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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276

Mem. Inst. Oswaldo Cruz vol.108 no.4 Rio de Janeiro June 2013 


Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis

Tiane Martin de MouraI  II  + 

Ana Paula Vaz CassenegoI  II 

Fabrício Souza CamposIII  IV 

Andrea Machado Leal RibeiroV 

Ana Cláudia FrancoI  IV 

Pedro Alves d'AzevedoVI 

Jeverson FrazzonVII 

Ana Paula Guedes FrazzonI  II 

IPrograma de Pós-Graduação em Microbiologia Agrícola e do Ambiente

IIIPrograma de Pós-Graduação em Microbiologia Agrícola e do Ambiente, Laboratório de Bacteriologia, Departamento de Microbiologia

IVLaboratório de Virologia, Departamento de Microbiologia, Instituto de Ciências Básicas da Saúde

IIIPrograma de Pós-Graduação em Ciências Veterinárias

VDepartamento de Zootecnia, Faculdade de Agronomia

VIIInstituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

VILaboratório de Cocos Gram-Positivos, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brasil


Here we report the presence and expression levels of the vanC1 and vanC2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC1 and vanC2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC1 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.

Key words: Enterococcus faecalis; vancomycin resistance; vanC gene; horizontal gene transfer

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 vanC1 and vanC2/3 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 vanC1 gene was recently detected in a vancomycin-susceptible strain of E. faecalis (Schwaiger et al. 2012). Here we report the presence of the vanC1 and vanC2/3 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).


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-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).

Determination of the minimal inhibitory concentration (MIC) of vancomycin - The MIC of vancomycin was determined by the broth microdilution method (BMM) (0.125-32 µg/mL) according to the Clinical and Laboratory Standards Institute (CLSI 2010) and by the Epsilometer-test (E-test) (0-256 µg/mL) (bioMérieux(r)), following 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 ddlE.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 vanC1,2/3 by PCR. The oligonucleotides and PCR conditions used in this study for vanA and vanC1 (Dutka-Malen et al. 1995), vanB (Depardieu et al. 2004) and vanC2/3 (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 vanC1,2/3 ) 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(r) (Invitrogen(r)), following the manufacturer's protocol. The total RNA was treated with RNase-free DNase I (Fermentas(r)) according to the manufacturer's recommendations.

Complementary DNA (cDNA) was synthesised from 1 µg of high-quality total RNA (A260nm/280nm of 1.80-2.0), following the manufacturer's instructions (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems(r)). Reverse transcriptase was omitted from the negative control. The cDNAs were used in the PCR amplification of the vanC1 and vanC2/3 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 ddlE.faecalis , vanC1 and vanC2/3 , 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(tm) 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 GenBank database using the Basic Local Alignment Search Tool (


Three E. faecalis isolates (CB114, CB356 and CB378) were identified by a PCR strategy using species-specific primers to amplify the 475 bp ddlE.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).

TABLE Phenotypic and genotypic characteristics of Enterococcus faecalis vanC-type isolates from cloacal swabs of broilers 

Isolate Phenotype a
E-test BMM ddl vanA vanB vanC 1 vanC 2/3
CB114 3.50 µg/mL 4.0 µg/mL + - - + -
CB356 2.25 µg/mL 1.0 µg/mL + - - + +
CB378 2.50 µg/mL 1.0 µg/mL + - - + +
Parameters ≤ 4 µg/mL b ≤ 4 µg/mL c

a: average values of triplicates; b: sensitive according to manufacturer; c: sensitive according to CLSI-M100-S20; BMM: broth microdilution method; ddl: D-alanine-D-alanine ligase; E-test: Epsilometer-test.

All isolates were positive for the vanC1 gene and the strains CB356 and CB378 harboured both the vanC1 and vanC2/3 genes. The vanC1 gene was detected in plasmid DNA and the vanC2/3 gene was present in the chromosomal DNA (data not shown).


The detection of more than one van-type gene in an Enterococcus strain has been reported in other studies, including the presence of vanC1 + vanA or vanC1 + 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 vanC1 gene in vancomycin-susceptible 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 (vanC1 ) and E. casseliflavus (vanC2/3 ) 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 vanC1 -specific mRNA in only one strain (CB356) and did not detect vanC2/3 mRNA (data not shown). Recently, a study using real-time RT-PCR assays also failed to detect a corresponding vanC1 transcript in a VanC1 genotype-positive 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).

The three partial ddlE.faecalis , vanC1 and vanC2/3 gene sequences were deposited in GenBank (accessions JX220983, JX220984 and JX220985, respectively). The alignment of the sequences showed 99% identity to ddlE.faecalis of E. faecalis (GenBank ID U00457), 99% to vanC1 of E. gallinarum (GenBank ID EU151770) and 99% to vanC2/3 of E. casseliflavus (GenBank ID EU151764), respectively.

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 vanC1 and vanC2/3 genes be used with caution as species-specific markers.


To the UFRGS and to the UFCSPA.


