research article molecular analysis of vana outbreak of...

Research ArticleMolecular Analysis of VanA Outbreak ofEnterococcus faecium in Two Warsaw HospitalsThe Importance of Mobile Genetic Elements

Ewa Wardal1 Katarzyna Markowska1 Dorota gabicka1 Marta Wroacuteblewska2

MaBgorzata Giemza2 Ewa Mik2 Hanna PoBowniak-Pracka3 Agnieszka Wofniak3

Waleria Hryniewicz1 and Ewa Sadowy1

1 National Medicines Institute ul Chełmska 3034 00-725 Warsaw Poland2 Institute of Hematology and Transfusion Medicine ul Ghandi 14 02-776 Warsaw Poland3 Institute of Oncology ul Roentgena 5 02-781 Warsaw Poland

Correspondence should be addressed to Ewa Sadowy ewasadowyclsedupl

Received 15 January 2014 Revised 21 March 2014 Accepted 4 April 2014 Published 9 June 2014

Academic Editor Mariano Pistorio

Copyright copy 2014 Ewa Wardal et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections Two outbreaks of thispathogen from neighboring Warsaw hospitals have been analyzed in this study Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA multilocus VNTR analysis (MLVA) and multilocus sequence typing (MLST) revealed a clonal variability ofisolates which belonged to three main lineages (17 18 and 78) of nosocomial E faecium All isolates were multidrug resistant andcarried several resistance virulence and plasmid-specific genes Almost all isolates shared the same variant of Tn1546 transposoncharacterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene In the majority of cases thistransposonwas located on 50 kb or 100 kb pRUM-related plasmids which lacked however the axe-txe toxin-antitoxin genes 100 kbplasmidwas easily transferred by conjugation andwas found in various clonal backgrounds in both institutions while 50 kb plasmidwas not transferable and occurred solely in MT159ST78 strains that disseminated clonally in one institution Although moleculardata indicated the spread of VRE between two institutions or a potential common source of this alert pathogen epidemiologicalinvestigations did not reveal the possible route by which outbreak strains disseminated

1 Introduction

Since the first isolation of vancomycin-resistant enterococci(VRE) in 1986 [1 2] this phenotype has spread rapidlyand now is present in hospitals worldwide [3] In Polandthe first VanA outbreak took place in the adult hematologyward of Gdansk Medical University in December 1996followed by outbreaks in other centers [4] The predominantspecies among VRE is Enterococcus faecium (VREfm) Themajority of worldwide VREfm belongs to the merocloneCC17 (ciprofloxacin- and ampicillin-resistant and enrichedin putative virulence traits) recently split into three distinctlineages 17 18 and 78 that evolved in hospital environmentthrough horizontal gene transfer (HGT) and recombination

processes [5] These hospital-adapted lineages play a crucialrole in the emergence and spread of VREfm

The vanA gene cluster is a widely studied vancomycinteicoplanin resistance determinant described as part ofTn1546-type transposons generally carried on plasmids andthus effectively disseminated by HGT [6] An acquisition ofvanA plasmid by a strain of E faecium representing hospital-adapted lineage may result in a spread of VREfm first colon-izing patients and then causing symptomatic infectionsTherefore both characterization of the Tn1546 structure andits linkage to particular plasmid groups is crucial for under-standing of VRE dissemination in hospital environmentsSeveral studies have shown the presence of various Tn1546

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 575367 12 pageshttpdxdoiorg1011552014575367

2 BioMed Research International

types on Inc18 pRUM-like pMG1-like and pLG1 plasmids[7ndash13] however our knowledge of vanA plasmids and theirepidemiology is still far from being satisfactory and thecommon presence of plasmids with Tn1546 belonging tounknown replicon types has been shown [10 14]

The aim of this study was to characterize E faeciumVanAisolates from the outbreaks that concomitantly took placein hospital wards of two neighboring medical centers TheInstitute of Oncology (IO) and The Institute of Hematologyand Transfusion Medicine in Warsaw (IH) The investigationfocused on the clonal relationships among isolates as well asanalysis of the Tn1546 transposon structure and colocaliza-tion of vanA with other plasmid genes in order to elucidatethe role of particular MGE during a VREfm outbreak inhospital settings

2 Materials and Methods

21 Outbreak Description Bacterial Isolates and Suscepti-bility Testing Forty-four vancomycin-resistant E faeciumoutbreak isolates were collected between February and June2009 in two neighboring hospitals inWarsawThe Institute ofOncology (IO) andThe Institute of Hematology and Transfu-sion Medicine (IH) 776- and 198-bed hospitals respectivelyFirst VREfmwas isolated from stool of 46-year-old patient on4th February at the Gastroenterology Clinic of IO Until theend of February eight more cases were reported in majorityfrom the Clinic of Lymphatic System Cancers of IO Fromthe 31st March till the 18th of April 18 VREfm were isolatedmainly from patients of this clinic (16 cases) and fromtwo patients of the Gastroenterology Clinic SimultaneouslyVREfm cases were reported in IH wards with the first twoisolations on the 5th February from rectum and stool of theHematologyWard patient and a patient from the ICU respec-tively One more isolate was obtained 10 days later in theSurgery Ward and till the end of June 14 other VREfm caseswere reported in the Hematology Ward of IH Altogetherthe outbreaks affected 42 patients including 27 patients ofIO (27 stool isolates) and 15 patients of IH (13 stool 1 urine3 blood isolates) Antimicrobial susceptibility of collectedisolates was determined using the Etest method (bioMerieuxMarcy lrsquoEtoile France) for glycopeptide susceptibility testingand broth microdilution method for other antimicrobialsThe results were interpreted following the breakpoints ofthe European Committee on Antimicrobial SusceptibilityTesting (EUCAST) [15] for chloramphenicol erythromycinciprofloxacin and tetracycline the Clinical and LaboratoryStandards Institute (CLSI) [16] breakpoints were applied andin the case of kanamycin and clindamycin the breakpointsproposed by the Societe Francaise de Microbiologie (SFM)[17] were used The Enterococcus faecalis strain ATCC29212was used for quality control purposes during testing Efaecium BM4147 was used as a control VanA strain in thisstudy

22 DNA Isolation and Genotyping of Isolates Total DNAof isolates was extracted using Genomic DNA Prep Plus kit(AampA Biotechnology Gdansk Poland) following the man-ufacturerrsquos instructions Additionally as the above method

may result in a low yield of small plasmids plasmid DNAwas isolated using the alkaline lysis method [18] Pulsed-field gel electrophoresis (PFGE) was performed accordingto de Lancastre et al [19] for agarose plugs preparationfollowedby the procedure ofClark et al [20] for total genomicDNA purification Purified DNA in plugs was digested withthe SmaI restriction enzyme (Fermentas Vilnius Lithua-nia) Electrophoresis was performed at 14∘C for 22 h witha pulse time of 1ndash30 s at 6Vcm2 in 05x TBE buffer andthe results were interpreted according to criteria proposedby Tenover et al [21] The Bionumeric software (AppliedMaths Kortrijk Belgium) was used to analyze the similarityof PFGE-banding patterns with an unweighted pair groupmethod with arithmetic average (UPGMA) algorithm andDice coefficient Multilocus variable-number tandem repeat(VNTR) analysis (MLVA) was performed as described byTop et al [22] with modifications given on the website(httpwwwmlvaumcutrechtnl) Multilocus sequence typ-ing (MLST) was performed as described previously [23]Allele numbers and sequence types (STs) were assigned usingE faecium MLST database (httpefaeciummlstnet 16thDecember 2013 date last accessed) PCRdetection of IS16wasperformed as described by Werner et al [24] The Simpsonindex andWallace indexwere calculated using the online toolavailable at httpdarwinphyloviznetComparingPartitions(14th January 2014 date last accessed)

23 Detection of Virulence Genes Antimicrobial ResistanceDeterminants and Plasmid Functional Modules by PCR Enterococcal virulence genes ℎ119910119897Efm 119890119904119901Efm gel asa and cylwere screened as described by Vankerckhoven et al [25]Genes representing four E faecium pilus gene clusters (PGC)were detected by the amplification of representative compo-nents of particular PGC namely fms21pilA (PGC-1) fms17(PGC-2) fms5 (PGC-3) and fms19 (PGC-4) [26] Antimicro-bial resistance determinants were investigated using primersand conditions described by others vanA [20] cat [27]erm(B) [28] tet(M) [29] tet(O) [30] aad6 [31] aac(61015840)-Ie-aph(210158401015840)-Ia aph(210158401015840)-Ib aph(210158401015840)-Ic aph(210158401015840)-Id aph(31015840)-IIIaand ant(41015840)-Ia [32] The presence of the vanA gene for 13isolates from the IO was established in our previous study[33] Detection of 19 rep families and the unique 119903119890119901pMG1gene was performed according to Jensen at al [34] PCRfor the 119903119890119901pLG1 [9] was performed with primers designedpreviously [35] The presence of plasmid addiction systemsrelaxase genes and the intA integrase gene of integrative andconjugative element ICEEfm1 was also verified by PCR [36ndash38]

