Faculty of Health Sciences
Mobile genetic elements causing plasticity
in E. faecium
—
Audun Sivertsen
A dissertation for the degree of Philosophiae Doctor – December 2016
Mobilegeneticelements
causingplasticityin
Enterococcusfaecium
AUDUNSIVERTSEN
AdissertationforthedegreeofPhilosophiaeDoctor
December2016
FacultyofHealthSciences
DepartmentofMedicalBiology
Host-MicrobeInteractionsgroup
UiT–TheArcticUniversityofNorway
1
2
TableofContents
Acknowledgements...........................................................................................................5
Preface...................................................................................................................................7
Thebasictraitsofenterococci......................................................................................8
ClinicalfeaturesofE.faecium........................................................................................8
Epidemiology...............................................................................................................................9
Virulence.......................................................................................................................................9
Host-microbeinteractions................................................................................................................10
Antimicrobialresistanceandtreatmentoptions................................................12
Vancomycin................................................................................................................................13
Beyondvancomycin–availableantibioticstotreatVREfmandtheirresistance
mechanisms...............................................................................................................................16
Whydoesresistanceaccumulationoccur?.....................................................................18
Horizontalgenetransferandmobilegeneticelements....................................20
Horizontalgenetransfermechanisms.........................................................................................21
Type4secretionsystems..................................................................................................................21
Plasmids......................................................................................................................................22
Transposons,integrativeconjugativeelementsandgenomicislands..................24
Howdomobilegeneticelementspersistinhosts?.......................................................25
Toxin/Antitoxinsystems...................................................................................................................25
Restriction/modification(R/M)systems...................................................................................26
CRISPR-Cassystems.............................................................................................................................27
Howdowecomparebacteria?...................................................................................28
DNAfingerprints......................................................................................................................29
ReasonstoperformcomparisonsofbacteriausingDNAsequences.....................29
Allele-basedclusteringmethods....................................................................................................30
Wholegenome-basedcomparisons..............................................................................................30
WhatdocomparativemethodstellaboutE.faecium?................................................32
Summaryofpapers........................................................................................................35
Paper1:AmulticentrehospitaloutbreakinSwedencausedbyintroductionofa
vanB2transposonintoastablymaintainedpRUM-plasmidinanEnterococcus
faeciumST192clone...............................................................................................................35
Paper2:SilencedvanAgeneclusteronatransferableplasmidcauseoutbreakof
Vancomycin-VariableEnterococci.....................................................................................35
Paper3:TheEnterococcusCassetteChromosome:anSCCmec-likemobilisable
elementinenterococci...........................................................................................................36
Discussion.........................................................................................................................38
EpidemiologyofE.faeciuminScandinavia.....................................................................38
Wholegenomesequencingenhancespathogensurveillance..................................39
Phylogenetic-andtransmissionanalysesusingPFGEand/orcoregenomeSNPs
onlytellpartsofthestory.....................................................................................................41
Challengeswithvancomycinsusceptibilitydetermination......................................43
MGEanalyses:wetgunpowderinthestartinggun......................................................45
DiscoveryandanalysisofMGEslikeECC:What’snext?..............................................48
References........................................................................................................................50
3
4
Acknowledgements
OnethingIhavelearnedduringtheseyearsof‘sciencing’,isthatproducingaPhD
candidateisinfactaresultofteamefforts.ThewholeenvironmentinwhichIhave
beengiventrusttoplayaroundinalab,tosuggestexperimentalandanalyticideas
likeI’mawalking&talkingbudgetdeficit,andhavethepossibilitytoerrwithout
theworldfallingapart,haspavedthewayforsubmissionofthisthesis.
I am greatly indebted to my main supervisor Kristin. Since 2010, when I first
walkedaroundaskingpeoplewhethertheyhadaresearchprojecttospareforme,
youhavealwaysknownwhattodoandhowtodoit.Iamalwaysbaffledbyyour
greattalentoflisteningtothechaosIsometimesperceivethestateofmyresearch
tobein,thinkforfiveseconds,andgiveapromptresolutiontoallmytroubles.
You’retheChaosPilot!
IalsoneedtogivegreatthankstomysecondsupervisorTorunn,whosetalentfor
precision writing and focus on the conceptual thinking and scientific reasoning
behindeverythingwedoreallyhelpedmestructuremythoughtsandimproved
mywritingskills.You’reoneofthe(elsewhererarelyencountered)benevolent3rd
reviewers!
Ihavehadagreattimewiththetwoofyouassupervisors.Weshareacommon
senseofhumourandgeneraljoiedevivre,whichledmetofeelthatthedoorsto
yourofficeswerealwaysopen,bothliterallyandfiguratively.
Next,Ihaveenjoyedtofollowofacollectionofvibrantresearchgroups.BothHostMicrobeinteractionsandK-Res,andthemembersofthesegroupsinadditionto
Micro-Popandthepaediatricsinfectiongroupshavebeenreallygreattotalkto
andbeinspiredby.We’vestarteddoingsomeseriousbioinformaticslately,andI
thankEspenÅ,Joachim,JessinandEspenTangenforexchangingideas,teaching
away what HPC is, and creating competition in the ‘heard it in the grapevine’exersiseof“Hey…seenthistoolyet?It’samazing!”.
5
Ialsohavetoextendahigh-fivetothefellow(andex)PhDcandidatesI’veshared
most time with: Theresa, Ilya, Clement, Vidar, and the late newcomer and
officemateAdriana.It’salwaysnicetohavepeoplewhounderstandtheprinciple
behind“don’tworkwhileI’mdisturbing”.
SinceI’mamedicaldoctor,Ididnotknowanythingaboutpipetting,mixinggels,
what‘hygieniclabconditions’are,wheretotossmys**twhenI’mdonewithitand
otherpresumablyessentiallab-relatedhoo-hahwhenIstarted.Luckily,theglass
house has some excellent elephant handlers in technicians, and if you’re lucky,
theymayhelpyououtwithdifficulttaskslike‘howtoweargloves–andwhich?
Really,which?’.Bettina,Bjørg,Tracy&Alena,you’rethebest!
Got to thank my social network for schticking around, and gracefully and
sarcasticallyshootmedownwhenmyintricatestoriesofgenomicislands,spooky
bugs and correct pipetting (thanks, technicians!) become too tedious to follow.
AndformyfellowassociatesinKjoSivDesIndustriesformanufacturingbeer!
Myparents.Mom,yourendlessrowsofbookshelvesleftmesittingaroundreading
ridiculousamountsofbooks.Theyprovideperspectiveandideasitishardtocome
byanyotherway,andwasimportantindevelopingmycuriousness.Nottoforget
reading speed – THAT is needed to complete a thesis! Dad: these endless
discussionsofpolitics,societyandlifewekeephavinghavesurelyhelpedmelearn
whatindependentthinkingis.Ithankyoubothforgreatadviceinlife.IthinkI’ve
donejustfine,yeah?
Lastly,Ineedtoappreciatehavingyouinmylife,Bente.Iloveyou!HowdidIever
manage to get such a lovely human being as my partner?! Let me dedicate this
thesis to you, to state that the end of my days/years/decades as a student are
finallyover.Now:timetolookforwardforbothofus.Exitingtimes.Iwannabring
youalong!
6
Preface
Clinicalmicrobiologyisavasttopicwithsomeoverarchingquestionsresearchers
aimtoanswer.Wewanttoknowhowtoidentifyandtreatinfections.Wewantto
avoid the spread of infectious agents by stopping transmission through
surveillanceandappropriatecontainmentmeasures.Wewanttounderstandthe
basalbiologicalprocessesexplaininghowpathogensspread,producedisease,and
evade treatment. Pathogen discovery, surveillance and disease prevention has
evolvedandmaturedgreatlythroughthelatestcenturies(1),andtheproblems
we encounter today and the methods we use to solve them are both in rapid
change. Infections is a major cause of morbidity and mortality in humans, and
during the last century, the revolution of the antimicrobial made us optimistic
abouteradicatingthisproblem.Indeed,manyeasilytreateddiseasesarenolonger
suchalikelycauseofdeath.Asantimicrobialshavebeenincreasinglyusedand
misused,thebugshaveevolvedthemselves,andsomearenolongerresponding
to antimicrobial treatment. WHO estimates that resistant bugs are again to
become a major cause of death relative to other diseases because of resistance
developmentinimportantpathogens(2)eventhoughtheseprojectionsstillneed
tobevalidated(3).Resistantorganismsposeathreattomodernmedicineaswe
know it, as many of the species which have developed the most resistance, are
particularly adapted to persist in health care facilities and cause disease in
patientswithotherseriousconditionswhootherwiseenjoysthebestandmost
advancedtreatmentmodernhealthcareisabletoprovide.
The research presented in this thesis focus on basal biological processes
governingthespread,evolutionandresistancetotreatmentinenterococci,and
Enterococcus faecium in particular. This is in nature a ubiquitous bug, which
recentlyhasemergedasamulti-resistantpathogenwithinhealthcareinstitutions.
7
Thebasictraitsofenterococci
E.faeciumwasfirstdescribedbydistinctphenotypictraitsin1899byThiercelin,
whoisolatedthebacteriumfromacaseofinfectiveendocarditis(4,5).E.faecium
wasdescribedasasturdyGram-positivediplococcusabletosurviveat60°C,grow
at10-45°C,growinsaltyconditionsupto6,5%NaCl,growinbasicmediaofupto
pH9,6,andtogrowinmediacontaining40%bile.Althoughtwodifferentspecies
today, E. faecium and E. faecalis, the two enterococci most prone to causing
infections in humans, weren’t considered two different species before the midsixties (6, 7). Enterococci reacted to serum of group D by the Lancefield’s
precipitintest,causingthemtobeclassifiedasgroupDstreptococci(8–10)until
theywerereclassifiedtoenterococciin1984(11)duetoadvancesinmolecular
classificationschemesshowingthatenterococciandstreptococciaretoodistantly
relatedgeneticallytobelongtothesamegenus.Althoughenterococcifirstwere
identified as parts of the human intestinal microbiota, we now know them as
ubiquitous in nature (12). Found in and on other animals, insects and fish, on
plants, in water and food, we now know of 54 species within the Enterococcus
genus(12–19).Mostofthemhavebeencharacterizedduringthelast20-30years.
Thus, this genus is likely to expand further as todays screening methods yield
substantiallyhigherthroughputs.
ClinicalfeaturesofE.faecium
Historically, enterococci has been isolated predominantly as causes of
bacteraemia,endocarditisandUTIs(20).Enterococciarealsocapableofinvading
otherfociasanopportunisticpathogen,ifapatient’sabilitytocleartheinfection
has been compromised. E. faecium has been included as one of the five major
multi-resistantpathogenspredominantlycausingthelion’sshareofnosocomial
infections–theESKAPEpathogens–theotheronesbeingStaphylococcusaureus,
Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa and
Enterobacterspecies(21).Thecomplexitiesofmodernmedicineleadtoaseverely
compromisedimmunedefenceinmanypatientsformanytreatments.Enterococci
and E. faecium in particular have become increasingly prevalent as causes of
nosocomial serious infections the last thirty years (22). This development is
8
pairedwithemergenceofahospital-associatedcladedemonstratinganincreasing
antimicrobialresistancepatternaswellaspresenceoffactorslikelyinvolvedin
virulenceandadaptationtohospitalenvironments(23,24).Thesethemeswillbe
discussedinlatersections.
Epidemiology
Enterococcihavebecomemoreprevalentascauseofnosocomialinfectionsthe
last30years.Thisdevelopmenthasbeenassociatedwithuseofthirdgeneration
cephalosporins in U.S.A., of which enterococci are naturally resistant (25). In
Europe,vancomycinresistantenterococcus(VRE)occurrencehasbeenlinkedto
the glycopeptide farm-animal growth-promoter avoparcin (26), but the overall
prevalenceisstilllowerthantheU.S.Thelargerimpactofenterococcalinfections
duringthelasttwodecadescanbeexplainedbyariseofE.faeciuminfections(22,
27),astherateofE.faecalisinfectionshasremainedstable.Increasednumbersof
enterococcalinfectionshasco-occurredwithincreasedantimicrobialresistance,
which mostly has occurred in E. faecium (27). The latest systemic surveys
assessing hospital-acquired infections described enterococci as the second or
thirdmostcommonpathogenintheU.S.A.andEuroperespectively(28,29).
Virulence
Causal factors of pathogenicity and virulence in E. faecium has to be seen in
connection to the extensive resistance levels this species demonstrates when
encountered in hospital settings, permitting survival and transfer to patients
susceptibletoinfection.Nosingledefiningvirulencefactorhasbeenidentifiedin
E. faecium, but several genes are enriched in clinical lineages that may aid in
invasiveness. As an opportunistic pathogen mostly causing severe disease in
immunocompromised hosts, E. faecium has proven to be elusive to accurately
characteriseintermsofvirulence.
It is difficult to assess the contribution of any particular putative virulence
determinant in E. faecium as there are few reliable ways of knocking out or
insertingageneintoexperimentalstrains(30).Thesearchforvirulencefactorsin
E.faeciumhasoftenconsistedoffindingsecretion-andcellwall-associatedgenes
9
enrichedinhospital-associatedstrains(31).Thisincludesgenesassociatedwith
biofilmformation[espandsgrA(32,33)],collagenadhesion[acm,(34)]andother
genes associated with the enterococcal cell-wall [efaAfm, hyl, ecbA, scm, orf903,
orf2010andorf2514(33,35,36)].Hospital-acquiredinfection(HAI)isolatesare
alsoenrichedwithgenesconstructingpili[pilA,pilB,ebpfm(37,38)].Somegenes
havebeenassociatedwithvirulenceinE.faecalisandcorrespondingvirulencein
E.faeciumisextrapolated,butofthefewgenesofwhichexperimentaldataexist,
acm,esp,andebpfmhavebeenshowntocontributetovirulencebyexertingthe
assumedphenotypesininvivosettings(38–40).Inaddition,twogenesassociated
with two different phosphotransferase systems (PTS) are associated with
improved intestinal colonization during antibiotic treatment [ptsD (41)] or
implicatedinbiofilmformationandpathogenesisofendocarditis[bepA(42)].
Althoughthesefactorsmaycontributetovirulence,itisimportanttoconsiderthat
infectionwithE.faeciumismostlikelytooccurindebilitatedhosts.Obtaininga
more comprehensive pathogenesis model would require examining the whole
system in which infection occurs, which includes health state of the host, the
intestinalenvironmentwhereE.faeciumresides,andobtainingaricherpictureof
howallpartsinteractwitheachother.
Host-microbeinteractions
Thereisadeepandcurrentlynotfullydefinedinteractionbetweenhumansand
themicrobiotaresidinginandonus.Manyrecentarticleshaveemergedthatrefer
totheimbalanceofbacterialspeciesinthegutwhereovergrowthofpathogenic
bacteria(ofwhichE.faeciumisone)leadstoincreasedriskofdisease,andrefer
tothisimbalanceas‘dysbiosis’(43).Disentanglinghowchangesingutmicrobiota
occur and what consequence they confer is a complex subject, and some
controversyexistsoverwhetherdysbiosis/imbalanceisasatisfactoryexplanation
or whether other models need to be used in order to gauge how bacterial
composition in the gut contributes to human diseases (44, 45). Outside the
hospital and the selective effect of antimicrobial exposure, E. faecium from the
commensal clade seem to outcompete nosocomial isolates in persistence over
time(46).Thissuggeststhatmodernadvancedmedicaltreatment,mostlydonein
medicalinstitutions,maycreateanignitionfordysbiosisandactsasa“virulence
10
enabler”byitself.Thepaperspresentedinthenextparagraphsaimtodescribe
howVREhavebeenimpliedindysbiosis,diseaseandprotectionfromdisease.
Brandletal.(47)presentsamodelofhowenterococcimayendupdominatingthe
gut flora as a result of broad-spectrum antibiotic use, thereby facilitating host
invasion. They experimented on a feedback-loop where Paneth cells in the gut
lining excretes an antimicrobial peptide with activity against Gram-positive
bacteriacalledRegIIIγinresponsetopresenceoflipopolysaccharidesoriginating
fromGram-negativebacteria(48).WhenGram-negativebacteriaareclearedfrom
the gut, the intraluminal concentration of RegIIIγ decreases, permitting VRE to
dominateandtherebycreatingadysbiosis.VREovergrowthisthusperceivedto
increasetheriskofinvasiveness.
Asacontinuation,Hendrickxetal.(49)createdasimilarVRE-dominantdysbiosis
andfounddramaticchangesofthegutliningofmiceasaresponsetoVREinjection
andconcurrentantimicrobialtreatment,withhostfactorssegregatingfromthe
gutwallandcreatinganextracellularmatrixaroundtheVREtoprotectthegut
epithelium. The apical cell wall mucus layer thickness decreased during VRE
dysbiosis. Epithelial architecture was also altered, which they were able to
connect to intraluminal biochemical changes resulting in displacement of
intercellular adherence junctions from the cell wall to the extracellular matrix
surrounding the VRE. All these intra-luminal changes were clinically observed
onlyasmildnon-inflammatorydiarrhoea.
Enterococciinhabitacrowdedenvironmentinsidethehumangutandcompete
with other species to survive. Their low virulence in immunocompetent hosts
perturbs clear assertions of whether enterococci and their hosts co-exists in
antagonistic,neutralorsynergisticfashion(50).Infact,VREintroducedpriorto
induced cecal puncture giving a polymicrobial invasive infection actually
protected mice by giving lower polymicrobial bacterial loads, milder
inflammatoryreactionsandswifterrecoverycomparedtoVREnegativecontrol
mice (51). E. faecium excretes the biofilm-associated peptidoglycan hydrolase
SagA (52), which according to recent studies is able to prevent Salmonella
pathogenesisthroughdegradingpartsoftheSalmonellacellwall(53).Thesecell
wall fragments induce changes in the immunological pathways of the gut
11
epithelium which prevent Salmonella pathogenesis during infection (54)
suggestingsynergybetweenpatientandE.faeciumduringSalmonellainfectionof
immunocompetenthosts.
Antimicrobialresistanceandtreatmentoptions
Already in 1929, Alexander Fleming noted that enterococci were resistant to
penicillin(55),asapreludetoreportsofthelimitedtreatmentoptionsoftoday.
Intrinsicresistancereferstoanantimicrobialdrugnotworkingduetoinherent
featuresinaspecies,likerestrictingdrugaccessibilitytotargetornothavingthe
drug target at all. Acquired resistance occurs when the bacterium is baseline
susceptible,butdevelopsresistanceeitherbysomaticmutationorbyacquisition
ofgenesbyhorizontaltransfer.Tostartwith,E.faeciumisonbaselineintrinsically
non-susceptibletopenicillin,ampicillin,cephalosporinsandotherß-lactams(25,
56)duetomutationsinthepenicillin-bindingproteinPBP5(57–60)(geneisalso
horizontallytransferrable(61))aswellaspresenceofothergeneticdeterminants
(62). Enterococci are in vivo resistant to clindamycin by efflux pumps,
trimethoprim-sulfamethoxazolebymissingtarget,andclinicallyachievablelevels
of most aminoglycosides due to enzymatic degradation (56, 63). E. faecium has
also swiftly gained resistance through mutations or by acquiring resistance
determinants towards antimicrobials such as quinolones, rifampicin and
chloramphenicol,precludingtheiruse(56,64–68).
Eventhoughenterococciareinherentlylow-levelresistanttowardsß-lactamsand
aminoglycosidessuchasgentamicin,invivothesedrugsincombinationappears
to have had synergistic effect as long as the bacterium does not harbour any
additional gentamicin resistance determinants providing high-level resistance
(60, 69–71), and has been considered standard treatment for decades. Mobileelement located enzymes modifying aminoglycosides are widespread in
enterococci (60, 72), effectively obstructing the ampicillin plus gentamicin
treatment alternative. In the case of high-level resistance and likely treatment
failureusingthisregimen,vancomycinhasbeenareliablealternative.
