Escherichia coli clonal group A causing bacteraemia of urinary tract origin.

Escherichia coli clonal group A (CgA) causes disease in humans. This is the first study investigating the prevalence of CgA among E. coli from non-urine, extraintestinal infections in a northern European country. E. coli blood (n = 196) and paired urine (n = 195) isolates from the same patients with bacteraemia of urinary tract origin were analysed. The isolates were collected from January 2003 through May 2005 at four hospitals in Copenhagen, Denmark. Pulsed-field gel electrophoresis (PFGE) patterns, antimicrobial resistance and patient characteristics were determined for all CgA isolates; presence of virulence-associated genes (VAGs) and serotypes were determined for the blood CgA isolates. Thirty blood isolates (15%) belonged to CgA. CgA blood isolates were associated with female patients and sulfamethoxazole-trimethoprim resistance and they harboured a distinctive VAG profile. The blood and urine isolates from each pair were found to be related in 26 of 27 CgA blood/urine pairs, confirming a urinary tract origin of infection. Furthermore, a relationship between the PFGE patterns of CgA blood/urine isolates and CgA isolates from UTI patients in general practice and a CgA isolate from a community-dwelling human reported previously, was found, suggesting a community origin of CgA. The finding of CgA strains in 15% of the E. coli bloodstream infections with a urinary tract origin in Denmark suggests that CgA constitutes an important clonal lineage among extraintestinal pathogenic E. coli. A reservoir of this pathogenic E. coli group in the community causing not only UTI but also more severe infections such as bacteraemia has implications for public health.

Enterococci of animal origin and their significance for public health.

Enterococci are commensal bacteria in the intestines of humans and animals, but also cause infections in humans. Most often, Enterococcus faecium isolates from clinical outbreaks belong to different types than E. faecium from animals, food, and humans in the community. The same variants of the vanA gene cluster (Tn1546) encoding vancomycin resistance can be detected in enterococci of both human and animal origin. This could indicate horizontal transfer of Tn1546 between enterococci of different origin. E. faecium isolates of animal origin might not constitute a human hazard in themselves, but they could act as donors of antimicrobial resistance genes for other pathogenic enterococci. Enterococcus faecalis of animal origin seems to be a human hazard, as the same types can be detected in E. faecalis from animals, meat, faecal samples from humans in the community, and patients with bloodstream infections.

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.

Faecal carriage of extended-spectrum ß-lactamase-producing and AmpC ß-lactamase-producing bacteria among Danish army recruits.

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum ß-lactamase (ESBL)-producing and AmpC ß-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla(CTX-M-14a) genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine.

Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite.

Two independent studies were conducted to describe symptoms and potential risk factors associated with Blastocystis infection. Isolates were subtyped by molecular analysis. In the NORMAT study (126 individuals randomly sampled from the general population) 24 (19%) were positive for Blastocystis. Blastocystis was associated with irritable bowel syndrome (P=0.04), contact with pigs (P<0.01) and poultry (P=0.03). In the Follow-up (FU) study (follow-up of 92 Blastocystis-positive patients), reports on bloating were associated with subtype (ST) 2 (P<0.01), and blood in stool to mixed subtype infection (P=0.06). ST1 was more common in FU individuals (32%) than in NORMAT individuals (8%), whereas single subtype infections due to ST3 or ST4 were seen in 63% of the NORMAT cases and 28% of the FU cases. Only FU individuals hosted ST7, and ST6/7 infections due to ST7 or ST9 were characterized by multiple intestinal symptoms. The data indicate subtype-dependent differences in the clinical significance of Blastocystis.

Emergence and spread of vancomycin resistance among enterococci in Europe.

Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

Gentamicin susceptibility in Escherichia coli related to the genetic background: problems with breakpoints.

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN(R)) genes aac(3)-II, aac(3)-IV or ant(2'')-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2'')-I had an MIC distribution of 8-64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN(R) mechanism. The MIC distribution, regardless of the GEN(R) mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (> or =16 mg/L).

Effect of pheromone induction on transfer of the Enterococcus faecalis plasmid pCF10 in intestinal mucus ex vivo.

The effect of synthetic sex pheromone on pheromone-inducible conjugation between the isogenic Enterococcus faecalis strains OG1RF and OG1SS was investigated in (i) Todd-Hewitt broth medium and (ii) intestinal mucus isolated from germ-free rats. In broth, the presence of synthetic pheromone cCF10 had no detectable effect on the transfer kinetics observed for the tetracycline resistance encoding plasmid pCF10. In mucus, presence of the same pheromone significantly increased the transfer efficiency observed during the first 2 h of conjugation, while the effect was less pronounced later in the experiment. We suggest that due to differences in diffusion rates and medium-binding of the pheromones, the effect of the synthetic cCF10 was immediately dominated by the effect of pheromones produced by the recipient E. faecalis strain in broth, while this happened later in mucus.

Indication of transposition of a mobile DNA element containing the vat(D) and erm(B) genes in Enterococcus faecium.

The vat(D) and erm(B) genes encoding streptogramin resistance in Enterococcus faecium transferred together, and a direct physical link between erm(B) and vat(D) was detected. Both the vat(D) and erm(B) probes hybridized to fragments of different sizes in the donor and transconjugants, which indicated a transposition event.

Associations between the use of antimicrobial agents for growth promotion and the occurrence of resistance among Enterococcus faecium from broilers and pigs in Denmark, Finland, and Norway.