24 Plasmid Profiling Hybridization Analyses and Tn1546Typing DNA in agarose plugs obtained as described abovewas treated with 14U of S1 nuclease (Takara Bio Japan) for15 minutes at 37∘C and separated by PFGE at 14∘C for 22 hwith pulse time 5ndash35 s at 6Vcm2 in 05x TBE buffer [39]This method allows visualization and determination of thenumber and size of plasmids larger than approximately 30 kbAfter electrophoresis DNA was blotted onto the Hybond-N+ membrane (GE Healthcare Buckinghamshire UK) by

BioMed Research International 3

capillary transfer Probe labeling and signal detection forPFGE-S1 membranes were carried out using the AmershamECL Random-Prime Labeling and Detection System (GEHealthcare) according to the manufacturerrsquos protocol

The 44 kb fragment of the vanRSHAX operon was ampli-fied using Expand Long Template System (Roche DiagnosticsGmbH Mannheim Germany) according to Palepou et al[40] with the following amplification conditions 94∘C for2min 10 cycles of 94∘C for 10 s 56∘C for 30 s and 68∘C for4min 20 cycles of 94∘C for 10 s 56∘C for 30 s and 68∘Cfor 4min (with the elongation time increased by 20 scycle)and 68∘C for 7min L-PCR amplicons were analyzed byrestriction fragment length polymorphism (RFLP) withDdeI(New England Biolabs UK) The whole Tn1546 transposonwas investigated by PCR mapping and sequencing (Table 1and references therein)

25 Conjugation Experiments Conjugation transfer of van-comycin resistancewas examined by cross-streakmating pro-cedure with E faecium strain 643 resistant to rifampin andfusidic acid as recipient Fresh colonies of donors were cross-streaked with recipient on BHI-Agar plates and incubatedovernight at 37∘C Bacterial cells from the streak crossing areawere then incubated overnight in 37∘C on selective mediaTransconjugants were then confirmed by MLVA For isolatesnegative for conjugation in this assay a technique specific forbacteria with low frequency of transfer was used [44]

3 Results

31 Antibiotic Resistance Phenotypes Antimicrobial ResistanceDeterminants and Virulence Genes All analyzed isolateswere resistant to vancomycin and teicoplanin and exhibitedthe presence of vanA determinant (Table 2) Additionallyall of them were penicillin- ampicillin- ciprofloxacin- clin-damycin- and erythromycin-resistant The vast majority ofisolates from both IO and IH showed resistance to rifampinHigh-level resistance to gentamicin (HLGR) kanamycin(HLKR) and streptomycin (HLSR) was more prevalentamong IH isolates which were particularly enriched inaminoglycoside resistance genes aac(61015840)-Ie-aph(210158401015840)-Iaaph(31015840)-IIIa and aad6 (Figure 1) The aph(210158401015840)-Ib geneoccurred in nine isolates and three other tested genes codingfor aminoglycoside resistance that is aph(210158401015840)-Ic aph(210158401015840)-Idand ant(41015840)-Ia were not detected Isolates from both groupscommonly carried erm(B) and tet(M) genes Resistance andintermediate susceptibility to tetracycline was typical for 61and 18 of isolates respectively Intermediate susceptibilityto chloramphenicol and quinupristin-dalfopristin was shownfor 51 and 29 of isolates respectively All isolates weresusceptible to linezolid and tigecycline

Among virulence determinants studied the ℎ119910119897Efm genewas prevalent in both outbreaks while the 119890119904119901Efm gene waspresent mainly in IH (Table 2 and Figure 1) All 119890119904119901Efm-positive isolates harbored the intA integrase gene PGC genesfms21 (PGC-1) fms5 (PGC-3) and fms19 (PGC-4) commonlyoccurred in the whole studied collection while the fms17

00100200300400500600700800900

1000

IO

IH

axe-txe

120596-120576

-120577rel p

AD

1rel p

CIZ

2rel p

EF1

rel p

HT120573

aad6

aph(3998400)hyl E

fmesp

Efm

rep

pMG

1rep

pLG

1

Isol

ates

()

-Ia

aac(6998400)-Ie-aph(2998400998400)- -IIIa

rep2

rep11

rep14

rep17

rep18

Figure 1 Distribution ( of isolates) of plasmid-specific genesselected virulence genes and resistance determinants among iso-lates from outbreak at the Institute of Oncology and Institute ofHematology Warsaw

(PGC-2) was more prevalent among IH than IO isolatesGenes gel asa and cyl were not detected

32 Clonal Relationships among Isolates The clonal structureof outbreak isolates was evaluated with the use of threetyping methods (Table 2 and Figure 2) PFGE analysis andMLVA were performed for the whole set of isolates yieldingaltogether 13 PFGE types (PTs) and eight MLVA types (MTs)Two PTs PT1 and PT6 were further diversified into threeand five subtypes respectively A single novel MT296 wasdetected for the isolate recovered from blood in the IHGenerally the IH isolates showed higher diversity of PTs andMTs compared to the isolates obtained from the IO Forboth hospitals the predominance of specific PTs and MTswas observed namely PT1MT3 and PT5MT10 among theIO isolates and PT6MT159 in the IH Isolates withMT1PT4were observed in the two institutions (a single isolate inboth IO and IH) In the case of two patients two VREfmisolates with different PFGE and MLVA types were obtainedfrom different body sites (rectum stool and blood) Thecomparison of MLVA and PFGE typing results showed agood correlation of both methods (Wallace indices PTMT0994 MTPT 0887) and a higher discriminatory power ofPFGE over MLVA (Simpsonrsquos indices 817 and 795 resp)Further MLST analysis for 20 representatives of different PTsand MTs yielded six sequence types (STs) all belonging tolineages 17 (STs 17 202) 18 (STs 18 262) and 78 (STs 78192) of meroclone CC17Themost common ST18 occurred inboth hospitals however in associationwith various PTsMTsthis ST was also characteristic for two MT1PT4 isolatesmentioned above


Table 1 Primers used in the analysis of Tn1546 transposon

Primer pair Primer names Sequence (51015840-31015840) Position in Tn1546 Application in this study Reference

1 vanRSHAX-1 AGACAAGTCTGAGATTGACCTTGCC 4141ndash4165 PCR [40]vanRSHAX-2 ATATGCTTCAAACCCACTGTTTTCC 8565ndash8589 PCR [40]

2 Tn1546 GGAAAATGCGGATTTACAACGCTAAG 13ndash38 PCR [40]ORF1-5 CACGTCCTGCCGACTATGATTATTT 1900ndash1876 PCR [41]

3 ORF2-F TCATTCCATTTCTGTATTTTCAATTT 3050ndash3086 PCR [42]ORF2-R GCCCATTAGCGGAATACAGA 3770ndash3751 PCR [42]

4 ORF2-F2 ACTAATGTATCTAGGGCTTCA 3710ndash3731 PCR [42]vanR-R GCAATTTCATGTTCATCATCCA 4000ndash3979 PCR [42]

5 vanS AACGCTATTCCAAACTAGAA 4690ndash4710 PCR sequencing [41]vanS-R GTCGGAAGCTCTACCCTAAA 5760ndash5741 PCR sequencing [41]

6 vanS1 ATTGTTCAGCATGGAGGGC 5700ndash5719 PCR sequencing [34]vanH2 GAGCATGGAATGCATCTGCC 6060ndash6041 PCR [34]

7 vanA1 CATGAATAGAATAAAAGTTGCAATA 6978ndash7002 PCR [20]vanX2 TTATTTAACGGGGAAATC 8600ndash8583 PCR sequencing [43]

8 vanX-F ATGGGTATTTTCAGAAGTCCC 8580ndash8601 PCR sequencing [42]vanZ2 AATGGGTACGGTAAACGAGC 10555ndash10536 PCR [34]

ORF1-4 GCATGTAGTGATGAAACACCTAGCTGC 960ndash987 sequencing [41]vanA2 CCCCTTTAACGCTAATACCATCAA 8007ndash7894 sequencing [20]vanY1 AGAGACGAACCATACCCCAA 9200ndash9181 sequencing [42]

vanY2-R AGTATGTGTTGATCCGGGAAAC 9900ndash9922 sequencing this study

991IO PT1-A3622IO PT1-B3612IO PT1-C

PT1-CPT1-C

PT2-BPT2-A

3623IO3628IO988IO3629IO3550IH PT83567IH PT133620IO PT53551IH PT9989IO PT33564IH PT123556IH PT6-C3563IH PT6-E3554IH PT6-A

PT7-BPT7-A

PT4-BPT4-A

PT6-BPT6-D

PT6-A3558IH3555IH3559IH3549IH3560IH990IO3562IH3561IH PT4-C3552IH PT103557IH PT11

60 70 80 90 100 Strain ID

PFGE typemedical centre

Figure 2 PFGE-based dendrogram of selected isolates from outbreaks in IO and IH representing all PTs Normalization performed bythe use of reference Lambda Ladder PFG Marker (New England BioLabs UK) The phylogenetic tree was constructed by the use of Dicecoefficient (optimization 05 band tolerance 13) and UPGMA clustering