12
Vancomycin
Vancomycin was isolated from Amycolatopsis orientalis in 1953 as a cell wall
compoundactiveagainstGram-positives,butwasconsideredsecondarytoother
antimicrobials with better bactericidal effects, and as initially thought, better
toxicity profiles (73). This changed in the 70s and 80s, as ß-lactam-resistant
staphylococci (MRSA) began to emerge, prompting better characterizations of
Figure1.Globalprevalenceofantimicrobialresistance,andtrendsinScandinavia
Left:maps showingglobalburdenofresistancetowardsvancomycin,gentamicinand
aminopenicillins from resistancemap.cddep.org. Data from 2014. Regions for which
datadoesnotexistismarkedingrey,resistancedataaspercentageofisolatesinblue
shades,thedarkershadethehigherpercentageareresistant.Right:Resistancetrends
aspercentageresistantisolates/year,relatingtoNorway,SwedenandDenmarkforthe
correspondingantimicrobials.NotedifferencesinY-axis.Surveillancedataandfigures
downloadedfromEARS-Nethttp://atlas.ecdc.europa.eu/public/index.aspx
13
vancomycinuse(74)andsubsequententryintotreatmentrecommendations(75)
as a last-resort antibiotic when other options were depleted. Vancomycin’s
glycopeptidecousin,teicoplanin,wasalsointroducedinthisperiod(76,77),and
several other less-used glycopeptides have appeared from the drug pipelines
since(78–81).
Normally,thecellwallisconstructedbyinterlinkingpeptidoglycansthroughthe
terminalD-alanyl-D-alanine(D-ala-D-ala)aminoacidsofthepeptidemoietiesby
transpeptidases (82). Vancomycin attaches to D-ala-D-ala through a hydrogen
bond and denies transpeptidases to access peptidoglycans and thus
polymerisation(82),seeFigure2.Vancomycinisabigmoleculelargelyinefficient
as an antimicrobial towards Gram-negative pathogens since it cannot cross the
Gram-negative outer membrane, but recent research on vancomycin analogues
show that increasing polarity through modification may circumvent this issue
(83).
Vancomycin became increasingly used in the 80s, and the first reports of a
vancomycin-resistant E. faecium (VREfm) outbreak (84) and first described
occurrenceofaplasmid-mediatedvancomycinresistancedeterminant(85)(both
in1988)signalledadevelopmenttothetroublingsituationwehavetoday.The
dissemination of vancomycin-resistant E. faecium occurred swiftly in North
American hospitals after that initial outbreak, while the later vancomycin
resistance spread in Europe has been associated to the use of the glycopeptide
avoparcinasagrowthpromoterinfarms(86–88).NorthAmericastillexperiences
higher prevalence of vancomycin resistance in clinical isolates compared to
Europe, as seen in the point prevalence figure obtained from
resistancemap.cddep.organdearssnet.org(Figure1).
E. faecium, called a “drug resistance trafficker”, has acquired no less than eight
different vancomycin resistance gene clusters – vanA, vanB, vanD, vanE, vanG,
vanL,vanMandvanNtodefenditself(89–92).Additionally,avanCgenecluster
has been found in E. casseliflavus and E. gallinarum chromosomes (89). The
functionofthesegenesistoprovideapathwaytoaltertheterminalD-alanineof
theD-ala-D-alavancomycinbindingsitetoD-lactate(D-lac-vanA,B,D,M)orDserine(D-ser-vanC,E,G,L,N).Inmodifiedpeptidoglycanaminoacidside-chain
productsterminatinginD-Lac,VanHproducesD-Lacfrompyruvate,VanXcleaves
14
Figure2.Thevancomycinresistancegeneclustersandresistancemechanism
Functionsof the proteins encodedby eachgene in thevanAgenecluster(upper).
Comparisons of several vancomycin resistance clusters found in enterococci,
showingthevariationinorganizationoftheoperonsofvanA,vanB,vanC,vanD,vanE,
andvanGgeneclusters(lower).Geneswithsimilarfunctionssharecolours.Upper
figurereprintedwithpermissionfromHughes(265),lowerfigurefromDepardieu
etal.(94).
15
offD-alafromthevancomycin-bindingregionoftheaminoacidside-chain,and
VanA/B adds D-lac as the terminal peptide (93). See Figure 2 for graphical
depictionoftheVanAresistancemechanismandvariationsinoperonstructureof
some of the vancomycin resistance clusters encountered in enterococci. The
terminalpeptidealterationprovidessignificantreductioninglycopeptidebinding
affinity.D-ala-D-lacprovideshigherresistancelevelthanD-ala-D-ser.Vancomycin
resistance generally occurs via activation of resistance effectors by a feedback
loop consisting of a sensor (VanS) that phosphorylates a regulator/activator
(VanR)whenvancomycinispresent.PhosphorylatedVanRsubsequentlybindsto
nucleotides in vicinity of the promoters of the resistance effector genes, and
activatestranscription.Vancomycinresistanceitselfismediatedbyseveralgenes
(vanH/T, vanA/B/C/D/E/G/L/M/N and vanX) that when expressed forms a
pathwayreplacingD-ala-D-alatoD-ala-D-lac(orD-ala-D-ser).Inadditiontothese
essential effector genes, accessory effectors that increases glycopeptide MIC
(VanY)orhavepoorlycharacterizedfunctions(vanZAVanWB)exist.SeeDepartieu
etal.foranextensivereviewofthetopic(94).Theoriginsofthesegeneclusters
seemtobediverse,anditislikelythatvancomycinresistancepredatesantibiotic
usebymillenniaaccordingtoDNAextractionsfromglaciers(95).
OnlyvanAandvanBseemtobeepidemiologicallysignificanttoprovideresistance
in clinical isolates (22). Globally, vanA has been and is still the most abundant
resistanceclusterinclinicalisolates,whereasvanBhasincreasinglybeenfound
the last decade in Europe, and is the most abundant vancomycin resistance
mechanism found in Australia (22, 23, 96). They are associated with mobile
genetic elements. The vanA gene cluster is normally part of the Tn3-family
transposonTn1546(89)andthemostprevalentvanBsubtypeisanintegralpart
of the integrative conjugative element Tn1549/5382 (61, 97). This may explain
their high relative abundance compared to the other vancomycin resistance
clusters.
Beyondvancomycin–availableantibioticstotreatVREfmandtheirresistance
mechanisms
Asvancomycinresistance,especiallyintheU.S.A.hasbecomerampant,several
recently approved drugs for which E. faecium often show susceptibility have
16
entered into treatment recommendations to clear VRE infections. Thus, several
recent reviews have addressed options beyond the ‘last resort treatment’
vancomycin(98,99).Iwillrefertotheseforfurtherdocumentation.
Daptomycin,alipopeptidebactericidalagainstenterococcithroughbacterialcell
membranedisturbance,isusedtotreatVREeitherbyitselforincombinationwith
ß-lactams. Resistance towards daptomycin has been shown to arise through
mutationsingenesassociatedwithcellmembraneconstructionpathways(liaFSR,
yycFG) after prolonged daptomycin exposure (98), and de novo resistance
developmentmayspontaneouslyariseovertimeduetoclinicaldaptomycinuse
(100).
Lipoglycopeptidedrugsliketelavancin,dalbavancinandoritavancinaremodified
versionsofglycopeptidessuchasvancomycin.Theybindtothesametargetasthe
glycopeptides but are thought to possess superior bactericidal action by closer
association with the cell membrane by appendage of a lipophilic moiety. Both
telavancinanddalbavancinhavepoorantimicrobialeffectsagainstVREduetothe
alteredbidingsiteprovidedbyvancomycinresistancegeneclusters,andarethus
notusedclinically.OritavancinontheotherhandshowactivityagainstbothvanA-
andvanB-containingenterococciduetowiderinteractionstothepeptidoglycan
precursors.Sinceithasrecentlybeenintroducedintothemarket,largestudies
describing oritavancin activity have not been published yet. Oritavancin is
consequentlynotinwidetherapeuticuse.
Otherantimicrobials,theoxazolidinoneslinezolidandtedizolid,actbybindingto
ribosomes and prohibiting mRNA-protein translation through abrogation of
aminoacyl-tRNAdocking.Thismechanismensuresbacteriostasisinenterococci
unlessspecificmutationsoccurinthe23SrRNAgene.Suchmutationsgenerally
confer cross-resistance to linezolid and tedizolid (101). Enterococci possess
severalcopiesof23SrDNA,andbecomeincreasinglyresistantasmoreofthegene
copies gain these mutations. The horizontally transmissible resistance
determinantscfrandoptrA,respectivelyencodinganrRNAmethylaseconferring
resistancetolinezolidandanABCtransporterpumpingoutbothlinezolidand
tedizolidhavealsobeenfoundinenterococci(102,103).
Streptograminsalsoattacktheribosomethroughbindingtothe50Ssubunit,and
the two drugs dalfopristin and quinopristin (Q/D) are delivered together since
17
theysynergisticallyprovidebactericidebyirreversibleinhibitionoftheribosome.
Resistance towards Q/D is mediated by multiple identified resistance
determinantsthataltertheribosome,providehindrancetotarget,pumpQ/Dout
of the cell or break either one or both Q and D thus hampering the synergistic
effectsandthereforebactericide.
Tigecycline also binds to the ribosomal subunit 16S and prohibits docking of
aminoacyl-transferRNAresultingintranslationhalt.Reservationsagainsttheuse
ofthisdrughasarisensinceithasahighvolumeofdistribution,whichcauseslow
concentrations of free tigecycline at infection sites. This becomes a problem as
observedmutationsinrelevantribosomalgenes,whichslightlyincreasesMICfor
thisantimicrobialrapidlycreateproblemssinceobtainableantibioticlevelsareso
low.
Whydoesresistanceaccumulationoccur?
In E. faecium, resistance development seems to occur in an additive manner in
which certain strains amass one resistance determinant after another, while
others stay susceptible and easier to manage during infection. As mentioned
above, rise in E. faecium infection incidence is able to explain the rise of the
Enterococcus(22).
AhighlyinterestingtheoreticalpaperbyChangetal.(104)attemptstoidentify
theoriginsandproliferationofmulti-resistanceinbacteriaandattempttosingle
out individual causes. Several of these multi-drug resistance development
mechanismsshowninFigure3havealreadybeenshowneffectiveinE.faecium.
Resistancegenesmaybeco-localisedonmobilegeneticelements[examples:(61,
103,105–107)]andmayassuchbesubjecttolinkageselection.Thenosocomial
clade of E. faecium is known to contain a wealth of MGEs and lack ‘immune
defences’againstthemsuchasrestriction/modificationsystemsandCRISPR-cas
(24),whichimplyapotentialforincreasedmutationrateintermsof
18
Figure 3. Mechanisms by which multiple resistance (MDR) patterns arise in
bacteria(A->D)aswellastheirdisseminationandpersistencewithinsusceptible
bacterial communities (E->I). Resistance genes or gene clusters may confer
resistance to multiple antibiotics or even multiple classes of antibiotics, here
exemplifiedbyamulti-drugeffluxpump(A).DiverseresistancemechanismsoftencoresideonMGEsand/orinproximityonthechromosome,andareco-inherited(B).High
mutation/recombination rates permit swifter acquisition of resistance trough
mutations or horizontally transferred DNA (C), but may also be an effect of
antimicrobialexposuresincebacterialstressincreasesmutationrates.The“slippery
slope” thesis states that minorities of a bacterial population statistically may be
resistanttooneorsomeoftheantimicrobialsusedduringmultidrugtherapy(using
severalantibioticsatoncetofightoffinfections)andhasahigherpropensitytoacquire
additionalresistance(D). Resistance determinantsmay persist withinbacteriaeven
withoutantibioticexposureduetogeneticlinkagetoothergenesencodingseparate
favorabletraitstothebacterium(E).Thebystandereffect(F)happensassystemically
administered antibiotics kill susceptible bacteria wherever they are present in the
body(notonlywherevertheinfectionisat),leavingresistantbystanderstoproliferate
intheirdeadcomrade’sabsence.Resistancephenotypesmayconferafitnesscostto
bacteria,meaningthattheygrowmoreslowlyandlosetheirplaceinthepopulationto
susceptibleones.Ontheotherhand,thecombinationoffitnesseffects(epistasis)may
be positive (leading to better fitness) in MDR strains compared to single-resistant
strains (G). Niche differentiation (H) refers to MDR bug proliferation in niche
environments(suchashospitals)thatcontainfrequentexposuretoselectingstressors
(like antibiotics). Finally, a MDR strain introduced into a bacterial population of
susceptiblestrains(inforinstanceapatient)arewellabletoreplacethemifthereis
antibioticselectionofwhichtheMDRstrainisresistant.Printedwithpermissionfrom
Changetal.(104).
19
MGEreorganisation.Inaddition,duetotheintrinsicresistancetowardsarangeof
commonlyusedantimicrobialsthatE.faeciumdemonstrates,bystanderselection
during treatment for other infections is intuitively a present factor. Use of
antimicrobials is more densely occurring in hospitals than in the community,
which may explain how high-risk E. faecium clones are often found in health
institutions. As demonstrated by Brandl et al. and Hendrickx et al. (47, 49), E.
faecium has a potential of ‘taking over’ the gut during broad spectrum
antimicrobial treatment as other bacteria succumb to the treatment while E.
faeciumsurvives.Finally,ahostandaresistancegenecontainingelementmaycoevolvetoalleviateanyinitialfitnesscostintroductionoftheelementmayhave
burdenedthehostwith(108,109).
Horizontalgenetransferandmobilegeneticelements
E. faecium is considered a master of HGT and understanding how genes move
aroundinthebacterialpopulationandwhichtraitstheyconferisacomplexbut
important task in order to reveal their basal biology. Mobile genetic elements
blendinaPandora’sboxwheremultiplemovementmechanismsoftenco-existin
vicinity of each other, creating multiple recombination- and gene arrangement
possibilities.
HGT as a phenomenon is a large field to embrace in some small sentences and
paragraphs,andhastobeseeninthelargerscopethanthespreadofantimicrobial
resistanceandputativevirulencegenes(110).IfweconsideranystretchofDNA
as under selection, following the thinking of Dawkins’ selfish gene (111) or
Baquero’sterm‘piece’–anorderedstructurethatformspartofaseparatehigherorderedstructure(112)–HGTenlargesthe‘playground’inwhichanyDNAisable
to exist, and contribute to disseminate genetic structures throughout different
lifeforms.Indeed,horizontaltransferisshownastheprimarydriverofexpansion
ofproteinfamiliesthroughprokaryotesasopposedtoslowerprocesseslikegene
duplication and subsequent specialisation (113). The transferred genetic
structuresthemselvesoftenseemtocodeforperipheralfunctionspredominately
underneutralselection(114)andtendinaggregatetobelessexpressedthancore
genes to mitigate the potential for reduced fitness in hosts that harbour them
(115, 116) in order to persist in new hosts. Gene exchange between bacteria
20
seemsdependentona‘habitat-specificgenepool’whereniche-adaptivegenesare
shared(114,117).ThemajorityofHGTeventsoccurbetweengenomesofhigh
sequencesimilarityandsimilarGCcontent(genomesvaryinamountofguanines
and cytosines compared to adenines and thymines), and these two traits
representthelargestbarrierofHGTbetweendifferentstrainsandspecies(118).
Taking this discussion down to the ground again, several mechanisms are
responsibleforshuttlinggenesfromonebacteriumtoanother,andthosewillbe
brieflyexplained.Inaddition,specificMGEtypeslikeplasmids,transposons,ICEs
and so forth will conceptually be described. Furthermore, bacterial immune
defencesaffectingthedynamicsbywhichMGEsmayormaynotenterandsurvive
in the cell are described, as the nosocomial clade of E. faecium seemingly lacks
thesesystems.
Horizontalgenetransfermechanisms
Horizontalgenetransferisaprocesspermittingtheexchangeofgeneticmaterial
betweenorganisms.Geneticexchangehappensthroughdifferentmechanisms.
•
ConjugationisaprocesswheretypeIVsecretionsystemscreatechannels
between bacterial cells through which DNA is transferrable, and is the
dominantHGTmechanismofenterococci.
•
Transformationoccurswhenbacteriaareabletoassembleanapparatus
abletointernalizenakedDNAfloatinginthecells’immediateenvironment.
•
Transduction happens when DNA is trapped within bacteriophages
(bacterialviruses)thathaveinfectedahost,andisreleasedandinserted
intothegenomeofanewhostafterbacteriophagetransmission.
In addition, several less characterized gene transfer mechanisms such as
nanotubes(119),micro-vesicles(120)andgene-transferagents(121)havebeen
described,nodocumentationfortheexistenceofthesemechanismsinenterococci
hasbeenpublishedtomyknowledge.
Type4secretionsystems
Type4secretionsystems(T4SS)arethechannelsbywhichplasmids,ICEsand
sometimesotherDNAmaypassthroughfromcelltocell.InE.faecium,conjugation
21
isconsideredthemostimportantHGTmechanism(122)andthusneedadditional
functional explanation. There are several system variants as determined by
comparison of T4SS from Gram-positive, Gram-negative and mobile element
sources (123–126), but most systems share some basal common features. The
process of conjugation starts with nicking the double-stranded (ds) DNA into
single-stranded(ss)DNAbyarelaxase,oftenreferredtoasaMOBprotein.The
relaxase recognises a specific site, the origin of transfer (oriT), which must be
present for initiation of transfer to occur (127). See Figure 4. Using the
nomenclatureofthemodelT4SSfoundinAgrobacteriumtumefaciens,therelaxase
then interacts with a Type 4 Coupling Protein (T4CP), called VirD4. VirD4, an
ATPase fuelling propagation of transfer-DNA, subsequently interact with other
ATPasessometimespresent(VirB4,VirB11)topasstheDNAthroughthechannel
(123,128).TheT4SSchannel,alsocalledMatingPairFormation(MPF)complex,
is a large cell membrane-spanning assembly of multi-copy proteins guiding the
DNAfromdonortorecipient(126).
Plasmids
Plasmidsareextrachromosomalstructuresabletoautonomouslyreplicatewithin
its host. They are widespread in enterococci, and enterococcal strains often
harbourseveralplasmids.Plasmidsvaryincomplexity,andmayrangeinsizefrom
afewkilobasesto0,2-0,3megabases(127).Thissizerangeisalsoreflectedinthe
numberofgenestheypossessandthuswhatrangeoffunctionstheyencode.The
simplestandsmallestplasmidsoftenjustencodetheirownreplicationapparatus,
are often present in many copies within a bacterial cell, and are referred to as
“cryptic”meaningthatthereisnoapparentfunctionalbenefittoharbourthem.
Plasmidsthataremorecomplexcontainadditionalgenesencodingfunctionssuch
as antimicrobial resistance genes, stability modules (discussed below) and
conjugation modules. They need other mechanisms to persist in the cell than
smaller ones, and by looking at gene content, are more likely to contain genes
encodingcentralfunctionstotheirhostssurvival(127).Plasmidsareconjugative
whentheyencodetheT4SSdescribedabove,andmobilisableiftheycontainoriT
and parts of the conjugation apparatus, most commonly the relaxase and
sometimes a T4CP. Recent compilations of plasmid sequences and attempts to
22
Figure4.Mobilityofplasmids(A,B),andorganizationofmobileelements(C-F).A:
basalelementsofamobilisableorconjugativeplasmid,andthesizedistributionofthese
plasmids (B). C: IS-element. D: typical transposon. E: composite transposon. F:
Integrative Conjugative element. A and B printed with permission fromSmillie et al.
(127),C-FfromDarmon&Leach(137).
classify them have shown that every scheme carries downsides due to the
observed complexity. As reported by Shintani et al. (129) which analysed over
4600plasmidsdepositedinNCBI,classificationbyreplicationgenesismuddled
byfrequentmosaicismwithpresenceofmultiplereplicons.Classificationsusing
relaxases/MOB genes excludes non-mobilisable plasmids, which accounted for
themajorityofthedataset.
Inenterococci,alargecompilationandclassificationofenterococcalplasmidshas
recentlybeenproduced(122),toserveasasubsetofthatreportedinShintaniet
al.Inshort,plasmidreplicationproteinsmaybeclassifiedbymodeofreplication,
sequence similarity and subdomains present within the translated gene.
