Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis

Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro,Vol. 108(4): 453-456, June 2013 Enterococcus faecalis is part of the normal microbiota inhabiting the gastrointestinal tract of humans and animals and it is also present in soil, plants and food (Moreno et al. 2006, Riboldi et al. 2008, Cassenego et al. 2011). This pathogen is responsible for serious health problems and causes the majority of human enterococcal infections (Franz et al. 2003). An important feature of this species is its resistance to a wide range of antimicrobial agents. Animals may be an important reservoir of vancomycin resistant enterococci (VRE) because of the possibility of resistance genes being transferred to the human gut bacteria through the food chain and/or animal husbandry (Poeta et al. 2005).
Nine VRE genotypes have been described in enterococci (vanA, -B, -C, -D, -E, -G, -L, -M and -N). These genes encode intrinsic or acquired resistance determinants that result in changes in the peptidoglycan binding site and significantly reduce the strength of vancomycin binding (Courvalin 2006, Boyd et al. 2008, Xu et al. 2010, Lebreton et al. 2011. The VanC phenotype is chromosomally encoded by the vanC 1 and vanC 2/3 genes, which are intrinsic to Enterococcus gallinarum and Enterococcus casseliflavus, respectively, and therefore can be used for species identification (Park et al. 1997, French 1998, Ramotar et al. 2000, Courvalin 2006). These genes confer low-level resistance to vancomycin (2-32 µg/mL) and susceptibility to teicoplanin (0.5-1 µg/ mL) (Dutka-Malen et al. 1992, 1995, Navarro & Courvalin 1994, Courvalin 2006. The vanC 1 gene was recently detected in a vancomycin-susceptible strain of E. faecalis (Schwaiger et al. 2012). Here we report the presence of the vanC 1 and vanC 2/3 genes and evaluate the expression of vanC genes by reverse transcription-polymerase chain reaction (RT-PCR) in E. faecalis isolates that were obtained from cloacal swabs of broilers; these strains were previously classified as vancomycin-intermediate resistant (Cassenego et al. 2011).

MATERIALS AND METHODS
E. faecalis strains -A total of 29 E. faecalis isolates from cloacal swabs of broilers that were classified as vancomycin-intermediate resistant by the disk diffusion method were screened for the presence of van genes by PCR (data not shown); three isolates (CB 114, CB 356 and CB 378) that were positive for the vanC gene were chosen for this study. The isolates were biochemically classified as E. faecalis, which was confirmed by PCR using species-specific primers for the D-alanine-D-ala-D-alanine-D-alanine ligase (ddl) E.faecalis gene (Depardieu et al. 2004). The strains were also tested to exclude the species E. casseliflavus and E. gallinarum by PCR using the species-specific primer pairs CA1/CA2 and GA1/GA2, respectively (Jackson et al. 2004). Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

Determination of the minimal inhibitory concentration (MIC) of vancomycin
lowing the manufacturer's recommendations. All tests were performed in triplicate and the E. faecalis strains ATCC 29212 (vancomycin-susceptible) and E. faecalis ATCC 51299 (resistant to vancomycin) were used for quality control.
Extraction of DNA and PCR assays -Genomic DNA was extracted following the standard method of phenol (Invitrogen) extraction and ethanol (Pro Analysis) precipitation (Sambrook & Russell 2001) with minor modifications, as previously described (Moura et al. 2012). Plasmid DNA was extracted using standard miniprep methods (Sambrook & Russell 2001). The species identification of the E. faecalis isolates was confirmed by PCR using species-specific primers for the ddl E.faecalis gene (Depardieu et al. 2004). E. faecalis ATCC 51299 and Enterococcus faecium ATCC 53519 were used as positive and negative control strains, respectively. All strains were retested for the presence of vanA, vanB and vanC 1,2/3 by PCR. The oligonucleotides and PCR conditions used in this study for vanA and vanC 1 (Dutka-Malen et al. 1995), vanB (Depardieu et al. 2004) and vanC 2/3 (Satake et al. 1997) followed those reported by their respective authors. Reactions were performed in an Eppendorf Mastercycler thermal cycler under the following cycle conditions: 3 min at 94ºC, 30 cycles of 1 min at 94ºC, 1 min at 50ºC (vanA) or 54ºC (vanB and vanC 1,2/3 ) and 1 min at 72ºC and 5 min at 72ºC.