Table2Clon

alrelatednessantim

icrobialresistancep

rofilesand

distrib

utionof

resistancea

ndvirulenced

eterminantsam

ongIO

andIH

outbreak

isolates

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

oncology

988

12-A17gt256

32RIF

ermB

ERY

mdashfm

s17fms5fms19

989lowast

159378gt256

256

CHLGEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Ia

cattetM

mdashesphyl

fms5fms19

990

14-A18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

991

31-A

18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

992

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

993

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

994

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

995

31-A

ndgt256

24RIFTE

TermB

tetM

ERY

hyl

fms5fms19

996

31-A

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3612

31-C

ndgt256

48GEN

RIFT

ETermB

tetM

ERY

hyl

fms5fms19

3613

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3614

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3615

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3616

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3617

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3618

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3620

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

hyl

fms17

fms5fms19

3621

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3622

31-B

18gt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3623

31-C

ndgt256

48RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3624

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3625

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3626

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3627

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

mdashfm

s17fms5fms19

3628

31-C

ndgt256

64RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3629

72-B18gt256

48GEN

KANT

ETaac(61015840)-Ie-aph(210158401015840

)-IaermB

tetM

ERY

hyl

fms17

fms5fms19

3630

105ndgt256

64GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERYTE

Tmdash

fms17

fms5fms19

IO(N

=27)

CHL(1)GEN

(9)KAN(8)

STR(6)TE

T(20)R

IF(26)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(8)aa

d6(6)erm

B(26)tetM

(26)

ERY(23)T

ET(1)

hyl(21)

esp(1)

fms17(8)fm

s5(27)fms19(27)


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom

ycinteicoplaninpenicillinampicillin

ciprofl

oxacinerythromycinand

clind

amycinbfiveisolatesw

ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und

onetetracycline-resistant

isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin

increased(512mgL)one

isolatew

ithcatsho

wed

aninterm

ediateresistancetochloramph

enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam

epatientsldquoXrdquo

andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets


33 Tn1546 Structures and Transferability of VancomycinResistance All isolates exhibited the presence of 44 kb L-PCR product containing the vanRSHAX operon and showedthat the DdeI restriction pattern is identical to the E faeciumBM4147 control VanA strain Further PCR mapping andsequencing showed the presence of ISEf1 inserted at the posi-tion 9147 nt of Tn1546 (numbering according to the GenBanksequence M97297) that is within the vanX-vanY intergenicregion The 51015840GACTGAAA duplication was observed at theinsertion site ISEf1 was present in all but two isolates withthe prototype Tn1546 One of the isolates was derived fromIO and the other from IH and each of them showed a uniquePFGE type PT3 and PT10 respectively (Table 2) Similarly allisolates except for the two mentioned above exhibited theG7747C point mutation of Tn1546 located within the vanAgene resulting in the amino acid substitution V257F Bothisolates with the prototype Tn1546 showed higher teicoplaninMIC values compared to the isolates with Tn1546 ISEf1

Conjugation experiments were performed for all 44isolates and 34 of them were able to transfer vancomycinresistance to the E faecium 643 recipient All donors pro-duced transconjugants with cross-streak mating except for asingle isolate which required use of the method designed forstrains with low-level conjugation frequencies [44] Suscep-tibility testing of transconjugants (a single transconjugant foreach donor) showed a concomitant transfer of erythromycinresistance in 32 cases One of these transconjugants showedalso HLGR phenotype and one was additionally resistant totetracycline

34 Diversity of Plasmid-Associated Gene Content PCRscreening revealed the presence of plasmid replication genesof the 11990311989011990114pRI and 11990311989011990117pRUM families as well as 119903119890119901pLG1in all isolates Four other rep groups 1199031198901199012pRE25 (119899 = 16)11990311989011990111pEF1071 (119899 = 15) 11990311989011990118pEF418 (119899 = 19) and 119903119890119901pMG1 (119899 =18) were also detected First three of themwere characteristicmainly for the IH outbreak while 119903119890119901pMG1 occurred mainlyin isolates from IO (Figure 1) The number of rep genes perisolate varied from three to seven and the average numberof plasmid rep genes per isolate was 450 however this valuewas lower for IO (407) compared to IH (518) Analysis ofdistribution of relaxase genes revealed the common pres-ence of two relaxases 119903119890119897pCIZ2 and 119903119890119897pEF1 while 119903119890119897pHT120573was predominantly detected in IO and the distribution ofthis gene was completely concordant with the presence of119903119890119901pMG1 Additionally one IH isolate had the 119903119890119897pAD1 geneScreening for plasmid toxin-antitoxin systems (TA) resultedin the detection of axe-txe and 120596-120576-120577 while other TA genesincluding ccd higBA mazEF par parDE phd-doc relBE andvagCD were absent in the studied group All but one 120596-120576-120577-positive isolates were also 1199031198901199012pRE25-positive and only one1199031198901199012pRE25-positive lacked the 120596-120576-120577 gene

35 Colocalization of vanA Determinant and Other PlasmidGenes Twenty-seven selected isolates (11 from IO and 16from IH) of various clonal types as defined byMLVAMLSTand PFGE were subjected to PFGE-S1 analysis followed

by Southern blot hybridization (Figure 3 and Table 3) withprobes specific for genes detected earlier by PCR such asvanA seven rep genes (1199031198901199012pRE25 11990311989011990111pEF1071 11990311989011990114pRI111990311989011990117pRUM 11990311989011990118pEF418 119903119890119901pLG1 and 119903119890119901pMG1) genes oftwo plasmid TA systems (120596-120576-120577 and axe-txe) and threeother plasmid-associated genes (pilA ℎ119910119897Efm and aac(61015840)-Ie-aph(210158401015840)-Ia) Altogether 122 plasmid bands were visualized inPFGE-S1 with 56 megaplasmids bands greater than 100 kbThe average number of plasmid bands in PFGE-S1 analysiswas 435 with very similar values for both IO and IHoutbreaks Thirty plasmid bands hybridized with the vanAprobe that is three of the analyzed isolates carried two vanAplasmids The majority of vanA plasmids were lt30ndash100 kbin size additionally four megaplasmids (170 200 240 and315 kb) were associated with the vanA determinant AmongvanA plasmids 24 were 11990311989011990117pRUM replicons mostly of 50 kband 100 kb The 100 kb plasmid was present in 15 isolates ofvarious clonal backgrounds in both IH and IO and most ofthese isolates easily produced transconjugants Moreover thefirst observed VREfm isolates in both IH and IO carried suchplasmids but in different clonal backgroundsThe 50 kb plas-mid was associated exclusively with MT159 isolates whichdiffered however in their PFGE patterns Six such isolatesoccurred exclusively in IH and all of them were deficientin conjugation Five of vanA-11990311989011990117pRUM plasmids of varioussizes hybridized also with other rep genes such as 119903119890119901pLG1(two 100 kb plasmids and one 315 kb plasmid) 11990311989011990118pEF418(one 100 kb plasmid) and both 1199031198901199012pRE25 and 11990311989011990118pEF418 (a40 kb plasmid) Two isolates of MT1PT4ST18 from IO andIH both had 100 kb 11990311989011990117pRUM plasmids but they differed inthe content of other plasmids (Table 3 isolates labeled C andX) moreover 100 kb plasmids from IO isolate additionallycarried 11990311989011990118pEF418 Among the remaining plasmids otherthan 11990311989011990117pRUM replicons vanA plasmids a single 70 kbplasmid had 1199031198901199012pRE25 and a 240 kb megaplasmid hybridizedwith 119903119890119901pLG1 and 11990311989011990118pEF418 Considering other tested genesthe pilA genewas associatedwith 18 vanA plasmids includingall the 100 kb plasmids with 11990311989011990117pRUM genes of the 120596-120576-120577TAsystemwere present on a single 40 kb plasmid with 1199031198901199012pRE2511990311989011990117pRUM and 11990311989011990118pEF418 and on a 70 kb plasmid carrying1199031198901199012pRE25 The 240 kb vanA megaplasmid carried also theaac(61015840)-Ie-aph(210158401015840)-Ia resistance gene Each of the two isolateswith the prototypeTn1546 carried two vanAplasmids (lt30 kbin both isolates and megaplasmids of 170 and 200 kb) thatdid not hybridize with any of probes used in this study Insummary almost all IO isolates showed the presence of 100 kbvanA plasmids with 11990311989011990117pRUM and pilA genes while in IHthe diversity of vanA plasmids was higher with pRUM-likereplicons of both 50 kb and 100 kb Information concerningother than vanA plasmids of E faecium that was obtainedduring the study is summarized in Table 3

4 Discussion

This study provides the molecular characteristics of VREfmoutbreak isolates with the special focus on the role of


Table 3 Plasmid profiles and colocalization of particular genes on vanA and other plasmids among selected 27 VREfm isolates

Lettercode Isolate MTPTST vanA plasmids in kb (hybridizing probes) Other plasmids in kb (hybridizing probes)

IO (119873 = 11)A 988 12-A17 100 (rep17 pilA) lt30 150 (pilA) 180 270

B 989lowast 159378 lt30 200110 (rep2 rep17 pilA axe-txe) 175 230 (rep18 119903119890119901

1199011198711198661

hyl aac (61015840)-Ie-aph (210158401015840)-Ia pilA) 310 gt400

C 990 14-A18 100 (rep17 rep18 pilA) 175 230 (rep18) 280 340D 991 31-A18 100 (rep17 pilA) 65 (119903119890119901

1199011198721198661

) 240 (1199031198901199011199011198711198661

hyl pilA) 295E 3612 31-Cnd 100 (rep17 pilA) 70 (119903119890119901

1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290F 3620 105262 100 (rep17 pilA) 65 150 (pilA) 290G 3622 31-B18 100 (rep17 pilA) 70 (119903119890119901