23
Replicationproteinsmayreplicateplasmidsbytoways–uni-directionalleading
strand Rolling Circle Replication (RCR) and bi-directional Theta (q) replication
(130,131).RCRplasmidsareoftencrypticandsmall,asthisreplicationmethodis
prone to mistakes and becomes unstable when they are over 10-15 kb. qreplicatingplasmidsaresubdividedintorepliconfamiliesinenterococci;Rep_3,
Inc18 and RepA_N. Briefly, Rep_3 plasmids are narrow host range plasmids of
similarsizeasRCRplasmidsandlikewiseareoftencryptic.Inc18plasmidsare
often conjugative 25-50 kb broad host-range plasmids frequently harbouring
resistancedeterminants.RepA_NplasmidsareprevalentinlowGCcontentGrampositives,andarepresentinawidesizerange(10-300kb).Individualplasmids
show a narrow host range. This classification scheme is often disturbed by
recombination and merging of especially Inc18 and RepA_N plasmids, creating
mosaicstructures(127,132–135).
Transposons,integrativeconjugativeelementsandgenomicislands
Ever since Barbara McClintock discovered genes which only apparent function
wastoencodetheirowntransposition(movement)fromonegenomiclocationto
another(136),multipleclassesoftransposableelementsusingadiversearrayof
transfer mechanisms located throughout all lifeforms including bacteria have
been described (137). As extensively reviewed by Darmon & Leach (137) and
Siguieretal.(138),transposasescomeinmanyshapesandformsinbacteria,and
combine to create an enormous amount of elements able to jump around the
genome through diverse mechanisms. The basal unit of mobile elements is the
transposase(Tnp)itself.Tnpsnormallybindstospecificinverted(IR)ordirect
(DR)repeatsup-anddownstreamofthetnpgene,andexcisesandintegratesthe
region between the repeats by two main mechanisms – cut-and-paste or copyand-paste.Tnpsare organizedinto differentfamiliesbasedontheirproperties.
FirstandforemostistheactiveproteinsitecleavingDNAduringtransposition,of
which there are five main motifs: DDE, DEDD, HUH, and site-specific
transposases/resolvases containing serine or tyrosine in the active site [also
functionally reviewed (139, 140)]. General mode of transposition, length and
sequence of IRs/DRs, functional domains in transposase proteins, and target
24
specificityarealsoamongthepropertiesconsideredtoplaceeachelementwithin
anIS/transposonfamily.
A unit transposon carries passenger genes in addition to the tnp within the
IRs/DRs.Compositetransposonsarerecognisedbytwotransposonsofthesame
ISfamilysurroundingastretchofDNAcontaininggenes,whichmaybepassengers
if the transposase recognises the IR’s on either side and thus moves both IS
elementsandtheintermediateDNAstretch.
Mobile elements may grow even larger, engulfing multiple passenger genes or
even complete pathways of gene clusters supporting complex functions. These
larger elements, often called genomic islands (GIs), integrative conjugative
elements(ICEs)iftheyareconjugativeorintegrativemobilisableelements(IMEs)
iftheyareabletohitch-hikewithotherconjugativesystems,areverydiverseand
likely more prevalent in nature than currently shown as we still struggle to
identify them (141). Evidence suggests that ICEs may be more abundant than
conjugativeplasmidsinprokaryotes(142).ICEsandIMEsmostoftendemonstrate
site-specific integration and excision mediated by tyrosine or serine
recombinases,mayrecombinebystackingthemselvesintheirinsertionsite,and
insomecasesdemonstratereplicativepropertiesoncetheyareexcisedandinan
extrachromosomal,circularform(141).
Howdomobilegeneticelementspersistinhosts?
BacteriaandtheMGEsthattransfersbetweenthemaretakingpartsinanarms
race where both parties harbour mechanisms designed to defend against MGE
inclusion for the bacterial host, or ensuring persistence for the MGE. These
systemsmayalsodemonstrateotherfunctionsinthecell,whichintroducesexiting
dynamics we do not fully understand. The three most studied attack/defence
mechanismsinteractingwithHGTaredescribedbelow.
Toxin/Antitoxinsystems
Toxin-Antitoxin (T/A) systems, also called post-segregational killing systems,
consistofaT/Apairdesignedtokillthebacteriumifthegenesencodingthem
becomesegregated(thatis:lost)fromthegenome.Thisworksastheantitoxin
inhibitstoxinfunction,butisinherentlylessstableinthecellthanthetoxin.Asthe
25
genes encoding T/A are lost, the unstable antitoxin will not be transcribed
anymoreandsinceitisswiftlydegraded,thestabletoxinwillkillthecell(122).
ThereexistfivetypesofT/Asystems,ofwhichonlyType2seemtobeprevalent
inenterococci.Briefly,T/Atypesaredividedbythetoxin/antitoxininteractions;
antitoxin binding to toxin for inhibition: mRNAantitoxin/mRNAtoxin (Type 1),
proteinantitoxin/proteintoxin (Type 2), and mRNAantitoxin/proteintoxin (Type 3),
antitoxinbindingtotoxinsubstrates(type4),andantitoxindegradingthetoxin
(143,144).EventhoughT/AsystemswereoriginallyassociatedwithMGEsasa
factor ensuring their persistence in cells, T/A systems are also found in
chromosomesofbacteriawithoutassociationtoMGEs.Thissurprisingfindinghas
ledtoanuancedviewofT/Asystemsfunction,astheyalsoseemtobeapartof
down-regulating cell growth and division under stressful conditions, apoptosis
and/or other cellular processes (144). Type 2 T/A-systems found in E. faecium
includesAxe/Txeandomega/epsilon/zeta(145,146).Theseplasmid-locatedT/A
systems are enriched in clinical MDR isolates (147, 148). Also, chromosomally
locatedHigBAEfandMazEFEfT/Asystemshavebeenassociatedwithexpression
patternsofseveralenterococcalvirulencegenes(149).
Restriction/modification(R/M)systems
R/M systems consists of a restriction enzyme cleaving unmethylated DNA in a
site-specificmannerandamethylationenzymeattachingamethylgrouptothe
samesitetherebyprohibitingDNAcleavage(150).Therearefourmaintypesof
R/Msystems,ofwhichtypeIIcomposedofaseparatemethylaseandrestriction
enzymeandtypeIconsistingofacomplexofmethylation/restriction/specificity
subunitsarethemostcommoninprokaryotes(151).3rdgenerationsequencing
byPacBioenablesidentificationofmethylatedsitesongenomesindicativeofR/M
activity, and a recent survey of 230 diverse bacteria and archaea found
methylationin93%ofthegenomes.Thesemethylationmotifscouldinmostcases
beconnectedtoaR/Msystem(151).BroadersearchesidentifyingR/Msystems
ingenomicdatasetsalsofounddispersalofR/Msystemsinprokaryotes,implying
thattheyserveimportantandcurrentlyunknownfunctionstoprokaryotebiology
(150).
26
R/MsystemshasbeenproposedasabacterialdefenceagainstforeignDNAasthe
hostgenomewillbeprotectedagainstcleavageduetomethylation,whereasthe
unmethylated foreign DNA will be cleaved at arrival (152–154). This genomic
defenceisonlyactiveaslongastheinvadingMGEdoesnotsucceedininhabiting
the cell for long enough to methylate its restriction-susceptible sites therefore
becomingimmune(155).R/MsystemsarealsofoundonmanyMGEtypes,and
are then thought to act as post-segregational killing systems by killing the new
host if it doesn’t allow persistence of the acquired MGE by methylation of its
chromosome(150,156).This“geneticaddiction”alsoappeartoalterdynamicsof
movementofMGEsthroughbacterialpopulations(157).TypeIImethylasesare
frequently found without their corresponding restriction enzyme, and these
appear to alter expression of genes selectively leaving gene-regulatory regions
unmethylated(151,158).
CRISPR-Cassystems
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas
enzymes work by cleaving foreign DNA and RNA through sequence specific
cleavage by Cas nucleases through hybridization of guiding sequences
(spacers/crRNA)totheDNAorRNA,seereviewbyHille&Charpentier(159).The
CRISPRregionconsistsofshortsequencesbearinghighidentitytoDNA/RNAof
phages and conjugative elements previously encountered by the isolate and its
ancestors.Spacersareseparatedbyshortrepeatsequencesthatservetoseparate
eachuniquespacerthroughcleavageofthefullytranscribedCRISPRarray,andto
provide interaction with the Cas nuclease apparatus. In addition to adaptive
immunedefence,CRISPR-CashasalsobeenimplicatedinDNArepair(160,161),
gene regulation (162) and structural genomic rearrangements (163) by
containingself-recognisingspacers.Thiswouldimplythatbacteriaareproneto
auto-immune disease (164) and that presence of CRISPR-Cas may have both
adaptiveandmaladaptiveeffects.
Interestingly,thenosocomialcladeofE.faeciumislargelydeficientoftheCRISPRCassystems(24,165),whichisassociatedwithincreasedpresenceofMGEs.This
poseaquestionofwhetheramountofHGTandactivityofCRISPR-Cassystemsis
negatively correlated, of which the answer seems to be no (166). Conversely,
27
CRISPR-Cas presence and activity is positively associated with resource
availability (food) (167) and inversely correlated to mesophilic (temperate
habitat) lifestyle (167, 168). Authors suggests mutation rate is correlated with
temperature,asbacteriaandMGEsinmesophilicenvironmentsaremorerapidly
mutating. High mutation rate would imply that spacers need to be frequently
exchangedtoaccommodatemutatedtargets,whichisastrenuoustasklikelyto
causenegativefitnesseffectsandsoadaptivesystemsarelikelylessefficientthan
innatedefenceslikeR/Msystems.Thisprovidesanalternativetheoryforabsence
ofCRISPR-CasinE.faecium.
Howdowecomparebacteria?
Animportantaspecttoconsiderwhendealingwithbacteriaisthenotionofhow
they evolve. Unlike us, who seemingly only accumulate mutations through the
vertical route – that is, from parent to child – many prokaryotes additionally
possesstheabilitytotransfergenesbetweenthemselvesandtherebytobothgain
andlosetheminaprocesscalledhorizontalgenetransfer(HGT).Treatinggenes
asacommodity,abacterialisolatemayrapidlyalteritsgenecontentandthereby
phenotypetoaccommodateadifferentenvironment,andassuch,evolve.
This aspect thus initiate thoughts of a concept where we no longer merely are
interestedinwhetherthereisanE.faeciuminfectionornot,butalsowhichisolate.
Canthisisolateberelatedtootherisolatesalsocausinginfection?Doesitcontain
specific traits able to at least give some indication of why exactly this isolate
invadedapatientandcausedseriousproblems?
Beingabletoanswersomeofthesequestionsrequirethehelpofcertainmethods.
Pathogen surveillance obviously require recording data of when and where a
pathogen caused an infection to seed suspicions of an outbreak – that one
particularlyvirulentbugwasabletodisseminateitselftoseveralpatientsfroma
commonsource.Earlymethodsforbacterialtypingincludedphenotypictyping
systems using serotypes, biotypes, phage-types and antibiograms (169). In
addition to that, there exist several methods able to assess genomic relations
betweenbacteria,whichhavefollowedthegeneralscientificdevelopment.Widely
used early adoptions in pathogen surveillance include wet-lab based molecular
28
methods which takes advantage of subtle genomic changes which can be
visualizedthroughpatternsonagelandthuscreatea“fingerprint”ofeachstrain.
DNAfingerprints
Pulse-Field Gel Electrophoresis (PFGE) (170, 171) has been the most widely
adopted method in enterococcal outbreaks since the nineties. It involves
immersingisolateculturesinagaroseplugs,chemicalandenzymaticcelllysisand
subsequent cutting of intact whole-genome DNA by a restriction enzyme that
cleaves DNA sequences in a sequence-specific manner, ideally fragmenting the
chromosome into 20-30 fragments. The resulting large genomic fragments are
separated by agarose electrophoresis overnight by an electrophoresis chamber
withelectricpulsefieldsalternatinginanglerespectiveofthedirectionDNAruns
throughthegeltoimproveseparationoftheselargefragments.TheDNAisthen
dyed, and the fragments appear as fingerprint patterns, which can be directly
compared to other isolates in the same gel. From there, comparison between
bands (number of matches and relative size and presence/absence of
mismatches)isusedtodeterminerelatednessbydefinedcriteria(170).
Other methods in the same “fingerprint” category includes amplified fragment
length polymorphism (AFLP) (172) and restriction fragment length
polymorphism(RFLP)(173)whicharevariationsofcuttingDNAwithrestriction
enzymesandamplificationofDNAbyPCR.Ribotypingisathirdmethod,involving
cutting whole-genome DNA with restriction enzymes, perform a gel
electrophoresis and then transfer the fragments onto a membrane (Southern
blotting) which is then hybridized with probes specific to labelled rRNAs,
producingfingerprints(174,175).
ReasonstoperformcomparisonsofbacteriausingDNAsequences
Giventhatthe“fingerprintmethods”mentionedabovearejustproxiesforactually
comparingtheDNAsequencesthemselvesandarecostlyandtime-consumingto
perform,furtherdevelopmenthaslargelycentredaroundcomparingsequences
directly.Otherdrawbackstoabove-mentionedmethodsincludepoorportability
between labs and maybe within labs, as well as difficulty assessing level of
29
difference between strains. Different methods needed to be used to address
differentquestions:methodswithhighdiscriminationsensitivityareneededto
group outbreak strains within a hospital, whereas lower discrimination
thresholdsareneededtoanalyseglobalevolution.
Allele-basedclusteringmethods
The most important early adoption of genomic global-scale comparisons of
bacteriaonstrainlevelcamewithmulti-locussequencetyping(MLST)(176),first
developedforNeisseriameningitidisandlateradoptedtomanyotherbacteria.The
method is based on choosing seven house-keeping genes ideally dispersed as
muchaspossiblethroughoutthebacterialchromosome,sequencethem,andthen
adopt them into an allele-based scheme. ‘House-keeping genes’ refer to genes
present in all strains of a species which are not super-conserved and never
mutating,andnotactivelyselectedupon–neutralselectionpressure.Eachallele
isassignedanumber,andifthealleleinyourgeneisdifferenttothosealready
present in the database by any means, it gets a new number. The allele
combinationscombinetocreateaSequenceType(ST),whichclustersbynumber
of allele differences between the STs. As clinical isolates are sequenced in
increasing numbers, additional genes are included in these schemes to gain
resolution. Other approaches extending the allele comparison principle include
MLSTofribosomalgenes(rMLST),genescommontoallormostoftheisolatesof
a species (core-gene/cgMLST), or comparisons of all encountered genes in the
entirebacterialpopulation(pan/wgMLST)(177).
Wholegenome-basedcomparisons
Many comparative studies in the microbiological field seek to explain what the
populationstructureofaspecieslookslike.Canstrainsbeseparatedbyecology,
geographicalboundaries,pathogenicity,amountofantimicrobialresistanceorby
othertraits?Canthephylogeny(’familytree’)ofaspeciestellanythingaboutthe
evolution of that species, and possibly anything about adaptation to certain
environments?
Mostly, such studies start with a phylogeny, which may be created by multiple
methods(178).ThebasalprocessistoidentifywhichstretchesofDNAispresent
30
in all isolates in the dataset (the core genome – interchangeable with common
genes),andthenidentifyindividualbasesthataredifferentbetweentheisolates
–so-calledsingle-nucleotidepolymorphisms,orSNPs.Asmostofthesestudiesuse
large amounts of isolates sequenced by short-read technologies like Illumina, a
common method to identify SNPs is to find one or a few well-assembled
reference(s),andmapalltheoutputfromthesequencertothatreferencetofind
theseSNPs.Theoutputfromthesequencerispaired-endreads–normally1000
baseslongDNAmoleculeswith≈100to300knownbasesineitherend.Thereare
several land-mark papers using this approach (179, 180). It is also possible to
assemblethereadstoobtainwholegenomesequences,andidentifycommonDNA
through whole-genome alignment, see Treangen et al. and references therein
(181).
SeveraladditionalanalysesmaybedoneontheseSNPstovalidatethem.Youmay
usestatisticsofeachSNPtoensurethatthisparticularSNPoccurinallreadsand
isnotproducedbyaflukeduringread-mapping,forexamplewithGATK(182).
PhylogeneticsoftwareproducingtreestendtoassumethatallSNPsarevertically
inherited,whichisoftennotthecaseasmany/mostofthespeciestransfergenes
horizontally, thus skewing inference of how they are related. Several methods
existtoremoveoraccountforSNPsthatlikelyoriginatefromhorizontaltransfer
andrecombination,tocreatealess‘polluted’collectionofSNPsfromwhichamore
correcttreecanbeinferred–forexampleGubbins(183),BratNextGen(184)and
ClonalFrameML (185). The finished dataset of SNPs will then be fed into a
phylogenetic software, of which there are several algorithms to choose from
(178).
Identifying common DNA content between strains is also possible to do by
clustering genes by similarity, subsequently gaining a matrix of which
encounteredgenesarepresentinwhichgenomesasoutput(186).Sizeofthecore
genome (genes present in all strains – interchangeable with common DNA
segments) and pan-genome (genes present in some or one strain) is then
computed (187). These questions are then often put in context with metadata
pertaining to each isolate to possibly say something about the evolution and
adaptationofthespeciesbasedonpresenceandabsenceofgenes.Manysoftware
packagesareavailabletoperformsuchanalyses(188–191),butnotallscalewell
31
tolargedatasetsduetodifferencesincomputationalefficiency.Asanextensionof
pan-genomics, bacterial Genome-Wide Association Studies (GWAS) pairing
phenotypeswithSNPs,presenceandabsenceofgenesandothergeneticregions
likeregulatorysequenceshasbeguntoemerge(192–194).
The field is rapidly changing with introduction of new softwares which do the
sametypeofanalysisasthepreviousones,butfaster,slightlymoreaccurately,
and/orwithadditionalavailableanalysisstepsandoutputs.
WhatdocomparativemethodstellaboutE.faecium?
Comparative analyses of E. faecium from different origins using molecular
fingerprintingmethodslikeAFLPandribotypingwereabletoshowthatE.faecium
could be divided by which hosts they originate from, as well as identifying a
subgroup associated with hospital-acquired infections (195–199). An MLST
schemeforE.faeciumwascreated(200),whichpermittedglobalcomparisonsof
which STs and ST clusters (clonal complexes - CCs) dominated in different
environmentsregionallyandglobally.Soonafter,reportsarrivedwhichindicated
thatSTslocatedinCC17wereoverrepresentedamongclinicalsamples,andlikely
weremoreresistanttoantimicrobials,andmorevirulentthanstrainsinotherCCs
(201–203). The method normally used to assess relatedness of STs is eBURST
(204,205),whichclusterSTstothelikelyancestorandcreaterelationnetworks
basedonnumberofsharedalleles.InacomparisonofeBURST-generatedrelation
networks,Turneretal.showedthatexcessiverecombinationwithinaspecieswill
distortthenetwork,withconsequentlystraggledeBURSTnetworkswherelikely
unrelatedCCsbecameincorrectlylinked(206).TheE.faeciumeBURSTnetwork
wasoneofthese.Asaresult,Willemsetal.addressedtheissuebyusingadifferent
analysis(BayesianAnalysisofPopulationStructure–BAPS)onthesameMLST
data.Thisanalysisrevealedthatnosocomialisolateslargelycouldbefoundintwo
subgroups,comprisedofthreeCCsoffoundersST17,18and78(207).Theyalso
performed an analysis aimed at assessing how much recombination occurred
between the different defined groups, and found it to occur rarely. The overall
conclusion was that eBURST-generated clusters were indeed artificially linked.
TheyalsofoundassociationofthenosocomialCCstoSTsoriginatingfromstrains
found in livestock. At this point, not many strains had been whole-genome
32
sequenced,butthefewthatweregotincludedinawholegenomesequence(WGS)
phylogenyshowingthatthenosocomialCCs(andthusthetwoassumedseparate
BAPSgroups)weredistinctfromlivestock-andcommensalisolates.Initialwholegenomestudiesassessingthepopulationstructureofenterococci(208,209)also
suggested a deep division between strains found in nosocomial settings and
elsewhere to the point where there was suggested to create a new subspecies
based on average nucleotide identity (ANI) scores dividing this clade from the
others.Inallthesestudies,thenosocomialstrainscontainedmoreresistance-and
virulence determinants, in addition to enrichment of certain mobile genetic
elements.