This study compares the susceptibility of Enterococcus faecium isolated from pigs and poultry in Denmark, Finland, and Norway to antimicrobial agents used for growth promotion. E. faecium was isolated from 211 broilers and 55 pigs in Denmark in 1997, from Norwegian 55 poultry farms (turkey and broiler farms) and 4 swine farms between 1995 and 1997, and Finnish poultry (52) and swine (43) in 1996 and examined for susceptibility to avilamycin, avoparcin, bacitracin, flavomycin, monensin, salinomycin, spiramycin, tylosin, and virginiamycin. Only a limited number of isolates were categorized as resistant to monensin or salinomycin. In general, an association between the usage of antimicrobial agents in the respective countries and the occurrence of associated resistance was observed. Resistance to avilamycin was frequently observed among isolates from broilers in Denmark, where avilamycin has been used, whereas all isolates from Finland and Norway, where these drugs have not been used, were susceptible. The same phenomenon could be observed for avoparcin, bacitracin, tylosin, and virginiamycin; resistance was frequently observed among isolates from where these antimicrobials have been widely used, but rarely among isolates from where the use has been limited. Also for avoparcin and bacitracin, an association between usage and occurrence of resistance was observed. All isolates categorized as avoparcin resistant contained the vanX gene; isolates from broilers had the T variant in position 8,234 and isolates from pigs the G variant. Three (1%) of the 222 isolates resistant to tylosin contained the ermA gene and 196 (88%) ermB. Sixteen (11%) of the 146 virginiamycin-resistant isolates from broilers and two (7%) of the 27 virginiamycin-resistant isolates from pigs in Denmark contained the satA gene, whereas satA was not observed among any of the virginiamycin-resistant isolates from Finland. A total of 72% of the virginiamycin-resistant E. faecium from broilers in Denmark and all nine virginiamycin-resistant E. faecium from Finland contained satG. This gene was also observed among two (7%) of the virginiamycin-resistant isolates from pigs in Denmark. This study indicates that the use of antimicrobial agents for growth promotion in Denmark, Finland, and Norway have selected for resistance to most of these drugs among E. faecium in food animals.

Characterization of vancomycin-resistant and vancomycin-susceptible Enterococcus faecium isolates from humans, chickens and pigs by RiboPrinting and pulsed-field gel electrophoresis.

Forty-eight vancomycin-resistant and 35 vancomycin-sensitive Danish Enterococcus faecium isolates obtained from pigs, chickens and humans, as well as the human vanA reference isolate BM4147, were characterized by EcoRI RiboPrinting and SmaI pulsed-field gel electrophoresis. RiboPrinting of the 84 isolates yielded 40 types whereas PFGE-typing yielded 57 types discriminated by differences in more than three bands. By molecular typing, both clonal spread of E. faecium as well as horizontal transmission of Tn1546 between animals and humans was supported. Furthermore, it was found that the population of E. faecium spreads freely between the animal and human reservoir.

Use of antimicrobial growth promoters in food animals and Enterococcus faecium resistance to therapeutic antimicrobial drugs in Europe.

Supplementing animal feed with antimicrobial agents to enhance growth has been common practice for more than 30 years and is estimated to constitute more than half the total antimicrobial use worldwide. The potential public health consequences of this use have been debated; however, until recently, clear evidence of a health risk was not available. Accumulating evidence now indicates that the use of the glycopeptide avoparcin as a growth promoter has created in food animals a major reservoir of Enterococcus faecium, which contains the high level glycopeptide resistance determinant vanA, located on the Tn1546 transposon. Furthermore, glycopeptide-resistant strains, as well as resistance determinants, can be transmitted from animals to humans. Two antimicrobial classes expected to provide the future therapeutic options for treatment of infections with vancomycin-resistant enterococci have analogues among the growth promoters, and a huge animal reservoir of resistant E. faecium has already been created, posing a new public health problem.

Detection of the satA gene and transferability of virginiamycin resistance in Enterococcus faecium from food-animals.

The satA gene encoding streptogramin A resistance was detected in virginiamycin-resistant Enterococcus faecium isolates from pigs and broilers. The satA gene was present in 22 of 89 (25%) virginiamycin-resistant E. faecium isolates. It was shown that the satA gene and other gene(s) encoding streptogramin resistance could be transferred between isogenic E. faecium strains at frequencies ranging from 2.3 x 10(-4) to 2.2 x 10(-3) transconjugants per donor.

Molecular analysis of Tn1546 in Enterococcus faecium isolated from animals and humans.

The internal areas and the position of integration of the glycopeptide resistance element Tn1546 were characterized by using PCR fragment length polymorphism, sequencing, and DNA hybridization techniques with 38 high-level vancomycin-resistant Enterococcus faecium isolates of human and animal origins from Europe and the United States. Only minor variations in the coding regions within Tn1546 were found, suggesting high genetic stability. The isolates originated from broilers (n = 5), a chicken (n = 1), a duck (n = 1), a turkey (n = 1), pigs (n = 8), a pony (n = 1), and humans (n = 23). A total of 13 different types were defined based on a single-nucleotide difference in the vanX gene, the presence of insertion sequences, and hybridization patterns. For some types more than one isolate were found. For type 1, 10 isolates of both human and animal origins were found. All were indistinguishable from the reference strain, BM4147. For type 2, 11 isolates of human and animal origins were found. Six human isolates from England were all of type 3. Two human isolates from the United States, indistinguishable from each other, were type 9. These results showed that vancomycin-resistant E. faecium of animal and human origins can contain indistinguishable genetic elements coding for vancomycin resistance, indicating either horizontal gene transfer between E. faecium organisms of human and animal origins or the existence of a common reservoir for glycopeptide resistance.