Camilla H. Lester

In Vivo Transfer of the vanA Resistance Gene from an Enterococcus faecium Isolate of Animal Origin to an E. faecium Isolate of Human Origin in the Intestines of Human Volunteers

ABSTRACT Transient colonization by vancomycin-resistant enterococci of animal origin has been documented in the intestines of humans. However, little is known about whether transfer of the vanA gene occurs in the human intestine. Six volunteers ingested a vancomycin-resistant Enterococcus faecium isolate of chicken origin, together with a vancomycin-susceptible E. faecium recipient of human origin. Transconjugants were recovered in three of six volunteers. In one volunteer, not only was vancomycin resistance transferred, but also quinupristin-dalfopristin resistance. This study shows that transfer of the vanA gene from an E. faecium isolate of animal origin to an E. faecium isolate of human origin can occur in the intestines of humans. It suggests that transient intestinal colonization by enterococci carrying mobile elements with resistance genes represents a risk for spread of resistance genes to other enterococci that are part of the human indigenous flora, which can be responsible for infections in certain groups of patients, e.g., immunocompromised patients.

Antimicrobial-Resistant Enterococci in Animals and Meat: A Human Health Hazard?

Enterococcus faecium and Enterococcus faecalis belong to the gastrointestinal flora of humans and animals. Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The use of avoparcin, gentamicin, and virginiamycin for growth promotion and therapy in food animals has lead to the emergence of vancomycin- and gentamicin-resistant enterococci and quinupristin/dalfopristin-resistant E. faecium in animals and meat. This implies a potential risk for transfer of resistance genes or resistant bacteria from food animals to humans. The genes encoding resistance to vancomycin, gentamicin, and quinupristin/dalfopristin have been found in E. faecium of human and animal origin; meanwhile, certain clones of E. faecium are found more frequently in samples from human patients, while other clones predominate in certain animal species. This may suggest that antimicrobial-resistant E. faecium from animals could be regarded less hazardous to humans; however, due to their excellent ability to acquire and transfer resistance genes, E. faecium of animal origin may act as donors of antimicrobial resistance genes for other more virulent enterococci. For E. faecalis, the situation appears different, as similar clones of, for example, vancomycin- and gentamicin-resistant E. faecalis have been obtained from animals and from human patients. Continuous surveillance of antimicrobial resistance in enterococci from humans and animals is essential to follow trends and detect emerging resistance.

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.

Natural transfer of sulphonamide and ampicillin resistance between Escherichia coli residing in the human intestine

OBJECTIVES The aim of this study was to investigate whether the sulphonamide resistance gene sul2 could be transferred between Escherichia coli in the human gut. METHODS Nine volunteers ingested a 10(9) cfu suspension of sulphonamide-susceptible, rifampicin-resistant E. coli recipients of human origin. Three hours later, they ingested a 10(7) cfu suspension of a sulphonamide-resistant (MIC>1024 mg/L) E. coli donor of pig origin. Stool samples were collected 24 h prior to ingestion, daily for 7 days and at days 14 and 35. Samples were plated on selective plates and monitored for the acquisition of sulphonamide-resistance by the recipient from the indigenous or administrated donor E. coli. Possible transconjugants were typed by PFGE and tested for the presence of plasmids containing the sul2 gene, which was also sequenced. RESULTS Concentrations of the human and animal E. coli reached a maximum of 7.5x10(6) cfu/g faeces and colonized for more than 7 days, and 2x10(8) cfu/g for more than 14 days, respectively. On day 2, a transconjugant was detected in one volunteer. This volunteer was colonized with sulphonamide-resistant E. coli at day 0. The transconjugant was sul2-positive, had an MIC>1024 mg/L for sulfamethoxazole and the same PFGE profile as the recipient. The resident E. coli transferred a plasmid (>63 kb), containing the sul2 gene, to the recipient. The sul2 sequence of the transconjugant was identical to that of the volunteers own E. coli from day 0, but differed from the animal strain. Co-transfer of ampicillin resistance was also demonstrated. CONCLUSIONS Transfer of sul2 was observed between E. coli bacteria in the human intestine. The transconjugants sul2 gene came from the volunteers own flora. The origin of the E. coli donor is unknown.

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.