Withaccesstomoreisolates,Lebretonetal.couldascertainwithbetterresolution
theresultsfrominitialWGSanalyses.Theyalsofurtherelaboratedtheassertions
ofhowlongagothedifferentE.faeciumlineagesdiverged,andfoundindications
that the livestock lineage departed from the commensal lineage roughly 3000
years ago, corresponding with humans domesticating animals, and that the
nosocomial lineage departed from the livestock lineage within a decade ago,
parallelwiththeuseofantimicrobials.TheyalsofoundthatHAIstendedtolack
CRISPR-cas9 and restriction/modification systems, commonly regarded as
bacterial immune defence systems (24), an aspect also specifically investigated
before(165,210).Furthercharacteristicsdividingtheselineagesaregenomesize.
Nosocomial isolates are on average larger with an additional gene content
associatedwithMGEs,resistanceandvirulencedeterminants(24,211).
ThereexistsacgMLST-schemeforE.faecium(212),butitisnotopensource,and
hasnotbeenwidelyimplementedyetsinceyouhavetopaytouseit.However,
cgMLSThasshownthatthenosocomial-andlivestocklineagemaybepartedinto
severalsub-clusterswherenosocomialstrainsclusterbythemselves,commensals
likewise, and some strains from nosocomial- and livestock origins cluster in
between(23).
33
34
Summaryofpapers
Paper1:AmulticentrehospitaloutbreakinSwedencausedbyintroductionof
avanB2transposonintoastablymaintainedpRUM-plasmidinanEnterococcus
faeciumST192clone
Inpaper1,theaimwastodeterminetheoriginandmolecularcharacteristicofa
vancomycin-resistant E. faecium clone spreading through hospitals in three
countiesinSweden.
•
A nosocomially adapted ST192 E. faecium clone caused an outbreak in
Sweden.Theclonespreadintodifferenthospitalsinthreecounties.
•
The outbreak clone was resistant to vancomycin at variable levels, in
additiontociprofloxacinandampicillinathighlevels.
•
VancomycinresistancewascausedbyavanB2resistanceclusterlocated
onaTn1549ICE,whichwasinsertedonareppRUMplasmidcontainingan
axe/txetoxin/antitoxinstabilizationmodule.
•
This plasmid was able to transfer between strains, and in the process
mergedwithareppRE25plasmidco-localisedintheclinicaldonor.Afterthe
merge, the reppRUM+reppRE25 plasmid was able to transfer at a higher
frequencyinsubsequentfiltermatingexperiments.
•
TheoccurrenceofclonalE.faeciumbothwithandwithoutvanBindicate
thatthecloneinitiallywasasuccessfulvancomycinsusceptiblecolonizer
ofhospitalizedpatientsinwhichTn1549containingvanB2wasintroduced,
likelyviaanaerobegutbacteriawithinthesameenvironment.
•
ThephenotypicmethodtoscreenforVREpriortotheoutbreakusedahigh
concentrationofvancomycin(32mg/L),whichproducedanunnecessary
risk of false negative results for low-MIC (4-32 mg/L) vanB VRE, thus
underestimatingfactualVREincidence.
Paper2:SilencedvanAgeneclusteronatransferableplasmidcauseoutbreak
ofVancomycin-VariableEnterococci
Intwopatients,anE.faeciumstraincontainingthevanAgenebutwassusceptible
to vancomycin gained resistance towards vancomycin during the course of
35
treatment.Asubsequentscreeningrevealeddisseminationofthisstraininseveral
hospitals.Theaiminpaper2wastodeterminewhythisstrainconvertedfrom
vancomycinsusceptibletoresistant.
•
A clonal outbreak of ST203 E. faecium occurred in multiple hospitals in
Trøndelag,Norway
•
All studied isolates carried the vanA gene cluster with additional IS
elementsinsertedintheintergenicregions.
•
One of these IS elements (an ISL3 element) silenced the vanHAX operon
resultinginsusceptiblephenotypesbyhamperingtranscriptionactivation
bythevanRSregulatorloop.
•
Prolongedinvitroexposuretovancomycinatsub-lethalconcentrationlead
to excision of the ISL3 element upstream of the vanHAX operon. This
restored the regulator loop and resulted in high-level glycopeptide
resistancewithintwodaysofvancomycinexposure.Twopatientsfromthe
startoftheoutbreaksufferedtreatmentfailureasaconsequenceofthisIS
elementsilencingtheresistanceexpression.
•
The vanA gene cluster was located on a reppRE25 plasmid, which was
transferrabletoanE.faeciumlabstrainandinvivointoclinicalE.faecium
isolatesnotbelongingtotheoutbreakclone.Thisplasmidwasevenfound
inaclinicalE.faecalisisolatesampledlateintheoutbreak.
Paper3:TheEnterococcusCassetteChromosome:anSCCmec-likemobilisable
elementinenterococci
TherearepossibleorthologuesofccrAB,theserinerecombinasesresponsiblefor
transpositionofSCCmec,inseveralspeciesoftheEnterococcusgenus.Theaimin
paper 3 was to determine if the ccrABEnt genes mobilised a larger MGE in
enterococci.
•
WesucceededtohorizontallytransferanSCCmec-likeelementfromanE.
faecium donor to two E. faecium recipients through letting the element
hitchhikewithaconjugativereppLG1megaplasmid.
36
•
In accordance with the mechanism of SCCmec, the enterococcal element
containedattsitespermittingsite-specificintegrationinthehostgenome
downstreamoftherlmHgene,andtheattsitemotifsbetweenS.aureusand
E.faeciumweresimilar.
•
ThedonorregionsurroundingccrABEntcontainedmanytransposons,and
thepresenceoftwoISEfm1elementscontainedwithina10kbrepeaton
eitherendofccrABEntalsoenabledthisregiontotransferontotheplasmid
throughhomologousrecombination.
•
AfterdownloadingallenterococciassembliesfromNCBI,wefoundccrAB
genes in 48 E. faecium genomes and one E. durans. Elements residing
downstreamofrlmHandcontainingattsiteswereextractablefrom25of
these49genomes.
•
Comparisons of identified elements show very high variation of gene
content suggesting high plasticity of this genomic region, in agreement
withsimilarstudiesdonewithSCCmec.
37
Discussion
EpidemiologyofE.faeciuminScandinavia
As reported from several sources [(213–219), paper 1, paper 2)] clinical
infectionswithVREinScandinaviafirstoccurredintheninetiesandhasnowbeen
thecauseofseveraloutbreaksinhospitalenvironmentsthroughoutthisregion.
Accordingtothenationalsurveillanceinstitutes(220–222)receivingreportsof
any encountered VRE, a surge of VRE has been seen from 2008 in Sweden and
2010 in Norway and Denmark, which seems to have flattened and started
declininginSwedenandNorwaysince2014.Itshouldbenotedthattherehave
occurred several outbreaks in this period, and that these numbers also include
identification of carriers during outbreak investigations, which outnumber
clinicalinfections.VREwererarelyencounteredbeforethelatenineties,andthe
outbreaks of VRE in Scandinavia described in paper 1, paper 2 and others
indicate that VRE are a problem in hospital environments likely to persist. The
sourcesofVREseemtobeacomplexsubjecttodisentangle.Severalreportshas
linkedVREtransmissiontofarm-animalrelatedsources,andidentifiedzoonotic
transmissionasalikelysource(109,223–225),inadditiontoimportofVREfrom
foreignpatients(220–222),internationalflighttravel(226)andhospital-related
reservoirs (227, 228). Another important pathway for multi-resistance
development relates to horizontal gene transfer of resistance genes from
environmental co-inhabitants into hospital-related lineages of enterococci, as
reportedinpaper1andbyothers(96,229).
The isolates originating from the two outbreaks were from ST192 and ST203.
TheseSTsaresinglelocusvariantsofST78,oneofthemajorlineageswhichhas
spreadgloballyandhavebecomemorecommonthanthelineagewhichemerged
anddominatedearlier,ST17(207).ST78anddaughterSTswerebyWillemsetal.
connected to BAPS group 2-1, which consisted of a mesh of clinical and farmanimalrelatedstrains.Pinholtetal.(213,214)findothersingleanddouble-locus
variants of ST78 (ST117, ST80) making a lion’s share of their data set. This
suggeststhatthislineageisprevalentinScandinavia,andcausesmostregionally
encounteredpathogenicVRE.
38
Wholegenomesequencingenhancespathogensurveillance
Paper1andpaper2usedahybridofPFGEofallisolatesandWGSofsubsampled
isolates as the foundation for genetic comparison, due to the high cost of
sequencing all strains at those time points. The WGS strains were thought to
provide the “prototype” of the strain and PFGE the larger analysis assessing
relationshipbetweenstrains.
In paper 1, we concluded that all representative isolates from the prolonged
outbreakwereclonalinspiteofmultiplebanddifferencesbetweensomestrains
andattributedthebanddifferencestothelongsamplingperiod.Wholegenome
sequencingofallstrainswouldhaveprovidedaricherdatasetbywhichonecould
strengthensuchconclusions.
Inpaper2,thepresenceofISelementsinthevanAgeneclusterwasdetermined
byPCRslinkingcontigstoeachother.WeinitiallyfoundtheISL3elementinsertion
by observing a contig break in the interoperonic region between vanRS and
vanHAXinsusceptibleisolatesbutnotinresistantonesafterWGSandassembly.
Maybeothervariantscouldhavebeenhiddenintheisolateswedidnotsequence?
Sequencingallthestrainsweanalysedinthisoutbreak,includingtheE.faecalis
VVE isolate, could have given more details than the PCRs, and possibly help
determinethewholesequenceofthevanA-containingplasmid.HGToftheplasmid
containing the silenced vanA gene cluster from E. faecium to E. faecalis was
confirmedbyS1nucleasePFGEandSouthernhybridizationofvanAandreplicon
probes. We knew the size of the plasmid (45-50 kb), but only 22 kb of the
sequence. WGS analyses of the presumably identical plasmid in two different
species backgrounds could possibly have identified the rest of the plasmid
sequence by DNA identity scores between contigs. Presently, long-read
technologiescanclosetheseplasmids,andwouldbethesuperioralternative.
Global population structures can be analysed by MLST (207, 230), but only
sampling seven genes is unlikely to provide necessary resolution for local and
regionaloutbreakinvestigations.Recently,theMLSTschemehasalsobeenshown
to be unreliable in certain cases, as one gets superior and zoomable resolution
usingthewholesequenceandmayseeuntypeableE.faeciumduetodeletionof
MLSTloci(229,231).
39
Importantly, the common unit for comparison of isolates has become the
sequenced genome (232). Data sharing on a global scale has not been
implementedyetduetochallengesrelatedtodatastorage,limitationsinanalytic
methodology and speed and form of submission among other things, see
Aarestrupetal.(233).Thisopinionarticlehighlightsthepotentialforglobalopen
accesstosequencedataandcurrentinitiativestryingtoaccomplishthis.Several
early papers using sequence data to compare global population structures in
bacteriabyMLST(forinstanceMaidenetal.(176))mentionedportabilityasakey
measureasassembledgenomicdataarecontainedinsmallfileswhichareeasily
transferred online. Portability of WGS data is currently not possible with the
envisionedease,asthedatapreferredsharedbetweenscientistsissequencereads
whichforeachstrainamasstohugefilesthatarenoteasilytransferred.Aarestrup
etal.suggestbringinganalysistoolstothedatathroughcloud-basedstorageand
analysis resources, which may provide a plausible pathway for allowing data
miningofcompletedatasetsandglobalsurveillanceofimportantpathogens.
DataminingbecameanissueduringsearchesforECCelementsinpaper3.We
decided to use published assemblies from NCBI as search queries and omitted
published raw data sets as downloading them, assembling them and thereafter
probingforcompleteECCelementswouldtakealongtimeandcreateissueswith
datastorageanddataanalysisandpresentation.Secondgenerationsequencing
withIlluminaiscurrentlydoneatamassivescale.Semi-automatedqualitychecks,
assemblyandannotationarealreadyimportantinitialstepsforanalysisofthese
datasets.Submissionoftheseannotatedassemblieswithproperdescriptionofthe
employedsoftwareshouldbepossibletodoatalargerscalethanwhatispresently
done. Publication of assemblies may become more common as long-read
technologies connecting the genomic fragments into full-size replicons gain
tractionbylowercostsandincreasedavailability.Wecouldthusimproveonboth
global,regionalorevenlocalpathogensurveillanceasinpaper1andpaper2,as
wellaseasingdataminingtoanswerbasalmicrobiologicalquestions,asinpaper
3.
40
Phylogenetic-andtransmissionanalysesusingPFGEand/orcoregenomeSNPs
onlytellpartsofthestory
Connectingaclonecausinganoutbreaklocallytoaglobalphylogenyinorderto
identify likely origins is a challenging task, and present methods seem to have
issueswiththis.Anobservationnotmentionedinpaper2wasanattempttofind
internal phylogenetic relations between the six sequenced strains, which
producedresultstoounlikelytobeincludedinthepaper.Wewantedtomapthe
six Vancomycin-Variable Enterococci (VVE) to Aus0085 (234) which was
genetically closest according to the parsnp WGS phylogeny. After the read
mapping, we got help from Marc Stegger at the Statens Serums Institut in
Copenhagen to identify probabilistically sound SNPs by validation through the
GATK pipeline (182). The result showed that the index isolate was identical (0
SNPs)toAus0085,andthattheotherswereseparabletothesetwostrainsby0-4
SNPs. The reads from our sequenced strains were able to cover 96% of the
Aus0085 chromosome, implying that these isolates indeed shared much of the
chromosome,andthatthechromosomalDNAdidnotdiffer.Thestrongrelation
was highly surprising as Aus0085 was isolated on the other side of the world
seven years before our isolates. We examined the accessory genome through
Roary (190), which found significant differences in gene content between
Aus0085 chromosome and plasmids on one side, and our fragmented VVE
assembliesontheother.Subsequently,weattemptedtousethecgMLSTscheme
ofcolleaguesinUtrecht(212),wheretheywereabletofindthatAus0085andthe
VVE were not particularly close in the network (163 alleles different from the
indexisolate),butactedwithsurprisewhenourisolateswerethesamecluster
type(under20allelesdifference)asanisolate(E7987)fromAmsterdam,isolated
inDecember2013.
It has been a challenge to create a comprehensive population structure of
nosocomial E. faecium. Constructing this with MLST data using the eBURST
algorithmproducedlikelyskewedresults,andperformingBayesiananalyseson
thesamedata(207)yieldedmoreaccurate,butstillcontestedresults.Thepresent
consensus states that there is a clear distinction between a commensal/
environmentallineageandanosocomiallineage(208).Therehasbeendebateon
whether livestock- and nosocomial strains are actually two separate clades, as
41
phylogenetic distinctions vary by the datasets used (23, 24). In addition to the
unpublished analyses done by us, others have remarked this seemingly lack of
core-genomediscriminationbetweenisolatesconnectedbyphylogenybutnotin
periodandlocation,andattributesuchobservationstosilentglobalcirculationof
E.faeciumclones(96,235).ArecentstudyofE.faeciumgatheredfromAustralia
(235) suggest extensive intra-species recombination as a prominent force of
speciesevolution,andalsofindslimiteddifferencemeasuredassinglenucleotide
variants between isolates gathered at different places within Australia. This
suggeststhatanalysisofpatienttransmissionofE.faeciumisadifficulttaskwith
presentmethods[exemplifiedbyBrodricketal.(236)],andthatrecruitmentof
currentlyun-useddatamayyieldbetterresolution.
AnexcellentexamplecomesfromMcNallyetal.(237),whohadaglobaldatasetof
ST131 E. coli found in different environments at different times, but appeared
clonal by SNPs extracted from the common genetic content. By differentiating
theseverysimilargenomesthroughaccessorygenomecontentandvariationsin
regulatory(inter-genic)regions,abetterresolvedpopulationstructurewithsubclusters could be achieved. Similar analyses are currently being pursued for a
largeE.faeciumdatasettoo(RobWillems,personalcommunication),andcould
provideadditiveknowledgehere.
Lastly, phylogenetic analyses that also take into account the accessory genome
couldenhancehowweviewoutbreaksandwhatweregardasclinicallyimportant
transmission events. Paper I and II describe that resistance markers are
horizontally transferrable. Plasmids harbouring resistance determinants may
transfertocreatemulti-resistantstrainswhichreducetreatmentoptions,andmay
crucially disturb clinical decisions made on basis of whether an outbreak is
occurringornot.AsSheppardetal.(238)found,rapidplasmiddisseminationand
frequent MGE rearrangements caused resistance genes to spread into several
MGEs residing in several strains of several species of Enterobacteriaceae. The
transferredresistancegeneblaKPCortheMGEonwhichitresidedcouldassuchbe
regardedthe“functionalunit”oftheoutbreak,andmolecularorgenetic/genomic
comparativeanalysesoftheharbouringstrainswouldgivemisleadingresults.As
E.faeciumisalsoknowntofrequentlyperformintra-andinterspeciesHGT,mobile
42
geneticelementanalysismayalsocontributetoexplaintherelativesuccessofthis
pathogeninnosocomialsettings,aswellastheoriginsofpathogenicstrains.
Challengeswithvancomycinsusceptibilitydetermination
As was already noted in the epidemiological paper by Söderblom et al. which
coupled paper 1 (215), the Swedish guidelines performed vancomycin
phenotypic susceptibility tests with enrichment broth containing 32 mg/L
vancomycin. This practice stopped in 2009 as vanB-containing isolates
phenotypicallymayhaveMICswellunder32mg/L,aswasseeninsomeofthe
isolatesincludedformolecularanalysisinpaper1andinSöderblometal.The
highconcentrationofvancomycinintheenrichmentbrothwasprimarilydesigned
to identify enterococci containing vanA, which phenotypically give consistently
higherMICs.
Little is known of the observed variability of vancomycin MICs in E. faecium
containingvanB,butsomestudiesexistonvanBactivationbyglycopeptides.An
importantfeatureofvanBistheinabilitytobeactivatedbyteicoplanin,thereby
only providing resistance to vancomycin (239). The failure of vanB protection
againstteicoplaninexposurerelatestopresenceofhydrophobicmoietiesonthis
glycopeptide which leads to impaired interaction with the vanB activator loop
(240, 241). Changes within VanSB has previously been shown to modulate the
activation loop thereby causing constitutive, teicoplanin-induced or variable
vanHAXBexpression(242,243).Tomyknowledge,nosystematicstudyassessing
themolecularreasonsforvariableMICsinvanBpositiveclinicalstrainshasbeen
done,althoughseveralstudiesindicatethatlow-MICvanB-containingstrainsmay
escape identification in standardized tests (244–247), making this issue
important.
The vancomycin resistance clusters contain a whole pathway consisting of
different genes, and changes in this pathway is likely to alter resistance
phenotype. As mentioned in paper 2, altered phenotypes associated with IS
elementinsertionsandotherrearrangementsofthevanAgeneclusterhadalready
beenseeninisolatesgatheredforTn1546typingpurposes(248–253),andshould
hinttowardsexistenceofVVE.
43
Aswecurrentlyreportvancomycinresistancebasedonphenotypictraits,silent
vancomycin resistance operons are not likely to be registered. Routine WGS of
both VSE and VRE for international pathogen surveillance has not been
implemented yet. We are incentivised to enable WGS as standard method of
pathogencharacterizationasthisbetterenablesdiscoveryofsusceptibleisolates
containing silent vancomycin resistance operons. Such discoveries are already
occurring.AccordingtoRavenetal.(229),vanB-containingVSEwerealsofound
intheirdataset,andintheseisolates,thevanRBandvanSBregulatorygeneswere
missing,likelyexplainingthesusceptiblephenotype.Theyfoundthatoutof256
VSE, 10 containedvanB and one contained vanA. Why the vanA-containingVSE
wassusceptiblewasnotdiscussed.Recently,Knightetal.discoveredaTn1549locatedvanB2operoninClostridiumdifficilefromaruminantcarcasssilencedby
aninsertioninthe5’endofvanRB,likelyabruptingtranscriptionoftheactivator
loop(254).Theseresults,inconjunctionwithourresultsfrompaper2,suggest
thatsilencedvancomycinresistanceclustersofbothmajortypes(vanAandvanB)
may exist, located on unknown MGEs in unknown bacteria. Future thirdgeneration sequencing technologies such as Oxford Nanopore may improve
genotypic resistance determination by providing real-time genetic content in
additiontostructuralcompositionbylongreads.Eventhoughalargereportby
Woodford et al. (255) recently cautioned against using WGS in general for
antimicrobial susceptibility testing (or rather – resistance testing) of strains in
clinical settings as the technology still appears slightly immature, testing for
vancomycin resistance – especially with long-read technologies – seems like a
goodidea.