1199011198721198661

) 240 (1199031198901199011199011198711198661

hyl pilA) 290H 3623 31-Cnd 100 (rep17 pilA) 70 (119903119890119901

1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290I 3624 105262 100 (rep17 pilA) 40 65 (rep2 120596-120576-120577) 150 (pilA) 290J 3628 31-Cnd 100 (rep17 pilA) 70 (119903119890119901

1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661

hyl aac (61015840)-Ie-aph (210158401015840)-Ia pilA)270

IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310

M 3550x 7818 45 (rep2 rep17 rep18 120596-120576-120577) 60 (rep17 pilA) 70 195 (1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA) 230 (rep18) 315

N 3551 159978 45 (rep17) 40 (rep2 120596-120576-120577) 70 (rep17 pilA axe-txe) 100 190(1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18) 290O 3552lowast 71018 lt30 170 65 (rep17 pilA) 240 (rep18 119903119890119901

1199011198711198661

pilA 120596-120576-120577)

P 3554 1596-A192 50 (rep17)40 (rep2 120596-120576-120577) 75 (rep17 pilA axe-txe) 100 (pilA) 220

(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)

Q 3555 1596-Bnd 50 (rep17) 40 (120596-120576-120577) 75 (rep17 pilA axe-txe) 100 (pilA) 220(1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)

R 3556 1596-Cnd 50 (rep17) 40 (120596-120576-120577) 75 (rep17 pilA axe-txe) 100 (pilA) 240(rep18 119903119890119901

1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)S 3557x 2961117 100 (rep17 pilA) 40 (120596-120576-120577) 235 (pilA)

T 3558 1596-And 50 (rep17) 40 (rep2 120596-120576-120577) 75 (rep17 pilA axe-txe) 100 (pilA) 240(rep18 119903119890119901

1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

U 3559 1596-Dnd 50 (rep17)40 (120596-120576-120577) 75 (rep17) 100 (rep17) 240 (rep18 aac(61015840)-Ie-aph (210158401015840)-Ia pilA) 330 (119903119890119901

1199011198711198661

axe-txe aac(61015840)-Ie-aph (210158401015840)-Ia pilA)

V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)X 3562 14-B18 100 (rep17 pilA) 240 (119903119890119901

1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)

40 (rep2 120596-120576-120577) 75 (rep17 pilA axe-txe) 100 (pilA) 240(rep18 119903119890119901

1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia) 250a 3567y 1441318 315 (rep17 119903119890119901

1199011198711198661

pilA) 75 (axe-txe aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)Letter code of each isolate corresponds to the designation used in Figure 2 nd not determined two isolates with the prototype Tn1546marked with an asteriskxyisolates from the same patients ldquoXrdquo and ldquoYrdquo isolates positive in conjugation


Outbreak A (IO) Outbreak B (IH)

120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z a

(a)A B C D E F G H I J K L MN O P Q R S T U VWX Y Z a

∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe


(d)A B C D E F G H I J K L MN O P Q R S T U VWX Y Z a

∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-

(g)A B C D E F G H I J K L MN O P Q R S T U VWX Y Z a

∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)

Figure 3 PFGEof S1-digested totalDNAof selected 27VREfm isolates visualized by ethidiumbromide staining (a) and subjected to Southernhybridization with the following probes vanA (b) 11990311989011990117pRUM (c) axe-txe (d) 119903119890119901pLG1 (e) pilA (f) aac(61015840)-Ie-aph(210158401015840)-Ia (g) 11990311989011990118pEF418 (h)1199031198901199012pRE25 (i) and 120596-120576-120577 (j) Lanes Andasha isolates designation as described in Table 3

MGE such as Tn1546-type transposons and vanA plasmidsacting as mediators of vancomycin resistance transfer Theinvestigated group of isolates originated from two hospitalsThe Institute of Oncology and The Institute of Hematologyand Transfusion Medicine in Warsaw where two VREfmoutbreaks occurred concomitantly Immunocompromisedpatients of oncological and hematological wards are knownto be of special risk for VRE colonization and infection [45]Such susceptibilitywas especially evident during the outbreakin the IH where three bloodstream infections caused byVRE were reported The proximity of the two hospitals inthe city and the simultaneous emergence of both outbreakswhich lasted for a few months suggested the possibility ofVRE transmission between the two institutions althoughthe investigation of available medical documentation doneindependently in IO and IH revealed no obvious routes suchas patient transfer between the two hospitals or from the same

third hospital or utilization of commondiagnostic equipmentjust before or during the outbreak periodThe involvement ofhospital personnel in VREfm transfer was also excluded

Molecular typing methods such as PFGE analysis andMLVA have shown a divergent clonal structure of isolatesSuch a situation is typical for VanA hospital outbreaks [46ndash49] MLST performed for representative isolates includedall of them into the hospital-associated lineages 17 18and 78 formerly described as CC17 complex [3] Isolatesbelonging to this meroclone display common features suchas ampicillin and ciprofloxacin resistance the prevalenceof IS16 119890119904119901Efm and ℎ119910119897Efm and enrichment in microbialsurface components recognizing adhesive matrix molecules(MSCRAMMs) including pili genes [26 50] However iso-lates from each institution showed some specific featuressuch as predominance of certain PTsMTs and differencesin distribution of virulence resistance and plasmid-specific


genes This observation would suggest the existence of twoseparate endemic subpopulations without much exchange ofstrains prior to the introduction of vanA-carrying MGE

VanA phenotype in both outbreaks was associated in thevast majority of cases with an acquisition of the same specificvariant of Tn1546 transposon with ISEf1 and a mutation inthe vanA gene Tn1546-type transposons show significantvariability due to point mutations deletions and presence ofvarious ISs [41 42 47 51] and thus analysis of transposonstructure provides valuable epidemiological information forinvestigation of VRE outbreaks The ISEf1 insertion in thevanX-vanY intergenic region was described previously forhospital VREfm in Portugal and Germany [47 51] and themutation in the vanA gene was not reported before Thepresence of the same type of Tn1546 in both outbreaksprovides a strong indication of either a common source ortransmission of VREfm between the two hospitals

Hybridization studies on representative isolates revealedthe presence of a 100 kb plasmid carrying the vanA determi-nant as well as rep17 typical for pRUM plasmid and the pilAgene in several isolates from both institutions Most of theseisolates readily produced transconjugants suggesting thatthis plasmidmight play the principal role in the outbreakTheobserved concomitant transfer of erythromycin resistance isin agreement with the colocalization of the ermB gene onpRUM[52] Other frequently encountered vanA plasmidwas50 kb in size and also represented the 11990311989011990117pRUM repliconhowever it lacked pilA and all isolates with this plasmid werenegative in conjugation The 50 kb plasmid was exclusivelyassociatedwith isolates ofMT159ST78 andobserved solely inIH The recently emerged lineage 78 of the hospital-adaptedE faecium shows increased epidemic properties and playsan important role in HAIs [53] Thus strains of this lineageharboring the 50 kb nonconjugative vanA plasmid werelikely to be spreading by efficient clonal dissemination duringthe outbreak in IH Association of van determinants withpRUM-type plasmids was described also by others [7 10 54]Both 100 kb and 50 kb plasmids lacked the axe-txe TA systemgenes typical for pRUM [10 52] suggesting the possiblecommon origin of these two plasmids Further studies basedon whole plasmid sequencing are indispensable to elucidatethe possible evolution of these plasmids during the outbreakAlthough 100 kb plasmids were found in two isolates of thesame clonal characteristics from IO and IH (Table 3 isolatesC and X) differences in the plasmid content do not allowus to indicate these isolates as a direct epidemiological linkbetween two hospitals In a few cases the vanA gene wasassociated with other replicons typical for pLG1 pRE25 andpEF418 Such vanA plasmids were observed also in otherstudies [7 10] The prevalent distribution of 119903119890119901pLG1 as wellas its predominant presence on plasmids over 200 kb in sizeis also in agreement with earlier studies which showed thatall VREfmmegaplasmids with the defined replicon type werealways pLG1-like [7 8]The association ofVanAdeterminantswith various plasmids during one outbreak may be causedby the Tn1546 transposition among plasmids andor plasmidrecombination The latter process may yield plasmids withmore than a single rep gene which was also observed in the

current study both for vanA- and other plasmids The roleof plasmid mosaics in the dissemination of Tn1546 amongVRE was emphasized recently by Freitas et al [7] Finally forsome vanA plasmids and other plasmids the replicon typescould not be established (13 and 34 of observed plasmidsresp) indicating that the pool of E faecium plasmids remainsonly partly explored [10] and that there is the need for furtherstudies of these epidemiologically important elements

5 Conclusions

Molecular analysis of VanA VREfm outbreaks revealed thatTn1546ISEf1 elements associated with pRUM-like plasmidswere the key mediators of vancomycin-resistant E faeciumdissemination among the investigated group of oncologi-calhematological patients Horizontal gene transfer of thewhole vanA plasmids andor Tn1546 transposons in endemicpopulations of nosocomial E faecium is suggested as thepotential way of VanA phenotype spread in the analyzedoutbreaks The enrichment in different plasmid-associatedgenes antimicrobial resistance and potential virulence deter-minants in the investigated population emphasizes theimpact of mobile genetic elements on the epidemiology andevolution of VREfm