Host range of enterococcal vanA plasmids among Gram-positive intestinal bacteria

OBJECTIVES The most prevalent type of acquired glycopeptide resistance is encoded by the vanA transposon Tn1546 located mainly on transferable plasmids in Enterococcus faecium. The limited occurrence in other species could be due to the lack of inter-species transferability and/or stability of Tn1546-containing plasmids in other species. We investigated the in vitro transferability of 14 pre-characterized vanA-containing plasmids hosted by E. faecium (n = 9), Enterococcus faecalis (n = 4) and Enterococcus raffinosus (n = 1) into several enterococcal, lactobacterial, lactococcal and bifidobacterial recipients. METHODS A filter-mating protocol was harmonized using procedures of seven partner laboratories. Donor strains were mated with three E. faecium recipients, three E. faecalis recipients, a Lactobacillus acidophilus recipient, a Lactococcus lactis recipient and two Bifidobacterium recipients. Transfer rates were calculated per donor and recipient. Transconjugants were confirmed by determining their phenotypic and genotypic properties. Stability of plasmids in the new host was assessed in long-term growth experiments. RESULTS In total, 282 enterococcal matings and 73 inter-genus matings were performed and evaluated. In summary, intra-species transfer was far more frequent than inter-species transfer, if that was detectable at all. All recipients of the same species behaved similarly. Inter-genus transfer was shown for broad host range control plasmids (pIP501/pAMβ1) only. Acquired resistance plasmids remained stable in the new host. CONCLUSIONS Intra-species transfer of enterococcal vanA plasmids was far more frequent than transfer across species or genus barriers and may thus explain the preferred prevalence of vanA-containing plasmids among E. faecium. A reservoir of vanA plasmids in non-enterococcal intestinal colonizers does not seem to be reasonable.

CorrespondenceGlobal spread of New Delhi metallo-β-lactamase 1

Karthikeyan Kumarasamy and colleagues express concern about the emergence of New Delhi metallo-βlactamase (NDM-1) in enterobacteria in India, Pakistan, and the UK. One of the fi rst acquired metallo-βlactamase-producing enterobacteria reported was an IMP-1-producing Klebsiella pneumoniae isolated in Singapore in 1996. Since then we have undertaken hospital surveillance for carbapenem-resistant enterobacteria. Until 2009, the only other metallo-β-lactamase enterobacteria we isolated was another unrelated IMP-1-producing K pneumoniae in 2004. In early 2010, we isolated two K pneumoniae with a high level of resistance (determined by Etest; bioMerieux, Marcy l’Etoile, France) to many antibiotics including to carbapenems (table). The fi rst—DU1301/10—was from the urine of a patient who had just returned from a 5-month stay in India where he had an indwelling catheter inserted. The second isolate, DU7433/10, was from THK, CTK, and TYK did the experimental work to identify the β-lactamase genes, and did the typing studies. LW, YLL, HNL, and LCL managed and provided background information about the two cases. All authors were involved in writing the letter. We declare that we have no confl icts of interest.

Vancomycin-resistant Enterococcus faecalis isolates from a Danish patient and two healthy human volunteers are possibly related to isolates from imported turkey meat

1. Mazel D. Integrons: agents of bacterial evolution. Nat Rev Microbiol 2006; 4: 608–20. 2. Hansson K, Sundström L, Pelletier A et al. IntI2 integron integrase in Tn7. J Bacteriol 2002; 184: 1712–21. 3. Ahmed AM, Furuta K, Shimomura K et al. Genetic characterization of multidrug resistance in Shigella spp. from Japan. J Med Microbiol 2006; 55: 1685–91. 4. Dubois V, Parizano MP, Arpin C et al. High genetic stability of integrons in clinical isolates of Shigella spp. of worldwide origin. Antimicrob Agents Chemother 2007; 51: 1333–407. 5. Pan JC, Ye R, Meng DM et al. Molecular characteristics of class 1 and 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri. J Antimicrob Chemother 2006; 58: 288–96. 6. Gassama Sow A, Diallo MH, Boye CS et al. Class 2 integron-associated antibiotic resistance in Shigella sonnei isolates in Dakar, Senegal. Int J Antimicrob Agents 2006; 27: 267–70. 7. White P, McIver C, Rawlinson W. Integrons and gene cassettes in the Enterobacteriaceae. Antimicrob Agents Chemother 2001; 45: 2658–61. 8. Ploy MC, Denis F, Courvalin P et al. Molecular characterization of integrons in Acinetobacter baumanii: description of a hybrid class 2 integron. Antimicrob Agents Chemother 2000; 44: 2684–8. 9. Prère MF, Chandler M, Fayet O. Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences. J Bacteriol 1990; 172: 4090–9. 10. Biskri L, Mazel D. Erythromycin esterase gene (ereA) is located in a functional gene cassette in an unusual class 2 integron. Antimicrob Agents Chemother 2003; 47: 3326–31.

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

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing and AmpC β-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.