Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) ofEnterococcus faecalisin the apparent absence of pheromone induction

Abstract

Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAM beta 1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2 x 10(-7) and 5 x 10(-7) per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae. Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.

A Multicentre Hospital Outbreak in Sweden Caused by Introduction of a vanB2 Transposon into a Stably Maintained pRUM-Plasmid in an Enterococcus faecium ST192 Clone

“…For comparison, one vancomycin resistant isolate (VRE0576) from 2006 was chosen because of its divergent PFGE pattern. E. faecium 64/3 [23], BM4105RF and BM4105-Str [24] were used as recipients in filter mating experiments. Isolates from a polyclonal cluster of vanB2 positive E. faecium from 2002–2004 in the Swedish county Örebro [18] were included in the ICE Slu van Q8 PCR to evaluate their vanB transposon signature.…”

Global Spread of the hyl Efm Colonization-Virulence Gene in Megaplasmids of the Enterococcus faecium CC17 Polyclonal Subcluster

“…Transferability of acquired antibiotic resistance genes frequently associated with CC17 isolates, vancomycin, and erythromycin was addressed by filter mating at a 1:1 donor/recipient ratio, using E. faecium strains GE-1 and BM4105RF as recipients and brain heart infusion (BHI) agar supplemented with 6 mg/liter of vancomycin and 20 mg/liter of erythromycin as selective media (20,38). The sizes of plasmids containing hyl Efm were determined by using a modified protocol of the technique described by Barton et al, followed by hybridization with specific probes in wild-type strains and transconjugants selected with antibiotics (4) (see below).…”

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences GenBank and GenPept accession numbers are given in Table 3 T3 .

“…E. faecalis BM4281 containing Tn1547 (Quintiliani & Courvalin, 1996) was vanB subtyped but not included in the other analyses. When screening for ISEnfa200-and ISEnfa110-like elements, the following strains were included : (i) 63 Enterococcus strains from the European VRE study (Schouten et al, 1999) (Poyart & Trieu-Cuot, 1994) was spread on a 45 µm nitrocellulose membrane filter (Millipore) placed on top of brain heart infusion (BHI) agar. After 18 h incubation at 37 mC, cells were resuspended in 1 ml BHI broth and spread on BHI agar containing 8 µg vancomycin ml − ", 20 µg rifampicin ml − " and 10 µg fucidic acid ml − ".…”