Conflict of Interests

The authors declare that they have no conflict of interestsregarding to the publication of this paper

Acknowledgments

The authors thank J Top for assigning new MTs This pub-lication made use of the Enterococcus faeciumMLST website(httpefaeciummlstnet) hosted at the Imperial College ofthe University of Oxford and funded by the Wellcome TrustThis work was supported by Grant no N401588540 fromthe National Science Centre Poland and by the NationalProgamme for the Protection of Antibiotics (NarodowyProgramOchrony AntybiotykowmdashNPOA) funding from theMinistry of Health Poland

References

[1] R Leclercq E Derlot J Duval and P Courvalin ldquoPlasmid-mediated resistance to vancomycin and teicoplanin in Entero-coccus faeciumrdquoThe New England Journal of Medicine vol 319no 3 pp 157ndash161 1988

[2] A H C Uttley C H Collins J Naidoo and R C GeorgeldquoVancomycin-resistant enterococcirdquoTheLancet vol 1 no 8575-8576 pp 57ndash58 1988

[3] R J L Willems J Top M van Santen et al ldquoGlobal spreadof vancomycin-resistant Enterococcus faecium from distinctnosocomial genetic complexrdquo Emerging Infectious Diseases vol11 no 6 pp 821ndash828 2005

[4] G Werner T M Coque A M Hammerum et al ldquoEmergenceand spread of vancomycin resistance among enterococci inEuroperdquo Eurosurveillance vol 13 no 47 p 19046 2008


[5] R J Willems J Top W van Schaik et al ldquoRestricted gene flowamong hospital subpopulations of Enterococcus faeciumrdquoMBiovol 3 no 4 pp e00151ndashe00112 2012

[6] M Arthur C Molinas F Depardieu and P Courvalin ldquoChar-acterization of Tn1546 a Tn3-related transposon conferringglycopeptide resistance by synthesis of depsipeptide peptido-glycan precursors in Enterococcus faecium BM4147rdquo Journal ofBacteriology vol 175 no 1 pp 117ndash127 1993

[7] A R Freitas C Novais A P Tedim et al ldquoMicroevolutionaryevents involving narrow host plasmids influences local fixationof vancomycin-resistance in Enterococcus populationsrdquo PLoSONE vol 8 no 3 Article ID e60589 2013

[8] A R Freitas T M Coque C Novais et al ldquoHuman andswine hosts share vancomycin-resistant Enterococcus faeciumCC17 and CC5 and Enterococcus faecalis CC2 clonal clustersharboring Tn1546 on indistinguishable plasmidsrdquo Journal ofClinical Microbiology vol 49 no 3 pp 925ndash931 2011

[9] J A Laverde Gomez W van Schaik A R Freitas et al ldquoAmultiresistance megaplasmid pLG1 bearing a hylEfm genomicisland in hospital Enterococcus faecium isolatesrdquo InternationalJournal ofMedicalMicrobiology vol 301 no 2 pp 165ndash175 2011

[10] T C S Rosvoll T Pedersen H Sletvold et al ldquoPCR-based plas-mid typing in Enterococcus faecium strains reveals widely dis-tributed pRE25- pRUM- pIP501- and pHT120573-related repliconsassociated with glycopeptide resistance and stabilizing toxin-antitoxin systemsrdquo FEMS Immunology and Medical Microbiol-ogy vol 58 no 2 pp 254ndash268 2010

[11] H Sletvold P J Johnsen O-G Wikmark G S SimonsenA Sundsfjord and K M Nielsen ldquoTn1546 is part of alarger plasmid-encoded genetic unit horizontally disseminatedamong clonal Enterococcus faecium lineagesrdquo Journal of Antimi-crobial Chemotherapy vol 65 no 9 pp 1894ndash1906 2010

[12] H Tomita C Pierson S K Lim D B Clewell and Y Ike ldquoPos-sible connection between a widely disseminated conjugativegentamicin resistance (pMG1-like) plasmid and the emergenceof vancomycin resistance in Enterococcus faeciumrdquo Journal ofClinical Microbiology vol 40 no 9 pp 3326ndash3333 2002

[13] W Zhu P R Murray W C Huskins et al ldquoDissemina-tion of an Enterococcus Inc18-like vanA plasmid associatedwith vancomycin-resistant Staphylococcus aureusrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 10 pp 4314ndash43202010

[14] W ZhuN C Clark L KMcDougal J Hageman L CMcDon-ald and J B Patel ldquoVancomycin-resistant Staphylococcus aureusisolates associated with Inc18-like vanA plasmids in MichiganrdquoAntimicrobial Agents and Chemotherapy vol 52 no 2 pp 452ndash457 2008

[15] European Committee on Antimicrobial Susceptibility Testing(EUCAST) ldquoBreakpoint tables for interpretation of MICsand zone diameters Version 30rdquo 2013 httpwwweucastorgfileadminsrcmediaPDFsEUCAST filesDisk test doc-umentsEUCAST Breakpoint table v 30pdf

[16] Clinical and Laboratory Standards Institute (CLSI) ldquoPerfor-mance standards for antimicrobial susceptibility testingrdquo 20thinformational supplement CLSI document M100-S20 CLSIWayne Pa USA 2010

[17] Recommandations 2011 du Comite de lrsquoantibiogramme de laSFM Societe Francaise de Microbiologie (SFM) Paris France

[18] S Handwerger J Skoble L F Discotto and M J PuccildquoHeterogeneity of the vanA gene cluster in clinical isolates ofenterococci from the northeasternUnited StatesrdquoAntimicrobialAgents and Chemotherapy vol 39 no 2 pp 362ndash368 1995

[19] H de Lencastre E P Severina R B Roberts et al ldquoTestingthe efficacy of a molecular surveillance network methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) genotypes in six hospi-tals in the metropolitan New York City areardquo Microbial DrugResistance vol 2 no 3 pp 343ndash351 1996

[20] N C Clark R C Cooksey B C Hill J M Swensonand F C Tenover ldquoCharacterization of glycopeptide-resistantenterococci from US hospitalsrdquo Antimicrobial Agents andChemotherapy vol 37 no 11 pp 2311ndash2317 1993

[21] F C Tenover R D Arbeit R V Goering et al ldquoInterpretingchromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis criteria for bacterial strain typingrdquoJournal of Clinical Microbiology vol 33 no 9 pp 2233ndash22391995

[22] J Top L M Schouls M J M Bonten and R J L WillemsldquoMultiple-locus variable-number tandem repeat analysis anovel typing scheme to study the genetic relatedness and epi-demiology of Enterococcus faecium isolatesrdquo Journal of ClinicalMicrobiology vol 42 no 10 pp 4503ndash4511 2004

[23] P Ruiz-Garbajosa M J M Bonten D A Robinson et alldquoMultilocus sequence typing scheme for Enterococcus faecalisreveals hospital-adapted genetic complexes in a background ofhigh rates of recombinationrdquo Journal of Clinical Microbiologyvol 44 no 6 pp 2220ndash2228 2006

[24] G Werner C Fleige U Geringer W van Schaik I Klareand W Witte ldquoIS element IS16 as a molecular screening toolto identify hospital-associated strains of Enterococcus faeciumrdquoBMC Infectious Diseases vol 11 article 80 2011

[25] V Vankerckhoven T van Autgaerden C Vael et al ldquoDevel-opment of a multiplex PCR for the detection of asaI gelEcylA esp and hyl genes in enterococci and survey for virulencedeterminants among european hospital isolates of Enterococcusfaeciumrdquo Journal of Clinical Microbiology vol 42 no 10 pp4473ndash4479 2004

[26] J R Galloway-Pena S R Nallapareddy C A Arias G MEliopoulos and B E Murray ldquoAnalysis of clonality and antibi-otic resistance among early clinical isolates of Enterococcusfaecium in the United Statesrdquo Journal of Infectious Diseases vol200 no 10 pp 1566ndash1573 2009

[27] C A Widdowson P V Adrian and K P Klugman ldquoAcqui-sition of chloramphenicol resistance by the linearization andintegration of the entire staphylococcal plasmid pC194 intothe chromosome of Streptococcus pneumoniaerdquo AntimicrobialAgents and Chemotherapy vol 44 no 2 pp 393ndash395 2000

[28] J Sutcliffe T Grebe A Tait-Kamradt and L WondrackldquoDetection of erythromycin-resistant determinants by PCRrdquoAntimicrobial Agents and Chemotherapy vol 40 no 11 pp2562ndash2566 1996

[29] N Doherty K Trzcinski P Pickerill P Zawadzki and C GDowson ldquoGenetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniaerdquo Antimi-crobial Agents and Chemotherapy vol 44 no 11 pp 2979ndash29842000

[30] K Trzcinski B S Cooper W Hryniewicz and C G Dow-son ldquoExpression of resistance to tetracyclines in strains ofmethicillin-resistant Staphylococcus aureusrdquo Journal of Antimi-crobial Chemotherapy vol 45 no 6 pp 763ndash770 2000

[31] C Poyart L Jardy G Quesne P Berche and P Trieu-CuotldquoGenetic basis of antibiotic resistance in Streptococcus agalactiaestrains isolated in a French hospitalrdquo Antimicrobial Agents andChemotherapy vol 47 no 2 pp 794ndash797 2003