Several mechanisms may contribute to phenotypic vancomycin resistance
variabilityinvanAandvanB-containingstrains.Insertionsofmobileelementsinto
geneclustersrepresentonepathway,eithercausingsilencingofanoperon(paper
2) or providing an accessory promoter giving constitutive expression (256).
PolymeraseslippageinpromoterregionsofvanHAXmaycreateabetterpromoter
forun-inducedexpressionofvanHAXgenes(257).SNPsinthevancomycingene
clusters or promoter regions may give rise to variable expression and protein
function.Vancomycinresistancegeneoperonscomingfromotherbacteriamay
containpromoters,whichduetovariableadaptiontothetranscriptionapparatus
44
in diverse strains or species can cause variable expression of vanRS and/or
vanHAX.PromoteradaptationislikelytoplayaroleinvanBresistanceoperons,
astheyfrequentlyseemtotransferbetweenanaerobespeciesinthegutandinto
E. faecium (96). Also, vancomycin is shown to act as a zinc chelator (258). It is
unclearifZn(II)-depletionhavedeleteriouseffectsonsurvivaland/orreplication
of E. faecium, and how transport and retention of this ion is affected by
vancomycinexposure.
ArecentarticlebySanMillanetal.(259)assessedtheevolutionoftheampicillin
resistance gene blaTEM-1 on the chromosome or a small multi-copy-number
plasmid given a challenge of ceftazidime against which blaTEM-1 may evolve to
additionallyprovideresistance.ResultsshowedthatplasmidlocatedblaTEM-1was
abletogainceftazidimeresistancefasterthanwhenchromosomallylocated,and
thatadaptationsleadingtoincreasingplasmidcopy-numberalsocontributedto
the quicker ceftazidime resistance development. The vancomycin resistance
clustersdescribedhere(vanAandvanB)arefrequentlyharbouredbyplasmids,as
seeninpaper1andpaper2.Theroleofplasmidsinacceleratingtheevolutionof
plasmid-encoded genes such as resistance determinants could further be
addressedinenterococci.Forinstance,inlargerlow-copy-numberplasmidslike
those frequently harbouring vanA or vanB, other adaptations may be at play.
These adaptations include structural rearrangements of genes caused by
homologous recombination and movement of replicative transposons, as seen
experimentallyinEnterobacteriaceae(260)andinvivoincollectionsofclinical
andsurveillanceisolates(261,262).
MGEanalyses:wetgunpowderinthestartinggun
Discovery of horizontal gene transfer of particular MGEs through WGS is
precludedbyshortreadtechnologies,asMGEsoftenbecomefragmenteddueto
presenceofrepeatedregionssuchasISelementswithinthem.E.faeciumcontain
a wealth of such repetitions, and circularization of chromosomes and large
plasmidsisthusimpossiblewithshort-readtechnologies.Thus,identificationof
horizontally transferred genetic elements is currently often done by extracting
and connecting contigs if (as most often is the case) the element has remained
fragmentedaftergenomeassembly.
45
Vancomycinresistancedeterminantsareoftenlocatedonconjugativeplasmidsas
seeninpaper1andpaper2.Interestingly,inpaper1weencounteredthatthe
plasmid harbouring vanB had merged with another plasmid after conjugation.
HGTandlargestructuralrearrangementsarenotrareevents(132,133),butthe
molecularmechanismsbehindaredifficulttopreciselydisentanglewithPFGEand
Southern hybridisation, or with short-read sequence data. In paper 2, we saw
HGTofthevanA-harbouringplasmidintobothE.faeciumandE.faecalis,which
suggeststhatthisplasmidposesahigherdisseminationriskduetoabroadhost
range.Eventhoughwewereabletolink22kbofgenomicsequencecontainingthe
vanA gene cluster, a cat chloramphenicol resistance gene and other plasmidrelatedgenesinallsixsequencedisolates,theplasmidwaslarger,about25kb
according to Southern hybridization of S1 nuclease PFGE separated plasmids.
Locatingapossiblesourceorspeciesdistributionanddegreeofconservationin
genomic content of this particular plasmid would have added a strength to the
analysis. Several scientific questions are difficult to assess due to hampered
analysisoflargerstructuralrearrangements.
One of the principal problems we encountered in paper 3 related to
circularizationoftheelement.First,whenwechosetheccrABEntcontainingstrain
UWECCcatformobilisationexperiments,wedidnotknowtherewasa10kbrepeat
on either side of the MGE. This repeat is visible in yellow in lower sequence
depictionsinFigure2Aand2Binpaper3,andshowthatanyproductwithinverse
primerslocatedadjacenttoattLandattRinUWECCcatwouldshowchromosomal
DNAsurroundingattRifitwassequenced.Thisproductwouldappearasaband
afterfewcycles,andwouldlikelybeafalsepositive,oratleastwoulddrownout
any PCR product originating from a circularized ECC. UWECCcat was thus not a
suitable isolate to test excision of ECCcat. The second feature of the observed
duplicationinUWECCcatwhichcreatedseveralproblems,wastheduoofISEfm1
elements which inconveniently also was present in the reppLG1 plasmid and
permitted homologous recombination of the entire ECC into the plasmid.
Remarkably,ifISEfm1wouldcreateacircularexcisedcompositetransposonby
excisingfromUWECCcatortheplasmidcontainingECCcat,theDNAcirclewouldbe
identical to ECCcat, except containing two adjacent ISEfm1. Unfortunately, how
ISEfm1movementoccursisstillanopenquestionandfindingouthowwouldbe
46
outsidethescopeofpaper3.Wethereforehadtodesignprimersfacingoutwards
fromthe5’and3’endsofECCcatandperformthePCRinBMECCcat.Weobserved
bandscorrespondingtoacircularizedelement,buttheyreachedsaturationinthe
PCR after <30 cycles, which was surprisingly early, but not indicative of any
specificexcisionrate.ApossiblewayofassessingrateofexcisioncouldbeqPCR
ashasbeendoneforSCCmecbyStojanovetal.(263),whichwehaveyettodo.
AnotherwayofdemonstratingandquantifyingECCexcisionhaspresenteditself
through identifying additional ECC elements in other strains by searching
publishedE.faeciumgenomesequences.Theseelementscouldbeverifiedtonot
containsequenceduplicationsbioinformatically.Specifically,wehavefourstrains
(E.faeciumDO,9-F-6,C68andK59-68)inourcollection,whosechromosomehave
beenclosedandECCboundarieshavebeenidentified.Apositivecircularization
qPCR of cultured cells would further show that CcrAB serine recombinases
recogniseandrecombineidentifiedattsitesinenterococciaswellasquantifying
excision in several isolates, and is thus something we intend to do before
publication.
WearealsoplanningtodoexperimentsaddingfunctionaldatatoCcrABEntsince
wehaveobservedtransferofanelementbutnotthattheccrABEntgenesaresolely
responsibleformobilizingtheECCcatelement.Intheory,knock-outofccrABEntin
UWECCcat and repetition of a filter mating experiment should yield no
transconjugants.Unfortunately,ECCmobilisationbyhomologousrecombination
into the mobilisation plasmid has already been demonstrated and results from
ccrABEntknock-outandfiltermatingthereforecannotbetrustedinthissystem.
Misiura et al. (264) have designed a reporter system which shows promise.
ExpressionofccrABEntinE.coliandCcrABEntactionagainstvectorscontainingatt
sites could show action of CcrABEnt on att sites in a cleaner system. Properly
designed vectors containing both attL/R1 and attL/R2 (see paper 3, figure 3)
could determine specifically whether CcrABEnt activity on att sites occur, and
whether base substitutions in the presumed central recognition motif affect
recombinationfrequency.AsUWECCcatcontainsattL1/R1andC68ECCcontains
attL2/R2,compatibilitybetweenattsitevariantsmayalsobeassessed.
47
DiscoveryandanalysisofMGEslikeECC:What’snext?
Inadditiontoissuesconcerningexperimentsandtheextentofconclusionstobe
drawn from them, we also leave a central question unanswered: what
mechanism(s)createtheobservedgenevariabilityintheECCregion?Howdoes
thegenesyntenyofthisregioncomparetotheoverallgenesyntenyofE.faecium
chromosomes,orevenothermobilegeneticelementsencounteredinthisspecies?
Prevalence of ECC elements is 9% in published genomes (paper 3). Does this
meanthatitisrarelyoccurring,orisitacommonGIinE.faecium?Itispossibleto
arguethatitiscommon,asECCmaybemoreprevalentthanforexampleelements
harbouringvancomycingeneclusters,forwhichactiveselectionoccursthrough
use of glycopeptides. As seen by the global resistance map (Figure 1) and
prevalence data from cddep.org, VRE are often occurring more rarely than ECC
elements.VREfromtheclinicalsetting(asisreportedtothedatabasescombined
in cddep.org) are skewed towards nosocomial clades which results in a higher
reportedprevalenceofvancomycinresistancesincecommensalisolatesarerarely
VRE. As we find ECC elements throughout several environments and dispersed
throughthewholeE.faeciumlineage,itispossibletostatethatitisasuccessful
elementabletoexistinmanydifferentbackgrounds.
Similarly, obtaining structural information of genomes can extend the scope of
mobilegeneticelementsfrom‘prototypes’aspublicsequencesnoware,toalarger
network of MGEs uniting the basal constituents they are now classified from –
transposase or replication gene, mobilization modules, resistance genes and so
forth–toamorecomprehensivenetworkshowinggeneticrelationandvariation
usingalltheDNA.
It is possible to gain knowledge by shifting methods, and new 3rd generation
sequencingtechnologiespermittingstructuralinformationofgenomesislikelyto
allowabetterviewoftheaccessorygenomeandthestructureofmobileelements
than has been possible with short-read technologies. Currently, the
commercializedtechnologies(PacificBiosciencesandOxfordNanopore)arestill
too expensive to allow for widespread sequencing and closure of genomes, but
thismayrapidlychangeasnewapparatusesandSMRTcell/flowcelldisposables
withimprovedthroughputsarereleased.
48
Anotherbenefitof3rdgenerationsequencersistheadditionofmethylationdata
tothemix(151),whichallowsforanalysisofR/Msystems.Asdescribedearlier,
R/MsystemsinfluencethedynamicsofbothhostdefenceagainstMGEsandMGE
stabilisationinprokaryotes.MGEsharbouringR/MsystemsincludeECC,SCCmec
andlargeconjugativeplasmidsencodingresistancedeterminants,anditwouldbe
interestingtoexperimentallyanalysewhetherthesesystemsareactive,andifthey
conferbenefitstotheseMGEs.
49
References
1.
GradYH,LipsitchM.2014.Epidemiologicdataandpathogengenome
sequences:apowerfulsynergyforpublichealth.GenomeBiol15:538.
2.
WHOpressWHO.2014.Antimicrobialresistance:globalreporton
surveillance.Report,WorldHealthOrganization.
3.
deKrakerMEA,StewardsonAJ,HarbarthS,O’NeillJ,KrakerMDe,
DaveyP,GrundmannH,StewardsonA,AllignolA,BeyersmannJ,
GravesN,SchumacherM,GavaldaL,MasuetC,BeltranJ,GarciaM,
GarciaD,SamoreM,HarbarthS,SchumacherM,AllignolA,
BeyersmannJ,BinderN,WolkewitzM,GaieskiD,EdwardsJ,KallanM,
CarrB,KaukonenK,BaileyM,SuzukiS,PilcherD,BellomoR.2016.
Will10MillionPeopleDieaYearduetoAntimicrobialResistanceby2050?
PLOSMed13:e1002184.
4.
ThiercelinM.E.1899.Morphologieetmodesdereproductionde
l’enterocoque.ComptesRendusdesSeanceslaSocBioldessesFil551–
553.
5.
ThiercelinM.E.JL.1899.Surundiplococquesaprophytedel’intestin
susceptiblededevenirpathogene.CRSocBiol269–271.
6.
BarnesEM.1956.Tetrazoliumreductionasameansofdifferentiating
StreptococcusfaecalisfromStreptococcusfaecium.JGenMicrobiol14:57–
68.
7.
DeibelRH.1964.ThegroupDStreptococci.BacteriolRev28:330–66.
8.
ShermanJM.1937.Thestreptococci.BacteriolRev1:3.
9.
ShermanJM.1938.TheEnterococciandRelatedStreptococci.JBacteriol
35:81–93.
10. AndrewesFW,HorderTJ.1906.AstudyoftheStreptococcipathogenic
forman.Lancet168:708–713.
11. SchleiferKH,Kilpper-BalzR.1984.TransferofStreptococcusfaecalis
andStreptococcusfaeciumtotheGenusEnterococcusnom.rev.as
Enterococcusfaecaliscomb.nov.andEnterococcusfaeciumcomb.nov.Int
JSystBacteriol34:31–34.
12. LebretonF,WillemsRJL,GilmoreMS.2014.EnterococcusDiversity,
OriginsinNature,andGutColonization,p.1–56.InEnterococci:From
50
CommensalstoLeadingCausesofDrugResistantInfection.Massachusetts
EyeandEarInfirmary,Boston.
13. FacklamRR,CarvalhoMdaGS,TeixeiraLM.2002.TheEnterococci.
AmericanSocietyofMicrobiology.
14. Lucena-PadrosH,GonzalezJM,Caballero-GuerreroB,Ruiz-BarbaJL,
Maldonado-BarraganA.2014.Enterococcusolivaesp.nov.,isolatedfrom
Spanish-stylegreen-olivefermentations.IntJSystEvolMicrobiol
64:2534–2539.
15. GuCT,WangF,LiCY,LiuF,HuoGC.2012.Lactobacillusxiangfangensis
sp.nov.,isolatedfromChinesepickle.IntJSystEvolMicrobiol62:860–
863.
16. CottaMA,WhiteheadTR,FalsenE,MooreE,LawsonPA.2013.Erratum
to:TwonovelspeciesEnterococcuslemaniisp.nov.andEnterococcus
eurekensissp.nov.,isolatedfromaswine-manurestoragepit.AntonieVan
Leeuwenhoek103:1409–1418.
17. KimJY,ShinN-R,NaH-K,HyunD-W,WhonTW,KimPS,YunJ-H,BaeJW.2013.Enterococcusdiestrammenaesp.nov.,isolatedfromthegutof
Diestrammenacoreana.IntJSystEvolMicrobiol63:4540–4545.
18. FrolkovaP,SvecP,SedlacekI,MaslanovaI,CernohlavkovaJ,GhoshA,
ZurekL,RadimerskyT,LiterakI.2013.Enterococcusalcedinissp.nov.,
isolatedfromcommonkingfisher(Alcedoatthis).IntJSystEvolMicrobiol
63:3069–3074.
19. KadriZ,SpitaelsF,CnockaertM,PraetJ,ElFarrichaO,SwingsJ,
VandammeP.2015.Enterococcusbullienssp.nov.,anovellacticacid
bacteriumisolatedfromcamelmilk.AntonieVanLeeuwenhoek
108:1257–1265.
20. AgudeloHiguitaNI,HuyckeMM.2014.EnterococcalDisease,
Epidemiology,andImplicationsforTreatment,p.1–36.InEnterococci:
FromCommensalstoLeadingCausesofDrugResistantInfection.
MassachusettsEyeandEarInfirmary,Boston.
21. RiceLB.2008.Federalfundingforthestudyofantimicrobialresistancein
nosocomialpathogens:noESKAPE.JInfectDis197:1079–81.
22. AriasCA,MurrayBE.2012.TheriseoftheEnterococcus:beyond
51
vancomycinresistance.NatRevMicrobiol10:266–78.
23. GuzmanPrietoAM,vanSchaikW,RogersMRC,CoqueTM,BaqueroF,
CoranderJ,WillemsRJL.2016.GlobalEmergenceandDisseminationof
EnterococciasNosocomialPathogens:AttackoftheClones?Front
Microbiol7:1–15.
24. LebretonF,vanSchaikW,McGuireAM,GodfreyP,GriggsA,
MazumdarV,CoranderJ,ChengL,SaifS,YoungS,ZengQ,WortmanJ,
BirrenB,WillemsRJL,EarlAM,GilmoreMS,MansonMcGuireA,
GodfreyP,GriggsA,MazumdarV,CoranderJ,ChengL,SaifS,YoungS,
ZengQ,WortmanJ,BirrenB,WillemsRJL,EarlAM,GilmoreMS.2013.
Emergenceofepidemicmultidrug-resistantEnterococcusfaeciumfrom
animalandcommensalstrains.MBio2013/08/22.4:e00534-13-e0053413.
25. MurrayBE.1990.ThelifeandtimesoftheEnterococcus.ClinMicrobiol
Rev3:46–65.
26. WernerG,CoqueTM,HammerumAM,HopeR,HryniewiczW,Johnson
A,KlareI,KristinssonKG,LeclercqR,LesterCH,LillieM,NovaisC,
Olsson-LiljequistB,PeixeLV,SadowyE,SimonsenGS,TopJ,VuopioVarkilaJ,WillemsRJ,WitteW,WoodfordN.2008.Emergenceand
spreadofvancomycinresistanceamongenterococciinEurope.Euro
Surveill13:1–11.
27. MendesRE,CastanheiraM,FarrellDJ,FlammRK,SaderHS,JonesRN.
2016.Longitudinal(2001-14)analysisofenterococciandVREcausing
invasiveinfectionsinEuropeanandUShospitals,includinga
contemporary(2010-13)analysisoforitavancininvitropotency.J
AntimicrobChemotherdkw319.
28. SievertDM,RicksP,EdwardsJR,SchneiderA,PatelJ,SrinivasanA,
KallenA,LimbagoB,FridkinS,NationalHealthcareSafetyNetwork
(NHSN)TeamandParticipatingNHSNFacilities.2013.Antimicrobialresistantpathogensassociatedwithhealthcare-associatedinfections:
summaryofdatareportedtotheNationalHealthcareSafetyNetworkat
theCentersforDiseaseControlandPrevention,2009-2010.InfectControl
HospEpidemiol34:1–14.
52
29. ECDC.2013.AntimicrobialresistancesurveillanceinEurope2013.Annual
ReportoftheEuropeanAntimicrobialResistanceSurveillanceNetwork
(EARS-Net).ECDC,Stockholm.
30. GarsinDa.,FrankKL,SilanpääJ,AusubelFM,HartkeA,ShankarN,
MurrayBE.2014.PathogenesisandModelsofEnterococcalInfection,p.
1–57.InEnterococci:FromCommensalstoLeadingCausesofDrug
ResistantInfection.MassachusettsEyeandEarInfirmary,Boston.
31. SillanpääJ,PrakashVP,NallapareddySR,MurrayBE.2009.
DistributionofgenesencodingMSCRAMMsandPiliinclinicalandnatural
populationsofEnterococcusfaecium.JClinMicrobiol47:896–901.
32. LeavisH,TopJ,ShankarN,BorgenK,BontenM,vanEmbdenJ,
WillemsRJL.2004.Anovelputativeenterococcalpathogenicityisland
linkedtotheespvirulencegeneofEnterococcusfaeciumandassociated
withepidemicity.JBacteriol186:672–82.
33. HendrickxAPA,vanLuit-AsbroekM,SchapendonkCME,vanWamel
WJB,BraatJC,WijnandsLM,BontenMJM,WillemsRJL.2009.SgrA,a
Nidogen-BindingLPXTGSurfaceAdhesinImplicatedinBiofilmFormation,
andEcbA,aCollagenBindingMSCRAMM,AreTwoNovelAdhesinsof
Hospital-AcquiredEnterococcusfaecium.InfectImmun77:5097–5106.
34. NallapareddySR,WeinstockGM,MurrayBE.2003.Clinicalisolatesof
Enterococcusfaeciumexhibitstrain-specificcollagenbindingmediatedby
Acm,anewmemberoftheMSCRAMMfamily.MolMicrobiol2003/03/08.
47:1733–47.
35. RiceLB,CariasL,RudinS,VaelC,GoossensH,KonstabelC,KlareI,
NallapareddySR,HuangW,MurrayBE.2003.Apotentialvirulence
gene,hylEfm,predominatesinEnterococcusfaeciumofclinicalorigin.J
InfectDis187:508–12.
