Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis.
OBJECTIVE
The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin.


METHODS
The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin.


RESULTS
The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic.


CONCLUSIONS
The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.
Danish Institute for Food and Veterinary Research, Copenhagen V, Denmark. hha@dfvf.dk
Henrik
H
Hasman
Frank M
FM
Aarestrup
Anders
A
Dalsgaard
Luca
L
Guardabassi
D000192:Actinomycetales / Q000235:genetics / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D013293:Enterococcus faecalis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006020:Glycopeptides / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D005810:Multigene Family; D017422:Sequence Analysis, DNA; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics / Q000378:metabolism; D012988:Soil Microbiology; D017334:Teicoplanin / Q000494:pharmacology; D014640:Vancomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006020:Glycopeptides; C071437:VanA ligase, Bacteria; C070700:VanH protein, Enterococcus faecium; D017334:Teicoplanin; D014640:Vancomycin; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase; D019729:Carbon-Oxygen Ligases