[32] S B Vakulenko S M Donabedian A M Voskresenskiy MJ Zervos S A Lerner and J W Chow ldquoMultiplex PCR fordetection of aminoglycoside resistance genes in enterococcirdquoAntimicrobial Agents and Chemotherapy vol 47 no 4 pp 1423ndash1426 2003

[33] D Zabicka J Strzelecki A Wozniak et al ldquoEfficiency of theCepheid Xpert vanAvanB assay for screening of coloniza-tion with vancomycin-resistant enterococci during hospitaloutbreakrdquo Antonie van Leeuwenhoek International Journal ofGeneral andMolecular Microbiology vol 101 no 3 pp 671ndash6752012

[34] L B Jensen L Garcia-Migura A J S Valenzuela M LoslashhrH Hasman and F M Aarestrup ldquoA classification system forplasmids from enterococci and other Gram-positive bacteriardquoJournal of Microbiological Methods vol 80 no 1 pp 25ndash432010

[35] E Wardal I Gawryszewska W Hryniewicz and E SadowyldquoAbundance and diversity of plasmid-associated genes amongclinical isolates of Enterococcus faecalisrdquo Plasmid vol 70 no 3pp 329ndash342 2013

[36] E M Moritz and P J Hergenrother ldquoToxin-antitoxin systemsare ubiquitous and plasmid-encoded in vancomycin-resistantenterococcirdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 1 pp 311ndash316 2007

[37] P Goicoechea M Romo T M Coque et al ldquoIdentificationof enterococcal plasmids by multiplex-PCR-based relaxasetypingrdquo Clinical Microbiology and Infection vol 14 supplement7 p S484 2008

[38] E Sadowy A Sienko I Gawryszewska A Bojarska K Mali-nowska and W Hryniewicz ldquoHigh abundance and diver-sity of antimicrobial resistance determinants among earlyvancomycin-resistant Enterococcus faecium in Polandrdquo Euro-pean Journal of Clinical Microbiology and Infectious Diseasesvol 32 no 9 pp 1193ndash1203 2013

[39] B M Barton G P Harding and A J Zuccarelli ldquoA generalmethod for detecting and sizing large plasmidsrdquo AnalyticalBiochemistry vol 226 no 2 pp 235ndash240 1995

[40] M-F I Palepou A-M A Adebiyi C H Tremlett L B Jensenand N Woodford ldquoMolecular analysis of diverse elementsmediating VanA glycopeptide resistance in enterococcirdquo Jour-nal of Antimicrobial Chemotherapy vol 42 no 5 pp 605ndash6121998

[41] J Y Huh W G Lee K Lee W S Shin and J H YooldquoDistribution ofinsertion sequences associated with Tn1546-like elements amongEnterococcus faecium isolates frompatientsin Koreardquo Journal of Clinical Microbiology vol 42 no 5 pp1897ndash1902 2004

[42] M Talebi M R Pourshafie M Oskouii and S S EshraghildquoMolecular analysis of vanHAX element in vancomycin resis-tant enterococci isolated from hospitalized patients in TehranrdquoIranian Biomedical Journal vol 12 no 4 pp 223ndash228 2008

[43] H-S Yu S-Y Seol and D-T Cho ldquoDiversity of Tn1546-likeelements in vancomycin-resistant enterococci isolated fromhumans and poultry in Koreardquo Journal of Clinical Microbiologyvol 41 no 6 pp 2641ndash2643 2003

[44] J M Manson L E Hancock and M S Gilmore ldquoMechanismof chromosomal transfer of Enterococcus faecalis pathogenicityisland capsule antimicrobial resistance and other traitsrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 107 no 27 pp 12269ndash12274 2010

[45] R Patel ldquoClinical impact of vancomycin-resistant enterococcirdquoJournal of Antimicrobial Chemotherapy vol 51 no 3 pp iii13ndashiii21 2003

[46] S Valdezate C Miranda A Navarro et al ldquoClonal outbreakof ST17 multidrug-resistant Enterococcus faecium harbouringan Inc18-likeTn1546 plasmid in a haemo-oncology ward of aSpanish hospitalrdquo Journal of Antimicrobial Chemotherapy vol67 no 4 pp 832ndash836 2012

[47] G Werner I Klare C Fleige and W Witte ldquoIncreasing ratesof vancomycin resistance among Enterococcus faecium isolatedfrom German hospitals between 2004 and 2006 are due towide clonal dissemination of vancomycin-resistant enterococciand horizontal spread of vanA clustersrdquo International Journal ofMedical Microbiology vol 298 no 5-6 pp 515ndash527 2008

[48] M Kawalec J Kedzierska A Gajda et al ldquoHospital outbreakof vancomycin-resistant enterococci caused by a single cloneof Enterococcus raffinosus and several clones of Enterococcusfaeciumrdquo Clinical Microbiology and Infection vol 13 no 9 pp893ndash901 2007

[49] M Kawalec M Gniadkowski and W Hryniewicz ldquoOutbreakof vancomycin-resistant enterococci in a hospital in GdanskPoland due to horizontal transfer of different Tn1546-liketransposon variants and clonal spread of several strainsrdquo Journalof Clinical Microbiology vol 38 no 9 pp 3317ndash3322 2000

[50] J Sillanpaa V P Prakash S R Nallapareddy and B EMurray ldquoDistribution of genes encoding MSCRAMMs and piliin clinical and natural populations of Enterococcus faeciumrdquoJournal of ClinicalMicrobiology vol 47 no 4 pp 896ndash901 2009

[51] CNovais A R Freitas J C Sousa F Baquero TMCoque andLV Peixe ldquoDiversity of Tn1546 and its role in the disseminationof vancomycin-resistant enterococci in PortugalrdquoAntimicrobialAgents and Chemotherapy vol 52 no 3 pp 1001ndash1008 2008

[52] R Grady and F Hayes ldquoAxe-Txe a broad-spectrum proteictoxin-antitoxin system specified by a multidrug-resistant clini-cal isolate of Enterococcus faeciumrdquoMolecularMicrobiology vol47 no 5 pp 1419ndash1432 2003

[53] M M Lam T Seemann N J Tobias et al ldquoComparativeanalysis of the complete genome of an epidemic hospitalsequence type 203 clone of vancomycin-resistant Enterococcusfaeciumrdquo BMC Genomics vol 1 no 14 p 595 2013

[54] E Bjoslashrkeng G Rasmussen A Sundsfjord L Sjoberg KHegstad and B Soderquist ldquoClustering of polyclonal VanB-type vancomycin-resistant Enterococcus faecium in a low-endemic area was associated with CC17-genogroup strainsharbouring transferable vanB2-Tn5382 and pRUM-like repAcontaining plasmids with axe-txe plasmid addiction systemsrdquoAPMIS vol 119 no 4-5 pp 247ndash258 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


types on Inc18 pRUM-like pMG1-like and pLG1 plasmids[7ndash13] however our knowledge of vanA plasmids and theirepidemiology is still far from being satisfactory and thecommon presence of plasmids with Tn1546 belonging tounknown replicon types has been shown [10 14]

The aim of this study was to characterize E faeciumVanAisolates from the outbreaks that concomitantly took placein hospital wards of two neighboring medical centers TheInstitute of Oncology (IO) and The Institute of Hematologyand Transfusion Medicine in Warsaw (IH) The investigationfocused on the clonal relationships among isolates as well asanalysis of the Tn1546 transposon structure and colocaliza-tion of vanA with other plasmid genes in order to elucidatethe role of particular MGE during a VREfm outbreak inhospital settings

2 Materials and Methods

21 Outbreak Description Bacterial Isolates and Suscepti-bility Testing Forty-four vancomycin-resistant E faeciumoutbreak isolates were collected between February and June2009 in two neighboring hospitals inWarsawThe Institute ofOncology (IO) andThe Institute of Hematology and Transfu-sion Medicine (IH) 776- and 198-bed hospitals respectivelyFirst VREfmwas isolated from stool of 46-year-old patient on4th February at the Gastroenterology Clinic of IO Until theend of February eight more cases were reported in majorityfrom the Clinic of Lymphatic System Cancers of IO Fromthe 31st March till the 18th of April 18 VREfm were isolatedmainly from patients of this clinic (16 cases) and fromtwo patients of the Gastroenterology Clinic SimultaneouslyVREfm cases were reported in IH wards with the first twoisolations on the 5th February from rectum and stool of theHematologyWard patient and a patient from the ICU respec-tively One more isolate was obtained 10 days later in theSurgery Ward and till the end of June 14 other VREfm caseswere reported in the Hematology Ward of IH Altogetherthe outbreaks affected 42 patients including 27 patients ofIO (27 stool isolates) and 15 patients of IH (13 stool 1 urine3 blood isolates) Antimicrobial susceptibility of collectedisolates was determined using the Etest method (bioMerieuxMarcy lrsquoEtoile France) for glycopeptide susceptibility testingand broth microdilution method for other antimicrobialsThe results were interpreted following the breakpoints ofthe European Committee on Antimicrobial SusceptibilityTesting (EUCAST) [15] for chloramphenicol erythromycinciprofloxacin and tetracycline the Clinical and LaboratoryStandards Institute (CLSI) [16] breakpoints were applied andin the case of kanamycin and clindamycin the breakpointsproposed by the Societe Francaise de Microbiologie (SFM)[17] were used The Enterococcus faecalis strain ATCC29212was used for quality control purposes during testing Efaecium BM4147 was used as a control VanA strain in thisstudy

