Emergence of VanB phenotype-vanA genotype Enterococcus faecium clinical isolate in Bulgaria

braz j infect dis.2014;18(6):693–695

The Brazilian Journal of

INFECTIOUS DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Letter to the Editor

Emergence of VanB phenotype-vanA genotype Enterococcus faecium clinical isolate in Bulgaria

DearEditor,

VanA glycopeptide resistance is characterized by acquired inducible resistance to both vancomycin and teicoplanin, whereas the VanB phenotype is characterized by variable levels of resistance to vancomycin but susceptibility to teicoplanin.¹ Herewereportavancomycin-resistantEntero- coccus faecium (VREFm) strain with VanB phenotype-vanA genotypeincongruence.

In the course of antimicrobial susceptibility testing of all Enterococcus spp. isolates, we identified a VREFm strain (BG139/2013)exhibitinghigh-levelresistancetovancomycin (MIC>256␮g/mL)and susceptibilitytoteicoplanin (4␮g/mL) accordingtotheClinicalandLaboratoryStandardsInstitute 2013recommendations,hence havingtheVanBphenotype.

BG139/2013strainwasisolatedon22February2013fromthe blood ofa male patient aged37 years ata Department of Nephrologyand DialysisinGabrovo,Bulgaria.Speciesiden- tificationwasdonebytheVITEK2system(bioMérieux)and confirmedusingmultiplexpolymerasechainreaction(PCR) aspreviouslydescribed.²

Antimicrobial susceptibility testing was performed by the Etest (LIOFILCHEM). BG139/2013 showed high- level gentamicin resistance; resistance toward ampicillin (MIC>256␮g/mL), erythromycin (>256), tetracycline (24), ciprofloxacin (>32); and susceptibility tolinezolid (1.5) and chloramphenicol(1.5).

BacterialDNAwasextractedusingtheISOLATEGenomic DNA Mini Kit (Bioline, UK) according to the manu- facturer’s instructions. The glycopeptide antibiotic resis- tance genes were amplified with specific primers as described before: A1/A2, B1/B2³ and vanD-F/vanD-R.⁴ The primers for the vanA and vanD genes were actualized:

vanAD-F 5-GARGAYGGMWSCATMCARGGY-3 and vanAD-R 5-MGTRAAWCCNGGCAKRGTRTT-3.Each25-␮LPCRmixture consistedof3␮LoftemplateDNA;a0.1␮Mofeachprimer;

12.5␮Lof MyTaq PCRmix (Bioline) and 7.5␮Lof ultrapure 18.2MPCRwater(Bioline).DNAamplificationwasperformed using the followingprotocol: initialdenaturation(95^◦C for 5min),followedby35cyclesofdenaturation(95^◦Cfor45s),

annealing (51.3–60^◦Cfor45s)andextension(72^◦Cfor60s), withasinglefinalextensionof7minat72^◦C.Forprimermodi- ficationandphylogeneticanalysesweusedtheGenBankNCBI (National Center for Biotechnology Information) sequence database:FJ545640,JN207933,JN207928,JN207930,EF206284, X56895 (VanA strains); AY665551, U35369, AF310956 and AY145441 (VanB);AF175293,AF153050,AF130997,AY489045, AY082011 and AF277571 (VanD). The sequence reactions and phylogenetic analyses were performed as previously described.⁵

A630bpPCRproductwasyieldedonlywiththe vanAD- F/vanAD-Rprimerpairatanannealingtemperatureof51.3^◦C.

After sequencing, the data for the phylogenetic analysis consistedofa450nucleotidesfragment, or150aminoacid (aa)residues,including125–274aaofvanA.Thepredictedaa sequenceoftheBG139/2013isolatewas100%identicaltothe knownvanAsequences(GenBankaccessionnoAAW56079.1 andAGU36307.1).ThealignmentwithJN207933,JN207928and JN207930 showed that our isolate had a non-synonymous nucleotide mutation leading tothe substitution ofalanine withvalineinposition227intheaminoacidchain.Thephy- logenetictreeshowedthatBG139/2013isolategroupstogether withclustervanAgeneisolatesofE.faecium(Fig.1).

TheVanA phenotyperesistance isdetermined bygenes locatedontransposonTn1546,whichincludesthevanRSreg- ulatorysystemthatactivatesthetranscriptionofresistance genes(vanHAXYZ).⁶ Thestudiedgenefragmentmakesupa part ofthe protein active site and was identified as vanA genotype in thephylogenetic analysis.On the other hand, BG139/2013hadVanBphenotype.Arelationhasbeenreported betweenanimpairment ofthe responsetoteicoplanin(i.e.

low-levelteicoplaninresistance)andaminoacidsubstitutions duetothreepointmutationsintheN-terminalregionofvanS.⁶ Thus,thesubstitutionfoundinposition227seemstobeunre- latedtothediscrepancybetweenthephenotypeandgenotype.

Thisdiscrepancycouldmostprobablybeattributedtonon- synonymousmutationsintheN-terminalregionofvanS.

Inconclusion,furtherstudiesonthegenesresponsiblefor the phenotyperesistanceofthe BG139/2013isolate,andon vanSinparticular,areneeded.Toourknowledge,thestudied

694

VanB2

VanA VanB

949

1000

0.1

1000

650

474

AY 1000 6655

51VanB U35369V

anB AF310956V

anB2

AF145441VanB2

JN20 7933

VanA

JN207928V anA

JN207930 VanA

BG139/2013

FJ 54564

0V anA EF20628

4VanA X56895VanA

AF175293vanD AF130997v AF anD

153050v AY489045V anXD

anD

AY082011VanD

AF277571V

anD

VanD

Fig.1–Phylogenetictreebasedonalignmentofa150aminoacidpredictedsequencefromisolateBG139/2013withE.

faeciumreferencestrains.ThetreewasconstructedfromalignedaminoacidpositionsinthefinaldatasetusingMUSCLE.

Onethousandbootstrapreplicationswereusedtobuildthephylogenetictree.

VanBphenotype-vanAgenotypeE.faeciumstrainisthefirstof itskindtobeisolatedinBulgaria.

Conflicts of interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ThisworkwassupportedbyagrantfromtheMedicalUniver- sityofSofia,Bulgaria(CouncilofMedicalScience,projectno.

12/2013,grantno.16/2013).

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TanyaStrateva^∗,DanielaAtanasova, IvanMitov DepartmentofMedicalMicrobiology,FacultyofMedicine, MedicalUniversityofSofia,Sofia,Bulgaria

IvoSirakov

NationalDiagnosticandResearchVeterinaryInstitute, Sofia,Bulgaria

AntoninaKatrandjieva

MultiprofileHospitalforActiveTreatment“Dr.TotaVenkova”, Gabrovo,Bulgaria

brazj infect dis.2014;18(6):693–695

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∗Corresponding author at: Department of Medical Microbiol- ogy,FacultyofMedicine,MedicalUniversityofSofia,2Zdrave Street,1431Sofia,Bulgaria.

E-mailaddress:[email protected](T.Strateva).

Received1June2014 Accepted11June2014

1413-8670/©2014ElsevierEditoraLtda.Allrightsreserved.

http://dx.doi.org/10.1016/j.bjid.2014.06.005