36. HendrickxAPA,vanWamelWJB,PosthumaG,BontenMJM,Willems
RJL.2007.FiveGenesEncodingSurface-ExposedLPXTGProteinsAre
EnrichedinHospital-AdaptedEnterococcusfaeciumClonalComplex17
Isolates.JBacteriol189:8321–8332.
37. HendrickxAPA,BontenMJM,vanLuit-AsbroekM,SchapendonkCME,
KragtenAHM,WillemsRJL.2008.Expressionoftwodistincttypesofpili
53
byahospital-acquiredEnterococcusfaeciumisolate.
Microbiology2008/10/04.154:3212–23.
38. SillanpääJ,NallapareddySR,SinghKV,PrakashVP,FothergillT,TonThatH,MurrayBE.2011.Characterizationoftheebp(fm)pilus-encoding
operonofEnterococcusfaeciumanditsroleinbiofilmformationand
virulenceinamurinemodelofurinarytractinfection.Virulence1:236–46.
39. NallapareddySR,SinghKV.,MurrayBE.2008.Contributionofthe
collagenadhesinAcmtopathogenesisofEnterococcusfaeciumin
experimentalendocarditis.InfectImmun76:4120–8.
40. HeikensE,SinghKV.,Jacques-PalazKD,vanLuit-AsbroekM,Oostdijk
EAN,BontenMJM,MurrayBE,WillemsRJL.2011.Contributionofthe
enterococcalsurfaceproteinEsptopathogenesisofEnterococcusfaecium
endocarditis.MicrobesInfect13:1185–90.
41. ZhangX,TopJ,deBeenM,BierschenkD,RogersM,LeendertseM,
BontenMJM,vanderPollT,WillemsRJL,vanSchaikW.2013.
IdentificationofageneticdeterminantinclinicalEnterococcusfaecium
strainsthatcontributestointestinalcolonizationduringantibiotic
treatment.JInfectDis207:1780–6.
42. PaganelliFL,HuebnerJ,SinghKV.,ZhangX,vanSchaikW,WobserD,
BraatJC,MurrayBE,BontenMJM,WillemsRJL,LeavisHL.2016.
Genome-wideScreeningIdentifiesPhosphotransferaseSystemPermease
BepAtoBeInvolvedinEnterococcusfaeciumEndocarditisandBiofilm
Formation.JInfectDis214:189–195.
43. HondaK,LittmanDR.2016.Themicrobiotainadaptiveimmune
homeostasisanddisease.Nature535:75–84.
44. OlesenSW,AlmEJ.2016.Dysbiosisisnotananswer.NatMicrobiol
1:16228.
45. HanageWP.2014.Microbiology:Microbiomescienceneedsahealthy
doseofscepticism.Nature512:247–8.
46. MontealegreMC,SinghKV,MurrayBE.2016.GastrointestinalTract
ColonizationDynamicsbyDifferentEnterococcusfaeciumClades.JInfect
Dis213:1914–1922.
47. BrandlK,PlitasG,MihuCN,UbedaC,JiaT,FleisherM,SchnablB,
54
DeMatteoRP,PamerEG.2008.Vancomycin-resistantenterococciexploit
antibiotic-inducedinnateimmunedeficits.Nature455:804–7.
48. CashHL,WhithamCV,BehrendtCL,HooperLV.2006.Symbiotic
bacteriadirectexpressionofanintestinalbactericidallectin.Science
313:1126–30.
49. HendrickxAPA,TopJ,BayjanovJR,KempermanH,RogersMRC,
PaganelliFL,BontenMJM,WillemsRJL.2015.Antibiotic-Driven
DysbiosisMediatesIntraluminalAgglutinationandAlternative
SegregationofEnterococcusfaeciumfromtheIntestinalEpithelium.MBio
6:e01346-15.
50. BerticsPJ,WiepzGJ.2009.NewDevelopmentswithVancomycinResistantEnterococci:E.faecium—FriendorFoe?JInfectDis200:679–
681.
51. LeendertseM,WillemsRJL,OeiGA,FlorquinS,BontenMJM,vander
PollT.2009.IntestinalEnterococcusfaeciumColonizationImprovesHost
DefenseduringPolymicrobialPeritonitis.JInfectDis200:735–744.
52. PaganelliFL,deBeenM,BraatJC,HoogenboezemT,VinkC,Bayjanov
J,RogersMRC,HuebnerJ,BontenMJM,WillemsRJL,LeavisHL.2015.
DistinctSagAfromHospital-AssociatedCladeA1Enterococcusfaecium
StrainsContributestoBiofilmFormation.ApplEnvironMicrobiol
81:6873–82.
53. RanganKJ,PedicordVA,WangY-C,KimB,LuY,ShahamS,MucidaD,
HangHC.2016.Asecretedbacterialpeptidoglycanhydrolaseenhances
tolerancetoentericpathogens.Science353:1434–1437.
54. PedicordVA,LockhartAAK,RanganKJ,CraigJW,LoschkoJ,RogozA,
HangHC,MucidaD.2016.Exploitingahost-commensalinteractionto
promoteintestinalbarrierfunctionandentericpathogentolerance.Sci
Immunol1:eaai7732-eaai7732.
55. FlemingA.1929.OntheAntibacterialActionofCulturesofaPenicillium,
withSpecialReferencetotheirUseintheIsolationofB.influenzæ.BrJExp
Pathol10:226–236.
56. KristichCJ,RiceLB,AriasCA.2014.EnterococcalInfection—Treatment
andAntibioticResistance,p.1–63.InEnterococci:FromCommensalsto
55
LeadingCausesofDrugResistantInfection.MassachusettsEyeandEar
Infirmary,Boston.
57. FontanaR,GrossatoA,RossiL,ChengYR,SattaG.1985.Transitionfrom
resistancetohypersusceptibilitytobeta-lactamantibioticsassociatedwith
lossofalow-affinitypenicillin-bindingproteininaStreptococcusfaecium
mutanthighlyresistanttopenicillin.AntimicrobAgentsChemother
28:678–83.
58. FontanaR,AldegheriM,LigozziM,LopezH,SucariA,SattaG.1994.
Overproductionofalow-affinitypenicillin-bindingproteinandhigh-level
ampicillinresistanceinEnterococcusfaecium.AntimicrobAgents
Chemother38:1980–3.
59. WilliamsonR,leBouguénecC,GutmannL,HoraudT.1985.Oneortwo
lowaffinitypenicillin-bindingproteinsmayberesponsiblefortherangeof
susceptibilityofEnterococcusfaeciumtobenzylpenicillin.JGenMicrobiol
131:1933–40.
60. MillerWR,MunitaJM,AriasCA.2014.Mechanismsofantibiotic
resistanceinenterococci.ExpertRevAntiInfectTher12:1221–36.
61. DahlKH,LundbladEW,RokenesTP,OlsvikO,SundsfjordA.2000.
GeneticlinkageofthevanB2geneclustertoTn5382invancomycinresistantenterococciandcharacterizationoftwonovelinsertion
sequences.Microbiology146:1469–79.
62. ZhangX,PaganelliFL,BierschenkD,KuipersA,BontenMJM,Willems
RJL,vanSchaikW.2012.Genome-wideidentificationofampicillin
resistancedeterminantsinEnterococcusfaecium.PLoSGenet8:e1002804.
63. HollenbeckBL,RiceLB.2012.Intrinsicandacquiredresistance
mechanismsinenterococcus.Virulence3:421–33.
64. LautenbachE,SchusterMG,BilkerWB,BrennanPJ.1998.Theroleof
chloramphenicolinthetreatmentofbloodstreaminfectiondueto
vancomycin-resistantEnterococcus.ClinInfectDis27:1259–65.
65. AndrewsJ,AshbyJ,JevonsG,MarshallT,LinesN,WiseR.2000.A
comparisonofantimicrobialresistanceratesinGram-positivepathogens
isolatedintheUKfromOctober1996toJanuary1997andOctober1997
toJanuary1998.JAntimicrobChemother45:285–93.
56
66. WernerG,FleigeC,EwertB,Laverde-GomezJa.,KlareI,WitteW.
2010.High-levelciprofloxacinresistanceamonghospital-adapted
Enterococcusfaecium(CC17).IntJAntimicrobAgents2009/12/17.
35:119–25.
67. HawkeyPM.2003.Mechanismsofquinoloneactionandmicrobial
response.JAntimicrobChemother51Suppl1:29–35.
68. DeshpandeLM,FritscheTR,MoetGJ,BiedenbachDJ,JonesRN.2007.
AntimicrobialresistanceandmolecularepidemiologyofvancomycinresistantenterococcifromNorthAmericaandEurope:areportfromthe
SENTRYantimicrobialsurveillanceprogram.DiagnMicrobiolInfectDis
58:163–70.
69. WilliamsonR,CalderwoodSB,MoelleringRC,TomaszA.1983.Studies
onthemechanismofintrinsicresistancetobeta-lactamantibioticsin
groupDstreptococci.JGenMicrobiol129:813–22.
70. RobbinsWC,TompsettR.1951.Treatmentofenterococcalendocarditis
andbacteremia;resultsofcombinedtherapywithpenicillinand
streptomycin.AmJMed10:278–99.
71. BaddourLM,WilsonWR,BayerAS,FowlerVG,BolgerAF,LevisonME,
FerrieriP,GerberMa,TaniLY,GewitzMH,TongDC,SteckelbergJM,
BaltimoreRS,ShulmanST,BurnsJC,FalaceDa,NewburgerJW,
PallaschTJ,TakahashiM,TaubertKa,CommitteeonRheumatic
Fever,EndocarditisandKD,CouncilonCardiovascularDiseaseinthe
Young,CouncilsonClinicalCardiology,StrokeandCSandA,
AmericanHeartAssociation,InfectiousDiseasesSocietyofAmerica.
2005.Infectiveendocarditis:diagnosis,antimicrobialtherapy,and
managementofcomplications:astatementforhealthcareprofessionals
fromtheCommitteeonRheumaticFever,Endocarditis,andKawasaki
Disease,CouncilonCardiovascularDiseaseintheYoung,.Circulation
111:e394-434.
72. ChowJW.2000.Aminoglycosideresistanceinenterococci.ClinInfectDis
31:586–9.
73. MoelleringRC.2006.Vancomycin:a50-yearreassessment.ClinInfectDis
42Suppl1:S3-4.
57
74. MoelleringRC,KrogstadDJ,GreenblattDJ.1981.Vancomycintherapyin
patientswithimpairedrenalfunction:anomogramfordosage.AnnIntern
Med94:343–6.
75. KoryM.1981.ReassessmentsofVancomycin-PotentiallyUseful
Antibiotic :BasedonSymposiaHeldinAtlanta,Georgia,November1-2,
1978SanFrancisco,California,November16-17,1978andChicago,
Illinois,.UniversityofChicagoPress,Chicago.
76. BardoneMR,PaternosterM,CoronelliC.1978.Teichomycins,new
antibioticsfromActinoplanesteichomyceticusnov.sp.II.Extractionand
chemicalcharacterization.JAntibiot(Tokyo)31:170–7.
77. ParentiF,BerettaG,BertiM,ArioliV.1978.Teichomycins,new
antibioticsfromActinoplanesteichomyceticusNov.Sp.I.Descriptionof
theproducerstrain,fermentationstudiesandbiologicalproperties.J
Antibiot(Tokyo)31:276–83.
78. DomenechO,FranciusG,TulkensPM,VanBambekeF,DufrêneY,
Mingeot-LeclercqMP.2009.Interactionsoforitavancin,anew
lipoglycopeptidederivedfromvancomycin,withphospholipidbilayers:
Effectonmembranepermeabilityandnanoscalelipidmembrane
organization.BiochimBiophysActa-Biomembr1788:1832–1840.
79. HigginsDL,ChangR,DebabovDV,LeungJ,WuT,KrauseKM,Sandvik
E,HubbardJM,KanigaK,SchmidtDE,GaoQ,CassRT,KarrDE,Benton
BM,HumphreyPP.2005.Telavancin,amultifunctionallipoglycopeptide,
disruptsbothcellwallsynthesisandcellmembraneintegrityin
methicillin-resistantStaphylococcusaureus.AntimicrobAgents
Chemother49:1127–34.
80. ChenAY,ZervosMJ,VazquezJA.2007.Dalbavancin:Anovel
antimicrobial.IntJClinPract61:853–863.
81. FarverDK,HedgeDD,LeeSC.2005.Ramoplanin:A
lipoglycodepsipeptideantibiotic.AnnPharmacother39:863–868.
82. ReynoldsPE.1989.Structure,biochemistryandmechanismofactionof
glycopeptideantibiotics.EurJClinMicrobiolInfectDis8:943–50.
83. YarlagaddaV,ManjunathGB,SarkarP,AkkapeddiP,
ParamanandhamK,ShomeBR,RavikumarR,HaldarJ.2016.
58
GlycopeptideAntibioticToOvercometheIntrinsicResistanceofGramNegativeBacteria.ACSInfectDis2:132–139.
84. UttleyAH,CollinsCH,NaidooJ,GeorgeRC.1988.Vancomycin-resistant
enterococci.Lancet1:57–8.
85. LeclercqR,DerlotE,DuvalJ,CourvalinP.1988.Plasmid-mediated
resistancetovancomycinandteicoplanininEnterococcusfaecium.NEngl
JMed319:157–61.
86. BagerF,MadsenM,ChristensenJ,AarestrupFM.1997.Avoparcinused
asagrowthpromoterisassociatedwiththeoccurrenceofvancomycinresistantEnterococcusfaeciumonDanishpoultryandpigfarms.PrevVet
Med31:95–112.
87. WillemsRJ,TopJ,vandenBraakN,vanBelkumA,MeviusDJ,
HendriksG,vanSanten-VerheuvelM,vanEmbdenJD.1999.Molecular
diversityandevolutionaryrelationshipsofTn1546-likeelementsin
enterococcifromhumansandanimals.AntimicrobAgentsChemother
43:483–91.
88. KruseH,JohansenBK,RørvikLM,SchallerG.1999.Theuseof
avoparcinasagrowthpromoterandtheoccurrenceofvancomycinresistantEnterococcusspeciesinNorwegianpoultryandswine
production.MicrobDrugResist5:135–9.
89. CourvalinP.2006.Vancomycinresistanceingram-positivecocci.Clin
InfectDis42Suppl1:S25-34.
90. BoydDA,WilleyBM,FawcettD,GillaniN,MulveyMR.2008.Molecular
characterizationofEnterococcusfaecalisN06-0364withlow-level
vancomycinresistanceharboringanovelD-Ala-D-Sergenecluster,vanL.
AntimicrobAgentsChemother52:2667–72.
91. LebretonF,DepardieuF,BourdonN,Fines-GuyonM,BergerP,
CamiadeS,LeclercqR,CourvalinP,CattoirV.2011.D-Ala-d-SerVanNtypetransferablevancomycinresistanceinEnterococcusfaecium.
AntimicrobAgentsChemother55:4606–12.
92. XuX,LinD,YanG,YeX,WuS,GuoY,ZhuD,HuF,ZhangY,WangF,
JacobyGA,WangM.2010.vanM,aNewGlycopeptideResistanceGene
ClusterFoundinEnterococcusfaecium.AntimicrobAgentsChemother
59
54:4643–4647.
93. WernerG,StrommengerB,WitteW.2008.Acquiredvancomycin
resistanceinclinicallyrelevantpathogens.FutureMicrobiol3:547–62.
94. DepardieuF,PodglajenI,LeclercqR,CollatzE,CourvalinP.2007.
Modesandmodulationsofantibioticresistancegeneexpression.Clin
MicrobiolRev20:79–114.
95. D’CostaVM,KingCE,KalanL,MorarM,SungWWL,SchwarzC,Froese
D,ZazulaG,CalmelsF,DebruyneR,GoldingGB,PoinarHN,WrightGD.
2011.Antibioticresistanceisancient.Nature477:457–61.
96. HowdenBP,HoltKE,LamMMC,SeemannT,BallardS,CoombsGW,
TongSYC,GraysonML,JohnsonPDR,StinearTP.2013.Genomic
InsightstoControltheEmergenceofVancomycin-ResistantEnterococci.
MBio2013/08/15.4:e00412-13-e00412-13.
97. UmedaA,GarnierF,CourvalinP,GalimandM.2002.Association
betweenthevanB2glycopeptideresistanceoperonandTn1549in
enterococcifromFrance.JAntimicrobChemother50:253–6.
98. MillerWR,MurrayBE,RiceLB,AriasCA.2016.Vancomycin-Resistant
Enterococci:TherapeuticChallengesinthe21stCentury.InfectDisClin
NorthAm30:415–39.
99. CrankC,O’DriscollT.2015.Vancomycin-resistantenterococcal
infections:epidemiology,clinicalmanifestations,andoptimal
management.InfectDrugResist217.
100. EgliA,SchmidH,KuenzliE,WidmerA,BattegayM,PlaggeH,FreiR,
AchermannR,WeisserM.2016.Associationofdaptomycinusewith
resistancedevelopmentinEnterococcusfaeciumbacteremia-A7-year
individualandpopulationbasedanalysis.ClinMicrobiolInfect.
101. Silva-DelToroSL,Greenwood-QuaintanceKE,PatelR.2016.Invitro
activityoftedizolidagainstlinezolid-resistantstaphylococciand
enterococci.DiagnMicrobiolInfectDis85:102–4.
102. LockeJB,ZurenkoGE,ShawKJ,BartizalK.2014.Tedizolidforthe
managementofhumaninfections:invitrocharacteristics.ClinInfectDis
58Suppl1:S35-42.
103. WangY,LvY,CaiJ,SchwarzS,CuiL,HuZ,ZhangR,LiJ,ZhaoQ,HeT,
60
WangD,WangZ,ShenY,LiY,FeßlerAT,WuC,YuH,DengX,XiaX,
ShenJ.2015.Anovelgene,optrA,thatconferstransferableresistanceto
oxazolidinonesandphenicolsanditspresenceinEnterococcusfaecalis
andEnterococcusfaeciumofhumanandanimalorigin.JAntimicrob
Chemother70:2182–90.
104. ChangH-H,CohenT,GradYH,HanageWP,O’BrienTF,LipsitchM.
2015.Originandproliferationofmultiple-drugresistanceinbacterial
pathogens.MicrobiolMolBiolRev79:101–16.
105. WernerG,HildebrandtB,WitteW.2003.Linkageoferm(B)andaadEsat4-aphA-3inmultiple-resistantEnterococcusfaeciumisolatesof
differentecologicalorigins.MicrobDrugResist9Suppl1:S9-16.
106. DavisIJ,RobertsAP,ReadyD,RichardsH,WilsonM,MullanyP.2005.
Linkageofanovelmercuryresistanceoperonwithstreptomycin
resistanceonaconjugativeplasmidinEnterococcusfaecium.Plasmid
54:26–38.
107. WernerG,HildebrandtB,KlareI,WitteW.2000.Linkageof
determinantsforstreptograminA,macrolide-lincosamide-streptogramin
B,andchloramphenicolresistanceonaconjugativeplasmidin
Enterococcusfaeciumanddisseminationofthisclusteramong
streptogramin-resistantenterococci.IntJMedMicrobiol290:543–8.
108. StarikovaI,Al-HaroniM,WernerG,RobertsAP,SorumV,NielsenKM,
JohnsenPJ.2013.Fitnesscostsofvariousmobilegeneticelementsin
EnterococcusfaeciumandEnterococcusfaecalis.JAntimicrobChemother
68:2755–2765.
109. JohnsenPJ,SimonsenGS,OlsvikØ,MidtvedtT,SundsfjordA.2002.
Stability,Persistence,andEvolutionofPlasmid-EncodedVanA
GlycopeptideResistanceinEnterococciintheAbsenceofAntibiotic
SelectionInVitroandinGnotobioticMice.MicrobDrugResist8:161–170.
110. SoucySM,HuangJ,GogartenJP.2015.Horizontalgenetransfer:building
theweboflife.NatRevGenet16:472–82.
111. DawkinsR.1976.TheSelfishGene.30thAnnivEdanewIntrodbyAuthor
384.
112. BaqueroF.2004.Frompiecestopatterns:evolutionaryengineeringin
61
bacterialpathogens.NatRevMicrobiol2:510–8.