22 DNA Isolation and Genotyping of Isolates Total DNAof isolates was extracted using Genomic DNA Prep Plus kit(AampA Biotechnology Gdansk Poland) following the man-ufacturerrsquos instructions Additionally as the above method

may result in a low yield of small plasmids plasmid DNAwas isolated using the alkaline lysis method [18] Pulsed-field gel electrophoresis (PFGE) was performed accordingto de Lancastre et al [19] for agarose plugs preparationfollowedby the procedure ofClark et al [20] for total genomicDNA purification Purified DNA in plugs was digested withthe SmaI restriction enzyme (Fermentas Vilnius Lithua-nia) Electrophoresis was performed at 14∘C for 22 h witha pulse time of 1ndash30 s at 6Vcm2 in 05x TBE buffer andthe results were interpreted according to criteria proposedby Tenover et al [21] The Bionumeric software (AppliedMaths Kortrijk Belgium) was used to analyze the similarityof PFGE-banding patterns with an unweighted pair groupmethod with arithmetic average (UPGMA) algorithm andDice coefficient Multilocus variable-number tandem repeat(VNTR) analysis (MLVA) was performed as described byTop et al [22] with modifications given on the website(httpwwwmlvaumcutrechtnl) Multilocus sequence typ-ing (MLST) was performed as described previously [23]Allele numbers and sequence types (STs) were assigned usingE faecium MLST database (httpefaeciummlstnet 16thDecember 2013 date last accessed) PCRdetection of IS16wasperformed as described by Werner et al [24] The Simpsonindex andWallace indexwere calculated using the online toolavailable at httpdarwinphyloviznetComparingPartitions(14th January 2014 date last accessed)

23 Detection of Virulence Genes Antimicrobial ResistanceDeterminants and Plasmid Functional Modules by PCR Enterococcal virulence genes ℎ119910119897Efm 119890119904119901Efm gel asa and cylwere screened as described by Vankerckhoven et al [25]Genes representing four E faecium pilus gene clusters (PGC)were detected by the amplification of representative compo-nents of particular PGC namely fms21pilA (PGC-1) fms17(PGC-2) fms5 (PGC-3) and fms19 (PGC-4) [26] Antimicro-bial resistance determinants were investigated using primersand conditions described by others vanA [20] cat [27]erm(B) [28] tet(M) [29] tet(O) [30] aad6 [31] aac(61015840)-Ie-aph(210158401015840)-Ia aph(210158401015840)-Ib aph(210158401015840)-Ic aph(210158401015840)-Id aph(31015840)-IIIaand ant(41015840)-Ia [32] The presence of the vanA gene for 13isolates from the IO was established in our previous study[33] Detection of 19 rep families and the unique 119903119890119901pMG1gene was performed according to Jensen at al [34] PCRfor the 119903119890119901pLG1 [9] was performed with primers designedpreviously [35] The presence of plasmid addiction systemsrelaxase genes and the intA integrase gene of integrative andconjugative element ICEEfm1 was also verified by PCR [36ndash38]

24 Plasmid Profiling Hybridization Analyses and Tn1546Typing DNA in agarose plugs obtained as described abovewas treated with 14U of S1 nuclease (Takara Bio Japan) for15 minutes at 37∘C and separated by PFGE at 14∘C for 22 hwith pulse time 5ndash35 s at 6Vcm2 in 05x TBE buffer [39]This method allows visualization and determination of thenumber and size of plasmids larger than approximately 30 kbAfter electrophoresis DNA was blotted onto the Hybond-N+ membrane (GE Healthcare Buckinghamshire UK) by





3 Results



00100200300400500600700800900

1000

IO

IH

axe-txe

120596-120576

-120577rel p

AD

1rel p

CIZ

2rel p

EF1

rel p

HT120573

aad6

aph(3998400)hyl E

fmesp

Efm

rep

pMG

1rep

pLG

1

Isol

ates

()

-Ia

aac(6998400)-Ie-aph(2998400998400)- -IIIa

rep2

rep11

rep14

rep17

rep18


















PT1-CPT1-C

PT2-BPT2-A


PT7-BPT7-A

PT4-BPT4-A

PT6-BPT6-D


60 70 80 90 100 Strain ID




Table2Clon

alrelatednessantim

icrobialresistancep

rofilesand

distrib

utionof

resistancea

ndvirulenced

eterminantsam

ongIO

andIH

outbreak

isolates

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

oncology

988

12-A17gt256

32RIF

ermB

ERY

mdashfm

s17fms5fms19

989lowast

159378gt256

256

CHLGEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Ia

cattetM

mdashesphyl

fms5fms19

990

14-A18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

991

31-A

18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

992

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

993

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

994

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

995

31-A

ndgt256

24RIFTE

TermB

tetM

ERY

hyl

fms5fms19

996

31-A

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3612

31-C

ndgt256

48GEN

RIFT

ETermB

tetM

ERY

hyl

fms5fms19

3613

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3614

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3615

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3616

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3617

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3618

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3620

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

hyl

fms17

fms5fms19

3621

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3622

31-B

18gt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3623

31-C

ndgt256

48RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3624

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3625

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3626

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3627

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

mdashfm

s17fms5fms19

3628

31-C

ndgt256

64RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3629

72-B18gt256

48GEN

KANT

ETaac(61015840)-Ie-aph(210158401015840

)-IaermB

tetM

ERY

hyl

fms17

fms5fms19

3630

105ndgt256

64GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERYTE

Tmdash

fms17

fms5fms19

IO(N

=27)

CHL(1)GEN

(9)KAN(8)

STR(6)TE

T(20)R

IF(26)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(8)aa

d6(6)erm

B(26)tetM

(26)

ERY(23)T

ET(1)

hyl(21)

esp(1)

fms17(8)fm

s5(27)fms19(27)


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom


ciprofl


clind


ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und


isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin


isolatew

ithcatsho

wed

aninterm


enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam


andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results



00100200300400500600700800900

1000

IO

IH

axe-txe

120596-120576

-120577rel p

AD

1rel p

CIZ

2rel p

EF1

rel p

HT120573

aad6

aph(3998400)hyl E

fmesp

Efm

rep

pMG

1rep

pLG

1

Isol

ates

()

-Ia

aac(6998400)-Ie-aph(2998400998400)- -IIIa

rep2

rep11

rep14

rep17

rep18


















PT1-CPT1-C

PT2-BPT2-A


PT7-BPT7-A

PT4-BPT4-A

PT6-BPT6-D


60 70 80 90 100 Strain ID




Table2Clon

alrelatednessantim

icrobialresistancep

rofilesand

distrib

utionof

resistancea

ndvirulenced

eterminantsam

ongIO

andIH

outbreak

isolates

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

oncology

988

12-A17gt256

32RIF

ermB

ERY

mdashfm

s17fms5fms19

989lowast

159378gt256

256

CHLGEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Ia

cattetM

mdashesphyl

fms5fms19

990

14-A18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

991

31-A

18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

992

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

993

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

994

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

995

31-A

ndgt256

24RIFTE

TermB

tetM

ERY

hyl

fms5fms19

996

31-A

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3612

31-C

ndgt256

48GEN

RIFT

ETermB

tetM

ERY

hyl

fms5fms19

3613

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3614

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3615

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3616

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3617

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3618

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3620

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

hyl

fms17

fms5fms19

3621

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3622

31-B

18gt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3623

31-C

ndgt256

48RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3624

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3625

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3626

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3627

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

mdashfm

s17fms5fms19

3628

31-C

ndgt256

64RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3629

72-B18gt256

48GEN

KANT

ETaac(61015840)-Ie-aph(210158401015840

)-IaermB

tetM

ERY

hyl

fms17

fms5fms19

3630

105ndgt256

64GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERYTE

Tmdash

fms17

fms5fms19

IO(N

=27)

CHL(1)GEN

(9)KAN(8)

STR(6)TE

T(20)R

IF(26)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(8)aa

d6(6)erm

B(26)tetM

(26)

ERY(23)T

ET(1)

hyl(21)

esp(1)

fms17(8)fm

s5(27)fms19(27)


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom


ciprofl


clind


ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und


isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin


isolatew

ithcatsho

wed

aninterm


enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam


andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















PT1-CPT1-C

PT2-BPT2-A


PT7-BPT7-A

PT4-BPT4-A

PT6-BPT6-D


60 70 80 90 100 Strain ID




Table2Clon

alrelatednessantim

icrobialresistancep

rofilesand

distrib

utionof

resistancea

ndvirulenced

eterminantsam

ongIO

andIH

outbreak

isolates

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

oncology

988

12-A17gt256

32RIF

ermB

ERY

mdashfm

s17fms5fms19

989lowast

159378gt256

256

CHLGEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Ia

cattetM

mdashesphyl

fms5fms19

990

14-A18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

991

31-A

18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

992

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

993

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

994

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

995

31-A

ndgt256

24RIFTE

TermB

tetM

ERY

hyl

fms5fms19

996

31-A

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3612

31-C

ndgt256

48GEN

RIFT

ETermB

tetM

ERY

hyl

fms5fms19

3613

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3614

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3615

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3616

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3617

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3618

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3620

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

hyl

fms17

fms5fms19

3621

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3622

31-B

18gt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3623

31-C

ndgt256

48RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3624

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3625

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3626

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3627

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

mdashfm

s17fms5fms19

3628

31-C

ndgt256

64RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3629

72-B18gt256

48GEN

KANT

ETaac(61015840)-Ie-aph(210158401015840

)-IaermB

tetM

ERY

hyl

fms17

fms5fms19

3630

105ndgt256

64GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERYTE

Tmdash

fms17

fms5fms19

IO(N

=27)