Boyd DA, Willey BM, Fawcett D, Gillani N, Mulvey MR 2008. Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel d-Ala-d-Ser gene cluster, vanL. Antimicrob Agents Chemother 52: 2667-2672. [ Links ]

Cassenego APV, d'Azevedo PA, Ribeiro AML, Frazzon J, Van Der Sand ST, Frazzon APG 2011. Species distribution and antimicrobial susceptibility of Enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements. Braz J Microbiol 42: 480-488. [ Links ]

CLSI - Clinical and Laboratory Standards Institute 2010. Performance standards for antimicrobial susceptibility testing, 20th informational supplement, CLSI, Wayne, 160 pp. [ Links ]

Courvalin P 2006. Vancomycin resistance in gram-positive cocci. Clin Infect Dis 42: 25-34. [ Links ]

Depardieu F, Perichon B, Courvalin P 2004. Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR. J Clin Microbiol 42: 5857-5860. [ Links ]

Dutka-Malen S, Evers S, Courvalin P 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33: 24-27. Erratum in J Clin Microbiol 1995 33: 1434. [ Links ]

Dutka-Malen S, Molinas C, Arthur M, Courvalin P 1992. Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine: D-alanine ligase-related protein necessary for vancomycin resistance. Gene 112: 53-58. [ Links ]

Elsayed S, Hamilton N, Boyd D, Mulvey M 2001. Improved primer design for multiplex PCR analysis of vancomycin-resistant Enterococcus spp. J Clin Microbiol 39: 2367-2368. [ Links ]

Fisher K, Phillips C 2009. The ecology, epidemiology and virulence of Enterococcus. Microbiology 155: 1749-1757. [ Links ]

Franz C, Stiles ME, Schleifer KH, Holzapfel WH 2003. Enterococci in foods - a conundrum for food safety. Int J Food Microbiol 88: 105-122. [ Links ]

French GL 1998. Enterococci and vancomycin resistance. Clin Infect Dis 27: S75-S83. [ Links ]

Hassan L, Getachew YM, Zunita Z, Kamaruddin MI 2008. Distribution of van genes of vancomycin-resistant Enterococcus isolated from broilers in Peninsular Malaysia. Available from: [ Links ]

Jackson CR, Fedorka-Cray PJ, Barrett JB 2004. Use of a genus - and species-specific multiplex PCR for identification of Enterococci. J Clin Microbiol 42: 3558-3565. [ Links ]

Lawrence JG, Hendrix RW, Casjens S 2001. Where are the pseudogenes in bacterial genomes? Trends Microbiol 9: 535-540. [ Links ]

Lebreton F, Depardieu F, Bourdon N, Fines-Guyon M, Berger P, Camiade S, Leclercq R, Courvalin P, Cattoir V 2011. d-Ala-d-Ser VanN-type transferable vancomycin resistance in Enterococcus faecium. Antimicrob Agents Chemother 55: 4606-4612. [ Links ]

Medeiros AW, d'Azevedo P, Pereira RI, Cassenego AP, Van Der Sand S, Frazzon J, Frazzon APG 2010. PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples. Rev Soc Bras Med Trop 43: 100-101. [ Links ]

Moreno MRF, Sarantinopoulos P, Tsakalidou E, De Vuyst L 2006. The role and application of enterococci in food and health. Int J Food Microbiol 106: 1-24. [ Links ]

Moura TM, Campos FS, d'Azevedo PA, Van Der Sand ST, Franco AC, Frazzon J, Frazzon APG 2012. Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding. Rev Soc Bras Med Trop 45: 579-585. [ Links ]

Navarro F, Courvalin P 1994. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrob Agents Chemother 38: 1788-1793. [ Links ]

Park IS, Lin CH, Walsh CT 1997. Bacterial resistance to vancomycin: overproduction, purification and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase. Proc Natl Acad Sci USA 94: 10040-10044. [ Links ]

Patel R, Uhl JR, Kohner P, Hopkins MK, Cockerill FR III 1997. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci. J Clin Microbiol 35: 703-707. [ Links ]

Patel R, Uhl JR, Kohner P, Hopkins MK, Steckelberg JM, Kline B, Cockerill FR III 1998. DNA sequence variation within vanA, vanB, vanC-1, and vanC-2/3 genes of clinical Enterococcus isolates. Antimicrob Agents Chemother 42: 202-205. [ Links ]

Poeta P, Antunes T, Rodrigues J 2005. Enterococcus spp resistentes à vancomicina isolados de fezes de frangos, pombos, gamos e ratos. Arq Bras Med Vet Zootec 57: 412-414. [ Links ]

Ramotar K, Woods W, Larocque L, Toye B 2000. Comparison of phenotypic methods to identify enterococci intrinsically resistant to vancomycin (VanC VRE). Diagn Microbiol Infect Dis 36: 119-124. [ Links ]

Riboldi GP, Mattos EP, Frazzon APG, d'Azevedo PA, Frazzon J 2008. Phenotypic and genotypic heterogeneity of Enterococcus species isolated from food in southern Brazil. J Basic Microbiol 48: 31-37. [ Links ]

Sambrook J, Russell DW 2001. Molecular cloning: a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York, 2344 pp. [ Links ]

Satake S, Clark N, Rimland D, Nolte FS, Tenover FC 1997. Detection of vancomycin-resistant Enterococci in fecal samples by PCR. J Clin Microbiol 35: 2325-2330. [ Links ]

Schwaiger K, Bauer J, Hörmansdorfer S, Mölle G, Preikschat P, Kämpf P, Bauer-Unkauf I, Bischoff M, Hölzel C 2012. Presence of the resistance genes vanC1 and pbp5 in phenotypically vancomycin and ampicillin susceptible Enterococcus faecalis. Microb Drug Resist 18: 434-439. [ Links ]

Xu X, Lin D, Yan G, Ye X, Wu S, Guo Y, Zhu D, Hu F, Zhang Y, Wang F, Jacoby GA, Wang M 2010. vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium. Antimicrob Agents Chemoter 54: 4643-4647. [ Links ]

Received: July 25, 2012; Accepted: April 02, 2013

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