| Enterococci are found in the gastrointestinal and female urinary tracts as part of the nomal host flora in healthy individuals, where they seldom cause infections. However, their role as the aetiological agents of endocarditis and urinary tract infection has been well documented. In recent years these organisms have emerged as a leading cause of nosocomial infections in hospitals. The majority (85-90%) of enterococcal infections are due to Enterococcus faecalis, while Enterococcus faecium is responsible for5-10%. Infections due to other Enterococcus specis such as Enterococcus durans, Enterococcus avium, Enterococcus raffinosus, Enterococcus gallinarum and Enterococcus casseliflavus are rarely encountered. Successful therapeutic regimes for serious enterococcal infections require a combination of β-lactam and aminoglycoside, typically gentamicin. Resistance to aminoglycosides usually occurs by enzymatic modification of the aminoglycoside modifying enzyme (AME), and AAC-APH is highly potent. The aac-aph gene has in most cases been identified on plasmids and being part of a transposon, which was designated as Tn5281. Meanwhile, the bifunctional enzyme gene aac(6’)-aph(2") is willing to transfer to strains which do not carry Gmr determinant with a transposon. Recently, there have been reports of Tn5281-truncated structures being identified in enterococcal species, where there has been no consistency in the location of IS256that flanks the aac-aph gene in these Tn5281-truncated structures.IS256is the founding member of IS256family of insertion sequence (IS) elements, and widespread in genomes of Gram-positive bacteria and Gram-negative bacteria. It is reported that IS256can activate transcription of resistance gene in Staphylococcus. In staphylococcus epidermidis, IS256has been related to Gm resistance. As IS256is part of the bifunctional modifying enzyme transposon, we speculate that IS256has relevance with Gm resistance in Enterococcus, probably with the transfer of the bifunctional modifying enzyme.In this work, we tried to clariy the role of IS256in the transfer of the bifunctional modifying enzyme gene aac(6’)-aph(2") using clinical Enterococcus isolated in Beijing from2006-2009. First, two groups of strains were screened out by HLGR determination and colony PCR determination of the bifunctional modifying enzyme gene:HLGR/aac-aph positive group and non-HLGR/aac-aph negative group. The HLGR/aac-aph positive group strains were subjected to PCR detections of the transposon structures and IS256, and the strains were further allocated to H1-H6subgroups based on the results, which were the "donor pool" for the conjugation. The non-HLGR/aac-aph negative group was first subjected to PCR detection of IS256followed by selection of the fusidic acid-resistant and rifampicin-resistant isolates by self mutation or induced mutation, the isolates were further divided into NH1-NH2groups, which were used as the "receptor pool". A filter mating method was used to investigate the transfer of gentamicin resistance by conjugation between "donor pool" strains and a laboratory-derived plasmid-free strain of Enterococcus faecalis, strain JH2-2; a laboratory-derived strain of Enterococcus faecalis, strain HH22and "receptor pool" strains; or "donor pool" strains and "receptor pool" strains. The role of IS256in the transfer of the aminolgycoside bifunctional modifying enzyme was analyzed based on the results of the transfer efficiency, transfer success rate of the Gmr marker in relate to the distribution status of IS256in the donor or receptor strains.Totally169randomly selected Enterococcus faecalis strains and21randomly selected Enterococcus faecium strains collected from hospitals in Beijing from2006to2009were included in the present study. Among the169Enterococcus faecalis,111(65.7%) were found to be high-level gentamicin resistant, and bifunctional enzyme gene was detected in108(97.3%) of the111HLGR isolates. Of the bifunctional enzyme gene positive isolates,11(10.2%) carried nontruncated transposon with IS256at both sides of the bifunctional enzyme gene, while the rest (89.8%) carried truncated transposon which showed negative results with at least one side colony PCR detection of the junction regions of bifunctional enzyme gene with IS256.71.4%Enterococcus faecium strains were high-level gentamicin resistant in our experiment, and all of them carried the aac(6’)-Ie-aph(2")-la, of which13.3%were nontruncated transposons,86.7%were truncated transposons. The result of IS256detection demonstrated that47.6%of the HLGR isolates carried IS256, while only9.4%of the non-HLGR clinical enterococci isolates showed positive result with IS256detection.The filter mating conjugation experiment demonstrated that when HLGR/aac-aph positive Enterococcus faecalis was used as the donor in conjugation with JH2-2,42of the108clinical isolates were able to transfer the HLGR marker into plasmid-free strain of Enterococcus faecalis JH2-2with transfer success rate of38.9%:9/57(15.8%) for H1group,1/3(33.3%) for H2group,1/4(25.0%) for H3group,9/16(56.3%) for H4group,13/17(76.5%) for H5group,9/11(81.8%) for H6 group.The bifunctional modifying enzyme marker in16.7%of the IS256negative strains (H1+H2) were transferable by conjugation, while the corresponding number for IS256posive strains (H3+H4+H5+H6) was68.8%. The gentamicin resistance marker was transferable in17.2%of strains carrying truncated transposons without IS256at both sides of the bifunctional enzyme gene (H1+H2+H3groups), while the corresponding gentamicin resistance marker transfer success rates for the strains carrying truncated transposons with IS256at one side (H4+H5groups) and the strains carrying nontruncated transposons with IS256at both sides of the bifunctional enzyme gene (H6group) were66.7%, and81.8%respectively. The results suggested that the presence of IS256contributed to the transfer of the aminoglycoside bifunctional modifying enzyme, with the transfer succes rate in the order of:the strains carrying nontruncated transposons> strains carrying truncated transposons with IS256at one side> strains carrying truncated transposons without IS256at both sides. The transfer efficiencies (normalized by control conjugation pair HH22::JH2-2) of the "donor pool" strains to JH2-2were in the order of:strains carrying nontruncated transposons (H6group,1.29×10-2～6.84×102)> strains carrying truncated transposons without IS256at both sides (H1group,1.78×10-3～6.21×101; H3group,1.02×100)> strains carrying truncated transposons with IS256at one side (H2group,1.30×10-3;H4group,1.32×10-3～1.85×10-1; H5group,1.30×10-3～5.46×100)Meanwhile,2(IS256negative) of12(IS256negative for11strains, IS256positive for1strain) clinical isolates were able to get the HLGR marker from strain of Enterococcus faecalis HH22in conjugation. The gentamicin resistance marker was also transferable between clinical isolates by conjugation,4clinical isolates were able to transfer the HLGR marker into strain of Enterococcus faecalis08-15and/or08-20.HLGR rate was high in the190enterococci clinical isolates collected from2006to2009in Beijing, China, and aac(6’)-aph(2") was detected in most of the HLGR strain. The transposons containing the bifunctional modifying enzyme gene were usually truncated. The presence of IS256flanking the bifunctional modifying enzyme gene in the transposons contributed to the transfer the Gmr determinant with nontruncated transposons of the highest transfer success rate and transfer efficiency. |
PDF Full Text Request