RNA extraction and analysis of vanC gene expression by RT-PCR -Briefly
, 500 µL of overnight culture was inoculated into 50 mL 2xYT broth and incubated with agitation at 37ºC to an optical density at 600 nm of 0.3. A 3 mL aliquot was harvested by centrifugation for 10 min at 10,000 g, the supernatant was discarded and total RNA was extracted using TRIzol ® (Invitrogen ® ), following the manufacturer's protocol. The total RNA was treated with RNase-free DNase I (Fermentas ® ) according to the manufacturer's recommendations.
Complementary DNA (cDNA) was synthesised from 1 μg of high-quality total RNA (A 260nm/280nm of 1.80-2.0), following the manufacturer's instructions (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems ® ). Reverse transcriptase was omitted from the negative control. The cDNAs were used in the PCR amplification of the vanC 1 and vanC 2/3 genes in a final volume of 25 µL. The cDNAs were also used for PCR amplification of the 16S rRNA gene (Medeiros et al. 2010).
Sequencing of samples -To confirm the presence of ddl E.faecalis , vanC 1 and vanC 2/3 , the amplified products were submitted to nucleotide sequence analysis. The primers and PCR followed the protocols previously described (Dutka-Malen et al. 1995, Satake et al. 1997, Depardieu et al. 2004. The DNA fragments were purified using an Illustra GFX™ PCR DNA and Gel Band Purification kit (GE Healthcare-Buckinghamshire, United Kingdom). Sequencing was carried out with the Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) in an ABI-PRISM 3100 Genetic Analyzer (ABI), according to the manufacturer's protocol. The nucleotide sequences obtained were compared with homologous nucleotide sequences deposited in the Gen-Bank database using the Basic Local Alignment Search Tool (blast.ncbi.nlm.nih.gov/Blast.cgi).

RESULTS
Three E. faecalis isolates (CB114, CB356 and CB378) were identified by a PCR strategy using species-specific primers to amplify the 475 bp ddl E.faecalis gene (data not shown). Moreover, the isolates were negative for the species E. casseliflavus and E. gallinarum.
The MIC determined by E-test exhibited an elliptical zone of inhibition within the range of 1.50-4.0 µg/mL and the strains were reclassified as vancomycin-susceptible (≤ 4.0 µg/mL). The BMM showed a MIC ranging from 4.0-1.0 µg/mL and all strains were also reclassified as vancomycin-susceptible by this method (Table).
All isolates were positive for the vanC 1 gene and the strains CB356 and CB378 harboured both the vanC 1 and vanC 2/3 genes. The vanC 1 gene was detected in plasmid DNA and the vanC 2/3 gene was present in the chromosomal DNA (data not shown).

DISCUSSION
The detection of more than one van-type gene in an Enterococcus strain has been reported in other studies, including the presence of vanC 1 + vanA or vanC 1 + vanB (Elsayed et al. 2001, Hassan et al. 2008. None of the strains tested positive by PCR for the vanA or vanB gene. The presence of the vanC 1 gene in vancomycinsusceptible E. faecalis strains isolated from pig manure samples was first described in Germany (Schwaiger et al. 2012). The detection of these vanC genes in E. faecalis is remarkable because they are thought to be intrinsic to E. gallinarum (vanC 1 ) and E. casseliflavus (vanC 2/3 ) and the vanC operon is chromosomally located in a transferable region, such as a transposon and/or integron (Dutka-Malen et al. 1992, Navarro & Courvalin 1994, Patel et al. 1997, 1998, Depardieu et al. 2004, Fisher & Phillips 2009). E. faecalis may have acquired vanC genes by horizontal transfer from E. gallinarum and E. casseliflavus, two natural inhabitants of the poultry gut. This flow of the vanC gene between species is important because the presence of this gene is often used to identify species and therefore, erroneous identification of species may be occurring. Furthermore, this flow also emphasises that the chromosomal location of a gene in intrinsically resistant strains does not necessarily protect against transfer to other species, thereby contributing to the diversification of species (Schwaiger et al. 2012). RT-PCR experiments detected vanC 1 -specific mRNA in only one strain (CB356) and did not detect vanC 2/3 mRNA (data not shown). Recently, a study using real-time RT-PCR assays also failed to detect a corresponding vanC 1 transcript in a VanC 1 genotypepositive strain (Schwaiger et al. 2012). A possible explanation for this result could be that a non-functional vanC gene cluster has been transferred from the bacterial community to CB114 and CB378 or it could reflect the action of a failed recombination event that inserted a non-functional gene and removed beneficial DNA (Lawrence et al. 2001).
In conclusion, we have reported the first identification and mRNA expression of the vanC gene in a vancomycin-susceptible E. faecalis strain that was isolated from cloacal swabs of broilers in Brazil. Our results suggest that E. faecalis may have acquired the vanC genes by horizontal transfer from E. gallinarum and E. casseliflavus. These results are significant because the detection of the vanC gene is a useful tool for the detection of E. gallinarum and E. casseliflavus. We recommend that the vanC 1 and vanC 2/3 genes be used with caution as species-specific markers.