition.

Limited similarity between plasmids encoding CTX-M-1 β-lactamase in Escherichia coli from humans, pigs, cattle, organic poultry layers and horses in Denmark

CTX-M-1 is a common extended-spectrum β-lactamase (ESBL) in Escherichia coli from animals and is often detected among human clinical isolates. The objective of this study was to investigate the epidemiological relationship between CTX-M-1-producing E. coli isolated from patients and animals in Denmark between 2006 and 2010. In total, 65 CTX-M-1-producing isolates from patients (n=22), pigs (n=21), cattle (n=4), organic poultry layers (n=3) and horses (n=15) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harbouring blaCTX-M-1 were characterised by S1 PFGE, PCR-based replicon typing, plasmid multilocus sequence typing, restriction fragment length polymorphism, and sequencing. Human and animal strains were unrelated based on PFGE. IncI1 was more common in human isolates (13/22) than in animal isolates (7/43), whereas the opposite trend was observed for IncN (5/22 human isolates and 24/43 animal isolates). Full characterisation of the plasmids harbouring blaCTX-M-1 revealed host-specific patterns in the distribution of plasmid types, with specific IncI1, IncN and IncH1 plasmid subtypes being predominant in humans, livestock and horses, respectively. Three indistinguishable human, bovine and porcine IncI1/ST49 plasmids had high nucleotide sequence homology and differed by the presence of IS66 elements in the bovine plasmid and the absence of one gene within the microcin-encoding operon in the human plasmid. In conclusion, this work suggests a minor contribution by animals to the occurrence of CTX-M-1 in human E. coli infections in Denmark during the study period.