113. TreangenTJ,RochaEPC.2011.HorizontalTransfer,NotDuplication,
DrivestheExpansionofProteinFamiliesinProkaryotes.PLoSGenet
7:e1001284.
114. SmillieCS,SmithMB,FriedmanJ,CorderoOX,DavidLA,AlmEJ.2011.
Ecologydrivesaglobalnetworkofgeneexchangeconnectingthehuman
microbiome.Nature480:241–244.
115. ParkC,ZhangJ.2012.Highexpressionhampershorizontalgenetransfer.
GenomeBiolEvol4:523–32.
116. DormanCJ.2014.H-NS-likenucleoid-associatedproteins,mobilegenetic
elementsandhorizontalgenetransferinbacteria.Plasmid75:1–11.
117. PolzMF,AlmEJ,HanageWP.2013.Horizontalgenetransferandthe
evolutionofbacterialandarchaealpopulationstructure.TrendsGenet
29:170–175.
118. PopaO,Hazkani-CovoE,LandanG,MartinW,DaganT.2011.Directed
networksrevealgenomicbarriersandDNArepairbypassestolateralgene
transferamongprokaryotes.GenomeRes21:599–609.
119. DubeyGP,Ben-YehudaS.2011.Intercellularnanotubesmediate
bacterialcommunication.Cell144:590–600.
120. FulsundarS,HarmsK,FlatenGE,JohnsenPJ,ChopadeBA,NielsenKM.
2014.GenetransferpotentialofoutermembranevesiclesofAcinetobacter
baylyiandeffectsofstressonvesiculation.ApplEnvironMicrobiol
80:3469–83.
121. LangAS,ZhaxybayevaO,BeattyJT.2012.Genetransferagents:phagelikeelementsofgeneticexchange.NatRevMicrobiol10:472–82.
122. ClewellDB,WeaverKE,DunnyGM,CoqueTM,FranciaMV,HayesF.
2014.ExtrachromosomalandMobileElementsinEnterococci:
Transmission,Maintenance,andEpidemiology,p.1–84.InGilmore,MS,
Clewell,DB,Ike,Y,Shankar,N(eds.),Enterococci:FromCommensalsto
LeadingCausesofDrugResistantInfection.BookSection,Massachusetts
EyeandEarInfirmary,Boston.
123. ChristiePJ.2016.TheMosaicTypeIVSecretionSystems.EcoSalPlus7.
124. Goessweiner-MohrN,ArendsK,KellerW,GrohmannE.2014.
62
ConjugationinGram-PositiveBacteria.MicrobiolSpectr2:PLAS-00042013.
125. ChandranDarbariV,WaksmanG.2015.StructuralBiologyofBacterial
TypeIVSecretionSystems.AnnuRevBiochem84:603–629.
126. Goessweiner-MohrN,ArendsK,KellerW,GrohmannE.2013.
ConjugativetypeIVsecretionsystemsinGram-positivebacteria.Plasmid
70:289–302.
127. SmillieC,Garcillán-BarciaMP,FranciaMV,RochaEPC,delaCruzF.
2010.Mobilityofplasmids.MicrobiolMolBiolRev74:434–52.
128. CascalesE,ChristiePJ.2004.DefinitionofabacterialtypeIVsecretion
pathwayforaDNAsubstrate.Science304:1170–3.
129. ShintaniM,SanchezZK,KimbaraK.2015.Genomicsofmicrobial
plasmids:classificationandidentificationbasedonreplicationand
transfersystemsandhosttaxonomy.FrontMicrobiol6:242.
130. KhanSA.2005.Plasmidrolling-circlereplication:highlightsoftwo
decadesofresearch.Plasmid53:126–136.
131. LillyJ,CampsM.2015.MechanismsofThetaPlasmidReplication.
MicrobiolSpectr3:PLAS-0029-2014.
132. FreitasAR,NovaisC,TedimAP,FranciaMV,BaqueroF,PeixeL,Coque
TM.2013.Microevolutionaryeventsinvolvingnarrowhostplasmids
influenceslocalfixationofvancomycin-resistanceinEnterococcus
populations.PLoSOne2013/04/05.8:e60589.
133. FreitasAR,TedimAP,FranciaMV.,JensenLB,NovaisC,PeixeL,
Sánchez-ValenzuelaA,SundsfjordA,HegstadK,WernerG,SadowyE,
HammerumAM,Garcia-MiguraL,WillemsRJ,BaqueroF,CoqueTM.
2016.MultilevelpopulationgeneticanalysisofvanAandvanB
Enterococcusfaeciumcausingnosocomialoutbreaksin27countries
(1986-2012).JAntimicrobChemother71:3351–3366.
134. SivertsenA,BillströmH,MeleforsÖ,LiljequistBO,WisellKT,Ullberg
M,ÖzenciV,SundsfjordA,HegstadK.2014.AMulticentreHospital
OutbreakinSwedenCausedbyIntroductionofavanB2Transposonintoa
StablyMaintainedpRUM-PlasmidinanEnterococcusfaeciumST192
Clone.PLoSOne2014/08/26.9:e103274.
63
135. SletvoldH,JohnsenPJ,SimonsenGS,AasnaesB,SundsfjordA,Nielsen
KM.2007.ComparativeDNAAnalysisofTwovanAPlasmidsfrom
EnterococcusfaeciumStrainsIsolatedfromPoultryandaPoultryFarmer
inNorway.AntimicrobAgentsChemother51:736–739.
136. McClintockB.1953.InductionofInstabilityatSelectedLociinMaize.
Genetics38:579–99.
137. DarmonE,LeachDRF.2014.Bacterialgenomeinstability.MicrobiolMol
BiolRev78:1–39.
138. SiguierP,GourbeyreE,VaraniA,Ton-HoangB,ChandlerM.2015.
Everyman’sGuidetoBacterialInsertionSequences.MicrobiolSpectr
3:MDNA3-0030-2014.
139. HickmanAB,DydaF.2015.MechanismsofDNATransposition.Microbiol
Spectr3:MDNA3-0034-2014.
140. RicePA.2015.SerineResolvases.MicrobiolSpectr3:MDNA3-0045-2014.
141. BellangerX,PayotS,Leblond-BourgetN,GuédonG.2014.Conjugative
andmobilizablegenomicislandsinbacteria:evolutionanddiversity.FEMS
MicrobiolRev38:720–760.
142. GuglielminiJ,QuintaisL,Garcillán-BarciaMP,delaCruzF,RochaEPC.
2011.TherepertoireofICEinprokaryotesunderscorestheunity,
diversity,andubiquityofconjugation.PLoSGenet7:e1002222.
143. FineranPC,BlowerTR,FouldsIJ,HumphreysDP,LilleyKS,Salmond
GPC.2009.Thephageabortiveinfectionsystem,ToxIN,functionsasa
protein-RNAtoxin-antitoxinpair.ProcNatlAcadSciUSA106:894–9.
144. Fernández-GarcíaL,BlascoL,LopezM,BouG,García-ContrerasR,
WoodT,TomasM.2016.Toxin-AntitoxinSystemsinClinicalPathogens.
Toxins(Basel)8:227.
145. GradyR,HayesF.2003.Axe-Txe,abroad-spectrumproteictoxinantitoxinsystemspecifiedbyamultidrug-resistant,clinicalisolateof
Enterococcusfaecium.MolMicrobiol47:1419–1432.
146. MeinhartA,AlonsoJC,SträterN,SaengerW.2003.Crystalstructureof
theplasmidmaintenancesystemepsilon/zeta:functionalmechanismof
toxinzetaandinactivationbyepsilon2zeta2complexformation.Proc
NatlAcadSciUSA100:1661–6.
64
147. HalvorsenEM,WilliamsJJ,BhimaniAJ,BillingsEA,HergenrotherPJ.
2011.Txe,anendoribonucleaseoftheenterococcalAxe-Txetoxinantitoxinsystem,cleavesmRNAandinhibitsproteinsynthesis.
Microbiology2010/10/30.157:387–97.
148. RosvollTCS,PedersenT,SletvoldH,JohnsenPJ,SollidJE,Simonsen
GS,JensenLB,NielsenKM,SundsfjordA.2010.PCR-basedplasmid
typinginEnterococcusfaeciumstrainsrevealswidelydistributedpRE25-,
pRUM-,pIP501-andpHTβ-relatedrepliconsassociatedwithglycopeptide
resistanceandstabilizingtoxin-antitoxinsystems.FEMSImmunolMed
Microbiol58:254–268.
149. SoheiliS,GhafourianS,SekawiZ,NeelaVK,SadeghifardN,
TaherikalaniM,KhosraviA,RamliR,HamatRA.2015.ThemazEF
toxin-antitoxinsystemasanattractivetargetinclinicalisolatesof
EnterococcusfaeciumandEnterococcusfaecalis.DrugDesDevelTher
9:2553–61.
150. OliveiraPH,TouchonM,RochaEPC.2014.Theinterplayofrestrictionmodificationsystemswithmobilegeneticelementsandtheirprokaryotic
hosts.NucleicAcidsRes42:10618–10631.
151. BlowMJ,ClarkTA,DaumCG,DeutschbauerAM,FomenkovA,FriesR,
FroulaJ,KangDD,MalmstromRR,MorganRD,PosfaiJ,SinghK,Visel
A,WetmoreK,ZhaoZ,RubinEM,KorlachJ,PennacchioLA,RobertsRJ.
2016.TheEpigenomicLandscapeofProkaryotes.PLoSGenet
12:e1005854.
152. ArberW,LinnS.1969.DNAModificationandRestriction.AnnuRev
Biochem38:467–500.
153. DupuisM-È,VillionM,MagadánAH,MoineauS.2013.CRISPR-Casand
restriction–modificationsystemsarecompatibleandincreasephage
resistance.NatCommun4:278–284.
154. PriceVJ,HuoW,SharifiA,PalmerKL.2016.CRISPR-CasandRestrictionModificationActAdditivelyagainstConjugativeAntibioticResistance
PlasmidTransferinEnterococcusfaecalis.mSphere1:e00064-16.
155. KoronaR,KoronaB,LevinBR.1993.Sensitivityofnaturallyoccurring
coliphagestotypeIandtypeIIrestrictionandmodification.JGen
65
Microbiol139:1283–1290.
156. NaitoT,KusanoK,KobayashiI.1995.Selfishbehaviorofrestrictionmodificationsystems.Science267:897–9.
157. OliveiraPH,TouchonM,RochaEPC.2016.Regulationofgeneticflux
betweenbacteriabyrestriction-modificationsystems.ProcNatlAcadSciU
SA113:5658–63.
158. MarinusMG,CasadesusJ.2009.RolesofDNAadeninemethylationin
host-pathogeninteractions:mismatchrepair,transcriptionalregulation,
andmore.FEMSMicrobiolRev33:488–503.
159. HilleF,CharpentierE.2016.CRISPR-Cas:biology,mechanismsand
relevance.PhilosTransRSocLondBBiolSci371.
160. LevyA,GorenMG,YosefI,AusterO,ManorM,AmitaiG,EdgarR,
QimronU,SorekR.2015.CRISPRadaptationbiasesexplainpreference
foracquisitionofforeignDNA.Nature520:505–510.
161. Ivančić-BaćeI,CassSD,WearneSJ,BoltEL.2015.Differentgenome
stabilityproteinsunderpinprimedandnaïveadaptationinE.coliCRISPRCasimmunity.NucleicAcidsRes43:10821–30.
162. SampsonTR,SarojSD,LlewellynAC,TzengY-L,WeissDS.2013.A
CRISPR/Cassystemmediatesbacterialinnateimmuneevasionand
virulence.Nature497:254–257.
163. VercoeRB,ChangJT,DyRL,TaylorC,GristwoodT,ClulowJS,Richter
C,PrzybilskiR,PitmanAR,FineranPC.2013.Cytotoxicchromosomal
targetingbyCRISPR/Cassystemscanreshapebacterialgenomesandexpel
orremodelpathogenicityislands.PLoSGenet9:e1003454.
164. SternA,KerenL,WurtzelO,AmitaiG,SorekR.2010.Self-targetingby
CRISPR:generegulationorautoimmunity?TrendsGenet26:335–340.
165. PalmerKL,GilmoreMS.2010.Multidrug-resistantenterococcilack
CRISPR-cas.MBio1:1–10.
166. GophnaU,KristensenDM,WolfYI,PopaO,DrevetC,KooninEV.2015.
NoevidenceofinhibitionofhorizontalgenetransferbyCRISPR–Cason
evolutionarytimescales.ISMEJ9:2021–2027.
167. WestraER,vanHouteS,Oyesiku-BlakemoreS,MakinB,Broniewski
JM,BestA,Bondy-DenomyJ,DavidsonA,BootsM,BucklingA.2015.
66
ParasiteExposureDrivesSelectiveEvolutionofConstitutiveversus
InducibleDefense.CurrBiol25:1043–9.
168. WeinbergerAD,WolfYI,LobkovskyAE,GilmoreMS,KooninEV.2012.
Viraldiversitythresholdforadaptiveimmunityinprokaryotes.MBio
3:e00456-12.
169. SabatAJ,BudimirA,NashevD,Sá-LeãoR,vanDijlJm,LaurentF,
GrundmannH,FriedrichAW,ESCMIDStudyGroupofEpidemiological
Markers(ESGEM).2013.Overviewofmoleculartypingmethodsfor
outbreakdetectionandepidemiologicalsurveillance.EurCommunDisBull
18:20380.
170. TenoverFC,ArbeitRD,GoeringRV,MickelsenPa,MurrayBE,Persing
DH,SwaminathanB.1995.InterpretingchromosomalDNArestriction
patternsproducedbypulsed-fieldgelelectrophoresis:criteriaforbacterial
straintyping.JClinMicrobiol33:2233–9.
171. SchwartzDC,CantorCR.1984.Separationofyeastchromosome-sized
DNAsbypulsedfieldgradientgelelectrophoresis.Cell37:67–75.
172. VosP,HogersR,BleekerM,ReijansM,LeeTvande,HornesM,Friters
A,PotJ,PalemanJ,KuiperM,ZabeauM.1995.AFLP:anewtechnique
forDNAfingerprinting.NucleicAcidsRes23:4407–4414.
173. SaikiRK,ScharfS,FaloonaF,MullisKB,HornGT,ErlichHA,Arnheim
N.1985.Enzymaticamplificationofbeta-globingenomicsequencesand
restrictionsiteanalysisfordiagnosisofsicklecellanemia.Science
230:1350–4.
174. GrimontF,GrimontPA.1986.Ribosomalribonucleicacidgene
restrictionpatternsaspotentialtaxonomictools.AnnInstPasteur
Microbiol137B:165–75.
175. StullTL,LiPumaJJ,EdlindTD.1988.Abroad-spectrumprobefor
molecularepidemiologyofbacteria:ribosomalRNA.JInfectDis157:280–
6.
176. MaidenMC,BygravesJA,FeilE,MorelliG,RussellJE,UrwinR,ZhangQ,
ZhouJ,ZurthK,CaugantDA,FeaversIM,AchtmanM,SprattBG.1998.
Multilocussequencetyping:aportableapproachtotheidentificationof
cloneswithinpopulationsofpathogenicmicroorganisms.ProcNatlAcad
67
SciUSA95:3140–5.
177. JolleyKA,MaidenMCJ.2014.Usingmultilocussequencetypingtostudy
bacterialvariation:prospectsinthegenomicera.FutureMicrobiol9:623–
30.
178. CollinsC,DidelotX.2017.ReconstructingtheAncestralRelationships
BetweenBacterialPathogenGenomes.MethodsMolBiol1535:109–137.
179. ChewapreechaC,MarttinenP,CroucherNJ,SalterSJ,HarrisSR,
MatherAE,HanageWP,GoldblattD,NostenFH,TurnerC,TurnerP,
BentleySD,ParkhillJ.2014.Comprehensiveidentificationofsingle
nucleotidepolymorphismsassociatedwithbeta-lactamresistancewithin
pneumococcalmosaicgenes.PLoSGenet10:e1004547.
180. CroucherNJ,HarrisSR,FraserC,QuailMA,BurtonJ,vanderLindenM,
McGeeL,vonGottbergA,SongJH,KoKS,PichonB,BakerS,ParryCM,
LambertsenLM,ShahinasD,PillaiDR,MitchellTJ,DouganG,Tomasz
A,KlugmanKP,ParkhillJ,HanageWP,BentleySD.2011.Rapid
pneumococcalevolutioninresponsetoclinicalinterventions.Science
331:430–4.
181. TreangenTJ,OndovBD,KorenS,PhillippyAM.2014.TheHarvestsuite
forrapidcore-genomealignmentandvisualizationofthousandsof
intraspecificmicrobialgenomes.GenomeBiol15:524.
182. McKennaA,HannaM,BanksE,SivachenkoA,CibulskisK,Kernytsky
A,GarimellaK,AltshulerD,GabrielS,DalyM,DePristoMA.2010.The
GenomeAnalysisToolkit:aMapReduceframeworkforanalyzingnextgenerationDNAsequencingdata.GenomeRes20:1297–303.
183. CroucherNJ,Pagea.J,ConnorTR,Delaneya.J,KeaneJa.,BentleySD,
ParkhillJ,HarrisSR.2015.Rapidphylogeneticanalysisoflargesamples
ofrecombinantbacterialwholegenomesequencesusingGubbins.Nucleic
AcidsRes43:e15–e15.
184. MarttinenP,HanageWP,CroucherNJ,ConnorTR,HarrisSR,Bentley
SD,CoranderJ.2012.Detectionofrecombinationeventsinbacterial
genomesfromlargepopulationsamples.NucleicAcidsRes40:e6.
185. DidelotX,WilsonDJ.2015.ClonalFrameML:efficientinferenceof
recombinationinwholebacterialgenomes.PLoSComputBiol
68
11:e1004041.
186. TettelinH,MasignaniV,CieslewiczMJ,DonatiC,MediniD,WardNL,
AngiuoliSV,CrabtreeJ,JonesAL,DurkinAS,DeboyRT,DavidsenTM,
MoraM,ScarselliM,MargarityRosI,PetersonJD,HauserCR,
SundaramJP,NelsonWC,MadupuR,BrinkacLM,DodsonRJ,Rosovitz
MJ,SullivanSA,DaughertySC,HaftDH,SelengutJ,GwinnML,ZhouL,
ZafarN,KhouriH,RaduneD,DimitrovG,WatkinsK,O’ConnorKJB,
SmithS,UtterbackTR,WhiteO,RubensCE,GrandiG,MadoffLC,
KasperDL,TelfordJL,WesselsMR,RappuoliR,FraserCM.2005.
GenomeanalysisofmultiplepathogenicisolatesofStreptococcus
agalactiae:implicationsforthemicrobial“pan-genome”.ProcNatlAcadSci
USA102:13950–5.
187. DonatiC,HillerNL,TettelinH,MuzziA,CroucherNJ,AngiuoliSV,
OggioniM,DunningHotoppJC,HuFZ,RileyDR,CovacciA,MitchellTJ,
BentleySD,KilianM,EhrlichGD,RappuoliR,MoxonER,MasignaniV.
2010.Structureanddynamicsofthepan-genomeofStreptococcus
pneumoniaeandcloselyrelatedspecies.GenomeBiol11:R107.
188. Contreras-MoreiraB,VinuesaP.2013.GET_HOMOLOGUES,aVersatile
SoftwarePackageforScalableandRobustMicrobialPangenomeAnalysis.
ApplEnvironMicrobiol79:7696–7701.
189. ChaudhariNM,GuptaVK,DuttaC.2016.BPGA-anultra-fastpan-genome
analysispipeline.SciRep6:24373.
190. PageAJ,CumminsCA,HuntM,WongVK,ReuterS,HoldenMTG,
FookesM,FalushD,KeaneJA,ParkhillJ.2015.Roary:rapidlarge-scale
prokaryotepangenomeanalysis.Bioinformatics31:3691–3.
191. SahlJW,CaporasoJG,RaskoDA,KeimP.2014.Thelarge-scaleblast
scoreratio(LS-BSR)pipeline:amethodtorapidlycomparegenetic
contentbetweenbacterialgenomes.PeerJ2:e332.