CHL(1)GEN

(9)KAN(8)

STR(6)TE

T(20)R

IF(26)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(8)aa

d6(6)erm

B(26)tetM

(26)

ERY(23)T

ET(1)

hyl(21)

esp(1)

fms17(8)fm

s5(27)fms19(27)


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom


ciprofl


clind


ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und


isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin


isolatew

ithcatsho

wed

aninterm


enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam


andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Clon

alrelatednessantim

icrobialresistancep

rofilesand

distrib

utionof

resistancea

ndvirulenced

eterminantsam

ongIO

andIH

outbreak

isolates

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

oncology

988

12-A17gt256

32RIF

ermB

ERY

mdashfm

s17fms5fms19

989lowast

159378gt256

256

CHLGEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Ia

cattetM

mdashesphyl

fms5fms19

990

14-A18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

991

31-A

18gt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

992

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

993

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

994

31-A

ndgt256

32RIF

ermB

tetM

ERY

hyl

fms5fms19

995

31-A

ndgt256

24RIFTE

TermB

tetM

ERY

hyl

fms5fms19

996

31-A

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3612

31-C

ndgt256

48GEN

RIFT

ETermB

tetM

ERY

hyl

fms5fms19

3613

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3614

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3615

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3616

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3617

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3618

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3620

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

hyl

fms17

fms5fms19

3621

31-C

ndgt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3622

31-B

18gt256

32RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3623

31-C

ndgt256

48RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3624

105262gt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3625

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3626

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

nomdash

fms17

fms5fms19

3627

105ndgt256

48GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERY

mdashfm

s17fms5fms19

3628

31-C

ndgt256

64RIFTE

TermB

tetM

ERY

hyl

fms5fms19

3629

72-B18gt256

48GEN

KANT

ETaac(61015840)-Ie-aph(210158401015840

)-IaermB

tetM

ERY

hyl

fms17

fms5fms19

3630

105ndgt256

64GEN

KANR

IFSTR

TET

aac(61015840)-Ie-aph(210158401015840

)-Iaaad6erm

BtetM

ERYTE

Tmdash

fms17

fms5fms19

IO(N

=27)

CHL(1)GEN

(9)KAN(8)

STR(6)TE

T(20)R

IF(26)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(8)aa

d6(6)erm

B(26)tetM

(26)

ERY(23)T

ET(1)

hyl(21)

esp(1)

fms17(8)fm

s5(27)fms19(27)


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom


ciprofl


clind


ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und


isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin


isolatew

ithcatsho

wed

aninterm


enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam


andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Con

tinued

Isolate

MTPT

ST

MIC

VAN

(mgL)

MIC

TEI

(mgL)

Resistancep

heno

typesa

bRe

sistanced

eterminantsb

cCotransferred

resistance

Virulence

genes

Piligenesd

Instituteof

hematolog

y3549

117-A202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

tetM

ERYGEN

esphyl

fms17

fms5fms19

3550

x7818

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)-IIIa

aad6

ermB

ERY

esphyl

fms17

fms5fms19

3551

159978gt256

32GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840

)IIIaaad6

ermB

ERY

esphyl

fms17

fms19

3552lowast

71018

gt256gt256

KANR

IFSTR

aph(31015840)-I

IIaaad6ermB

tetM

ERY

esphyl

fms17

fms5fms19

3553

117-Andgt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)Ibaph(31015840

)IIIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3554

1596-

A19

2gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3555

1596-

Bnd

gt256

48GEN

KANR

IFSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3556

1596-

Cnd

gt256

32GEN

KANSTR

aac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3557

x2961117gt256

48KA

NR

IFaph(31015840)-I

IIaaad6catermB

tetM

ERY

esphyl

fms17

fms19

3558

1596-

Andgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5fms19

3559

1596-

Dndgt256

48GEN

KANP

ENaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaaad6ermB

noesphyl

fms17

fms5

3560

17-B18

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

ERY

hyl

fms5fms19

3561

114-C

202gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5

3562

14

-B18gt256

32GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(31015840)-I

IIaerm

BtetM

nohyl

fms5fms19

3563

1596-

E192gt256

48GEN

KANR

IFaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaaad6

ermB

noesphyl

fms17

fms5fms19

3564

y71218

gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-Ibaph(31015840)-I

IIaerm

BtetM

ERY

esphyl

fms17

fms5fms19

3567

y1441318gt256

48GEN

KANR

IFT

ETaac(61015840)-Ie-aph(210158401015840

)-Iaaph(210158401015840

)-IbermB

ERY

hyl

fms17

fms5fms19

IH(N

=17)

GEN

(15)K

AN(17)STR

(6)

TET(7)RI

F(15)

cat(1)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(15)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(10)erm

B(17)tetM

(8)

ERY(11)G

EN(1)

hyl(17)

esp(14)

fms17(15)fms5

(15)fms19(15)

IOan

dIH

(N=44

)CH

L(1)GEN

(24)K

AN

(25)STR

(12)T

ET(27)R

IF(41)

cat(2)aac(61015840

)-Ie-ap

h(210158401015840

)-Ia(23)aph

(210158401015840

)-Ib(10)

aph(31015840)-I

IIa(15)aad

6(16)erm

B(43)tetM

(34)

ERY(34)T

ET(1)

GEN

(1)

hyl(38

)esp(15)

fms17(23)fms5

(42)fms19(42)

ndnot

determ

inednono

transcon

jugantso

btainedtwoiso

latesw

iththep

rototype

Tn1546

markedwith

anasteris

k isolatesfrom

bloo

da alliso

lates

resis

tant

tovancom


ciprofl


clind


ithtet(M

)sho

wed

interm

ediatesusceptib

ility

totetracyclin

eand

oneisolatewith

tet(M

)was

susceptib

leto

thiscompo

und


isolate(3567)

was

negativ

efor

thed

eterminantsteste

dforfou

risolatesw

ith1198861198861198896theM

ICvalues

forstre

ptom

ycin


isolatew

ithcatsho

wed

aninterm


enicol

c alliso

latesw

erep

ositive

forthe

vanA

genedallisolatesp

ositive

forthe

pilA

genex

yiso

latesfrom

thesam


andldquoY

rdquoRIFrifam

pinCH

Lchoram

phenicolG

ENgentamicin

(HLG

R)STR

stre

ptom

ycin

(HLSR)K

ANkanam

ycin

(HLK

R)T

ETtetracyclineFo

rthe

summarized

resultsthe

numbero

fisolates

isgivenin

brackets







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







4 Discussion







1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






1199011198711198661



1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 240 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661


1199011198721198661

) 235 (1199031198901199011199011198711198661

hyl pilA) 290

K 3629 72-B18 100 (rep17 pilA) 235 (rep18 1199031198901199011199011198711198661


IH (119873 = 16)

L 3549 117-A202 100 (rep17 pilA)70 (119903119890119901

1199011198721198661

pilA) 240 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph(210158401015840)-Ia pilA) 310





1199011198711198661

pilA 120596-120576-120577)


(aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18 1199031198901199011199011198711198661

aac(61015840)-Ie-aph (210158401015840)-Ia pilA)


aac (61015840)-Ie-aph (210158401015840)-Ia) 240 (rep18)


1199011198711198661



1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)


1199011198711198661


V 3560 17-B18 100 (rep17 1199031198901199011199011198711198661

pilA) 85 (pilA) 235 (rep18 1199031198901199011199011198711198661

aac (61015840)-Ie-aph (210158401015840)-IapilA)

W 3561 114-C202 100 (rep17 1199031198901199011199011198711198661

pilA) 240 (1199031198901199011199011198711198661


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Y 3563 1596-E19250 (rep17) 240 (rep18 119903119890119901

1199011198711198661

pilAaac(61015840)-Ie-aph(210158401015840)-Ia)


1199011198711198661

aac (61015840)-Ie-aph (210158401015840)-Ia pilA)

Z 3564y 71218 70 (rep2 pilA 120596-120576-120577)60 (119903119890119901

1199011198711198661

) 75 (rep17 axe-txe) 130 (rep18) 200(1199031198901199011199011198711198661


1199011198711198661




120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



120582120582

∽ 4365kb∽ 388kb∽ 3395kb

∽ 1455kb

∽ 291kb∽ 242kb∽ 194kb

∽ 97kb∽ 485 kb



∽ 4365 kb∽ 388kb∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

anA

(b)


rep17pRUM

(c)

axe-txe



∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb∽ 485 kb

reppLG1

(e)


pilA

(f)


-Iaaac(6998400)-Ie-aph(2998400998400)-


∽ 388kb∽ 4365 kb

∽ 3395kb∽ 291kb∽ 242kb∽ 194kb

∽ 1455kb

∽ 97kb

∽ 485 kb

rep18pEF418

(h)


rep2pRE25

(i)

120596-120576-120577


(j)










5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusions




Acknowledgments


References
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article molecular analysis of vana outbreak of...

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