192. HallBG.2014.SNP-AssociationsandPhenotypePredictionsfrom
HundredsofMicrobialGenomeswithoutGenomeAlignments.PLoSOne
9:e90490.
193. LeesJA,VehkalaM,VälimäkiN,HarrisSR,ChewapreechaC,Croucher
NJ,MarttinenP,DaviesMR,SteerAC,TongSYC,HonkelaA,ParkhillJ,
69
BentleySD,CoranderJ.2016.Sequenceelementenrichmentanalysisto
determinethegeneticbasisofbacterialphenotypes.NatCommun
7:12797.
194. EarleSG,WuC-H,CharlesworthJ,StoesserN,GordonNC,WalkerTM,
SpencerCCA,IqbalZ,CliftonDA,HopkinsKL,WoodfordN,SmithEG,
IsmailN,LlewelynMJ,PetoTE,CrookDW,McVeanG,WalkerAS,
WilsonDJ.2016.Identifyinglineageeffectswhencontrollingfor
populationstructureimprovespowerinbacterialassociationstudies.Nat
Microbiol1:16041.
195. CoqueTM,WillemsRJL,FortúnJ,TopJ,DizS,LozaE,CantónR,
BaqueroF.2005.PopulationstructureofEnterococcusfaeciumcausing
bacteremiainaSpanishuniversityhospital:settingthesceneforafuture
increaseinvancomycinresistance?AntimicrobAgentsChemother
49:2693–700.
196. WillemsRJ,TopJ,vanDenBraakN,vanBelkumA,EndtzH,MeviusD,
StobberinghE,vanDenBogaardA,vanEmbdenJD.2000.Host
specificityofvancomycin-resistantEnterococcusfaecium.JInfectDis
182:816–23.
197. JureenR,TopJ,MohnSC,HarthugS,LangelandN,WillemsRJL.2003.
Molecularcharacterizationofampicillin-resistantEnterococcusfaecium
isolatesfromhospitalizedpatientsinNorway.JClinMicrobiol41:2330–6.
198. BorgenK,WastesonY,KruseH,WillemsRJL.2002.VancomycinresistantEnterococcusfaecium(VREF)fromNorwegianpoultrycluster
withVREFfrompoultryfromtheUnitedKingdomandTheNetherlandsin
anamplifiedfragmentlengthpolymorphismgenogroup.ApplEnviron
Microbiol68:3133–7.
199. VancanneytM,LombardiA,AndrighettoC,KnijffE,TorrianiS,
BjörkrothKJ,FranzCMAP,FoulquiéMorenoMR,RevetsH,DeVuystL,
SwingsJ,KerstersK,DellaglioF,HolzapfelWH.2002.Intraspecies
genomicgroupsinEnterococcusfaeciumandtheircorrelationwithorigin
andpathogenicity.ApplEnvironMicrobiol68:1381–91.
200. HomanWL,TribeD,PoznanskiS,LiM,HoggG,SpalburgE,Van
EmbdenJDA,WillemsRJL.2002.Multilocussequencetypingschemefor
70
Enterococcusfaecium.JClinMicrobiol2002/05/31.40:1963–71.
201. WillemsRJL,TopJ,vanSantenM,RobinsonDA,CoqueTM,BaqueroF,
GrundmannH,BontenMJM.2005.GlobalspreadofvancomycinresistantEnterococcusfaeciumfromdistinctnosocomialgeneticcomplex.
EmergInfectDis11:821–8.
202. LeavisHL,WillemsRJL,TopJ,BontenMJM.2006.High-level
ciprofloxacinresistancefrompointmutationsingyrAandparCconfinedto
globalhospital-adaptedclonallineageCC17ofEnterococcusfaecium.JClin
Microbiol44:1059–64.
203. HeikensE,vanSchaikW,LeavisHL,BontenMJM,WillemsRJL.2008.
Identificationofanovelgenomicislandspecifictohospital-acquiredclonal
complex17Enterococcusfaeciumisolates.ApplEnvironMicrobiol
74:7094–7.
204. FeilEJ,LiBC,AanensenDM,HanageWP,SprattBG.2004.eBURST:
inferringpatternsofevolutionarydescentamongclustersofrelated
bacterialgenotypesfrommultilocussequencetypingdata.JBacteriol
186:1518–30.
205. SprattBG,HanageWP,LiB,AanensenDM,FeilEJ.2004.Displayingthe
relatednessamongisolatesofbacterialspecies--theeBURSTapproach.
FEMSMicrobiolLett241:129–34.
206. TurnerKME,HanageWP,FraserC,ConnorTR,SprattBG.2007.
AssessingthereliabilityofeBURSTusingsimulatedpopulationswith
knownancestry.BMCMicrobiol7:30.
207. WillemsRJ,TopJ,vanSchaikW,LeavisH,BontenM,SirenJ,Hanage
WP,CoranderJ.2012.Restrictedgeneflowamonghospital
subpopulationsofEnterococcusfaecium.MBio2012/07/19.3:e00151-12.
208. Galloway-PeñaJ,RohJH,LatorreM,QinX,MurrayBE.2012.Genomic
andSNPanalysesdemonstrateadistantseparationofthehospitaland
community-associatedcladesofEnterococcusfaecium.PLoS
One2012/02/01.7:e30187.
209. PalmerKL,GodfreyP,GriggsA,KosVN,ZuckerJ,DesjardinsC,
CerqueiraG,GeversD,WalkerS,WortmanJ,FeldgardenM,HaasB,
BirrenB,GilmoreMS.2012.Comparativegenomicsofenterococci:
71
variationinEnterococcusfaecalis,cladestructureinE.faecium,and
definingcharacteristicsofE.gallinarumandE.casseliflavus.MBio
3:e00318-11.
210. vanSchaikW,TopJ,RileyDR,BoekhorstJ,VrijenhoekJE,
SchapendonkCM,HendrickxAP,NijmanIJ,BontenMJ,TettelinH,
WillemsRJ.2010.Pyrosequencing-basedcomparativegenomeanalysisof
thenosocomialpathogenEnterococcusfaeciumandidentificationofa
largetransferablepathogenicityisland.BMCGenomics2010/04/20.
11:239.
211. KimEB,MarcoML.2014.NonclinicalandclinicalEnterococcusfaecium
strains,butnotEnterococcusfaecalisstrains,havedistinctstructuraland
functionalgenomicfeatures.ApplEnvironMicrobiol80:154–65.
212. deBeenM,PinholtM,TopJ,BletzS,MellmannA,vanSchaikW,
BrouwerE,RogersM,KraatY,BontenM,CoranderJ,WesthH,
HarmsenD,WillemsRJL.2015.CoreGenomeMultilocusSequence
TypingSchemeforHigh-ResolutionTypingofEnterococcusfaecium.JClin
Microbiol53:3788–97.
213. PinholtM,Larner-SvenssonH,LittauerP,MoserCE,PedersenM,
LemmingLE,EjlertsenT,SøndergaardTS,HolzknechtBJ,JustesenUS,
DzajicE,OlsenSS,NielsenJB,WorningP,HammerumAM,WesthH,
JakobsenL.2015.MultiplehospitaloutbreaksofvanAEnterococcus
faeciuminDenmark,2012-13,investigatedbyWGS,MLSTandPFGE.J
AntimicrobChemother70:2474–82.
214. PinholtM,GumpertH,BaylissS,NielsenJB,VorobievaV,PedersenM,
FeilE,WorningP,WesthH.2016.Genomicanalysisof495vancomycinresistantEnterococcusfaeciumrevealsbroaddisseminationofavanA
plasmidinmorethan19clonesfromCopenhagen,Denmark.JAntimicrob
Chemotherdkw360.
215. SoderblomT,AspevallO,ErntellM,HedinG,HeimerD,HokebergI,
Kidd-LjunggrenK,MelhusA,Olsson-LiljequistB,SjogrenI,
SmedjegardJ,StruweJ,SylvanS,Tegmark-WisellK,ThoreM.2010.
AlarmingspreadofvancomycinresistantenterococciinSwedensince
2007.EuroSurveill15:pii=19620.
72
216. BjørkengEK,RasmussenG,SundsfjordA,SjöbergL,HegstadK,
SöderquistB.2011.ClusteringofpolyclonalVanB-typevancomycinresistantEnterococcusfaeciuminalow-endemicareawasassociatedwith
CC17-genogroupstrainsharbouringtransferablevanB2-Tn5382and
pRUM-likerepAcontainingplasmidswithaxe-txeplasmidaddiction
system.APMIS119:247–258.
217. GranlundM,CarlssonC,EdebroH,EmanuelssonK,LundholmR.2006.
NosocomialoutbreakofvanB2vancomycin-resistantEnterococcus
faeciuminSweden.JHospInfect62:254–6.
218. BillströmH,TopJ,EdlundC,LundB.2010.Frequentoccurrenceof
multidrug-resistantCC17Enterococcusfaeciumamongclinicalisolatesin
Sweden.JApplMicrobiol108:1810–1816.
219. LanderslevKG,JakobsenL,OlsenSS,PedersenMB,KristensenB,
LemmingLE,WangM,KjærsgaardM,SteggerM,HasmanH,
HammerumAM.2016.PolyclonalspreadofvanAEnterococcusfaecium
inCentralDenmarkRegion,2009-2013,investigatedusingPFGE,MLST
andWGS.IntJAntimicrobAgents.
220. Meldingssystemforsmittsommesykdommer(MSIS).
http://www.msis.no.
221. Folkhälsomyndigheten.
https://www.folkhalsomyndigheten.se/folkhalsorapporteringstatistik/statistikdatabaser-ochvisualisering/sjukdomsstatistik/vancomycinresistenta-enterokocker-vre/.
222. StatensSerumInstitut.2016.
http://www.ssi.dk/Smitteberedskab/Sygdomsovervaagning.aspx.
223. JohnsenPJ,ØsterhusJI,SletvoldH,SørumM,KruseH,NielsenK,
SimonsenGS,SundsfjordA.2005.Persistenceofanimalandhuman
glycopeptide-resistantenterococciontwoNorwegianpoultryfarms
formerlyexposedtoavoparcinisassociatedwithawidespreadplasmidmediatedvanAelementwithinapolyclonalenterococcusfaecium
population.ApplEnvironMicrobiol2005/01/11.71:159–68.
224. FreitasAR,CoqueTM,NovaisC,HammerumAM,LesterCH,ZervosMJ,
DonabedianS,JensenLB,FranciaMV,BaqueroF,PeixeL.2011.Human
73
andswinehostssharevancomycin-resistantEnterococcusfaeciumCC17
andCC5andEnterococcusfaecalisCC2clonalclustersharboringTn1546
onindistinguishableplasmids.JClinMicrobiol49:925–31.
225. Garcia-MiguraL,Sanchez-ValenzuelaAJ,JensenLB.2011.Presenceof
Glycopeptide-EncodingPlasmidsinEnterococcalIsolatesfromFoodand
HumansinDenmark.FoodbornePathogDis8:1191–1197.
226. NordahlPetersenT,RasmussenS,HasmanH,CarøeC,BælumJ,
CharlotteSchultzA,BergmarkL,SvendsenCa.,LundO,SicheritzPonténT,AarestrupFM.2015.Meta-genomicanalysisoftoiletwaste
fromlongdistanceflights;asteptowardsglobalsurveillanceofinfectious
diseasesandantimicrobialresistance.SciRep5:11444.
227. HansenTA,JoshiT,LarsenAR,AndersenPS,HarmsK,MollerupS,
WillerslevE,FuurstedK,NielsenLP,HansenAJ.2016.Vancomycingene
selectioninthemicrobiomeofurbanRattusnorvegicusfromhospital
environment.EvolMedpublicHeal2016:219–26.
228. IversenA,KühnI,FranklinA,MöllbyR.2002.Highprevalenceof
vancomycin-resistantenterococciinSwedishsewage.ApplEnviron
Microbiol68:2838–42.
229. RavenKE,ReuterS,ReynoldsR,BrodrickHJ,RussellJE,TörökME,
ParkhillJ,PeacockSJ.2016.AdecadeofgenomichistoryforhealthcareassociatedEnterococcusfaeciumintheUnitedKingdomandIreland.
GenomeRes26:1388–1396.
230. NumminenE,GutmannM,ShubinM,MarttinenP,MéricG,vanSchaik
W,CoqueTM,BaqueroF,WillemsRJL,SheppardSK,FeilEJ,Hanage
WP,CoranderJ.2016.Theimpactofhostmetapopulationstructureon
thepopulationgeneticsofcolonizingbacteria.JTheorBiol70:156–7.
231. CarterGP,BuultjensAH,BallardSA,BainesSL,TomitaT,StrachanJ,
JohnsonPDR,FergusonJK,SeemannT,StinearTP,HowdenBP.2016.
EmergenceofendemicMLSTnon-typeablevancomycin-resistant
Enterococcusfaecium.JAntimicrobChemother71:3367–3371.
232. LomanNJ,PallenMJ.2015.Twentyyearsofbacterialgenomesequencing.
NatRevMicrobiol13:787–794.
233. AarestrupFM,KoopmansMG.2016.SharingDataforGlobalInfectious
74
DiseaseSurveillanceandOutbreakDetection.TrendsMicrobiol24:241–5.
234. LamMMC,SeemannT,TobiasNJ,ChenH,HaringV,MooreRJ,Ballard
S,GraysonLM,JohnsonPDR,HowdenBP,StinearTP.2013.
Comparativeanalysisofthecompletegenomeofanepidemichospital
sequencetype203cloneofvancomycin-resistantEnterococcusfaecium.
BMCGenomics14:595.
235. vanHalSJ,IpCLC,AnsariMA,WilsonDJ,EspedidoBA,JensenSO,
BowdenR.2016.EvolutionarydynamicsofEnterococcusfaeciumreveals
complexgenomicrelationshipsbetweenisolateswithindependent
emergenceofvancomycinresistance.Microbgenomics2.
236. BrodrickHJ,RavenKE,HarrisonEM,BlaneB,ReuterS,TörökME,
ParkhillJ,PeacockSJ.2016.Whole-genomesequencingreveals
transmissionofvancomycin-resistantEnterococcusfaeciumina
healthcarenetwork.GenomeMed8:4.
237. McNallyA,OrenY,KellyD,PascoeB,DunnS,SreecharanT,VehkalaM,
VälimäkiN,PrenticeMB,AshourA,AvramO,PupkoT,DobrindtU,
LiterakI,GuentherS,SchauflerK,WielerLH,ZhiyongZ,SheppardSK,
McInerneyJO,CoranderJ.2016.CombinedAnalysisofVariationinCore,
AccessoryandRegulatoryGenomeRegionsProvidesaSuper-Resolution
ViewintotheEvolutionofBacterialPopulations.PLoSGenet
12:e1006280.
238. SheppardAE,StoesserN,WilsonDJ,SebraR,KasarskisA,AnsonLW,
GiessA,PankhurstLJ,VaughanA,GrimCJ,CoxHL,YehAJ,
ModernisingMedicalMicrobiology(MMM)InformaticsGroup,Sifri
CD,WalkerAS,PetoTE,CrookDW,MathersAJ.2016.NestedRussian
Doll-LikeGeneticMobilityDrivesRapidDisseminationoftheCarbapenem
ResistanceGeneblaKPC.AntimicrobAgentsChemother60:3767–78.
239. HillCM,KrauseKM,LewisSR,BlaisJ,BentonBM,MammenM,
HumphreyPP,KinanaA,JancJW.2010.Specificityofinductionofthe
vanAandvanBoperonsinvancomycin-resistantenterococcibytelavancin.
AntimicrobAgentsChemother54:2814–8.
240. DongSD,OberthürM,LoseyHC,AndersonJW,EggertUS,PeczuhMW,
WalshCT,KahneD.2002.ThestructuralbasisforinductionofVanB
75
resistance.JAmChemSoc124:9064–5.
241. KwunMJ,HongH-J.2014.Theactivityofglycopeptideantibioticsagainst
resistantbacteriacorrelateswiththeirabilitytoinducetheresistance
system.AntimicrobAgentsChemother58:6306–10.
242. BaptistaM,DepardieuF,ReynoldsP,CourvalinP,ArthurM.1997.
Mutationsleadingtoincreasedlevelsofresistancetoglycopeptide
antibioticsinVanB-typeenterococci.MolMicrobiol25:93–105.
243. AslangulE,BaptistaM,FantinB,DepardieuF,ArthurM,CourvalinP,
CarbonC.1997.Selectionofglycopeptide-resistantmutantsofVanB-type
EnterococcusfaecalisBM4281invitroandinexperimentalendocarditis.J
InfectDis175:598–605.
244. WijesuriyaTM,PerryP,PryceT,BoehmJ,KayI,FlexmanJ,Coombs
GW,IngramPR.2014.LowvancomycinMICsandfecaldensitiesreduce
thesensitivityofscreeningmethodsforvancomycinresistancein
Enterococci.JClinMicrobiol52:2829–33.
245. HegstadK,GiskeCG,HaldorsenB,MatuschekE,SchonningK,
LeegaardTM,KahlmeterG,SundsfjordA.2014.Performanceofthe
EUCASTDiskDiffusionMethod,theCLSIAgarScreenMethod,andthe
Vitek2AutomatedAntimicrobialSusceptibilityTestingSystemfor
DetectionofClinicalIsolatesofEnterococciwithLow-andMedium-Level
VanB-TypeVancomycinResistanc.JClinMicrobiol2014/03/07.52:1582–
1589.
246. KlareI,FleigeC,GeringerU,WitteW,WernerG.2012.Performanceof
threechromogenicVREscreeningagars,twoEtest(®)vancomycin
protocols,anddifferentmicrodilutionmethodsindetectingvanBgenotype
EnterococcusfaeciumwithvaryingvancomycinMICs.DiagnMicrobiol
InfectDis74:171–6.
247. WernerG,KlareI,FleigeC,GeringerU,WitteW,JustH-M,ZieglerR.
2012.Vancomycin-resistantvanB-typeEnterococcusfaeciumisolates
expressingvaryinglevelsofvancomycinresistanceandbeinghighly
prevalentamongneonatalpatientsinasingleICU.AntimicrobResistInfect
Control1:21.
248. ChaJO,YooJI,KimHK,KimHS,YooJS,LeeYS,JungYH.2013.Diversity
76
ofTn1546invanA-positiveEnterococcusfaeciumclinicalisolateswith
VanA,VanB,andVanDphenotypesandsusceptibilitytovancomycin.JAppl
Microbiol115:969–76.
249. SongJ-H,KoKS,SuhJY,OhWS,KangC-I,ChungDR,PeckKR,LeeNY,
LeeWG.2008.Clinicalimplicationsofvancomycin-resistantEnterococcus
faecium(VRE)withVanDphenotypeandvanAgenotype.JAntimicrob
Chemother61:838–844.
250. GagnonS,LevesqueS,LefebvreB,BourgaultA-M,LabbeA-C,RogerM.
2011.vanA-containingEnterococcusfaeciumsusceptibletovancomycin
andteicoplaninbecauseofmajornucleotidedeletionsinTn1546.J
AntimicrobChemother66:2758–2762.
251. HashimotoY,TanimotoK,OzawaY,MurataT,IkeY.2000.Aminoacid
substitutionsintheVanSsensoroftheVanA-typevancomycin-resistant
enterococcusstrainsresultinhigh-levelvancomycinresistanceandlowlevelteicoplaninresistance.FEMSMicrobiolLett185:247–254.
252. SzakacsTA,KalanL,McConnellMJ,EshaghiA,ShahinasD,McGeerA,
WrightGD,LowDE,PatelSN.2014.Outbreakofvancomycin-susceptible
Enterococcusfaeciumcontainingthewild-typevanAgene.JClin
Microbiol2014/02/14.52:1682–1686.
253. GuL,CaoB,LiuY,GuoP,SongS,LiR,DaiH,WangC.2009.Anew
Tn1546typeofVanBphenotype-vanAgenotypevancomycin-resistant
EnterococcusfaeciumisolatesinmainlandChina.DiagnMicrobiolInfect
Dis63:70–5.
254. KnightDR,AndrogaGO,BallardSA,HowdenBP,RileyTV.2016.A
PhenotypicallySilentvanB2OperonCarriedonaTn1549-LikeElementin
Clostridiumdifficile.mSphere1.
255. EllingtonMJ,EkelundO,AarestrupFM,CantonR,DoumithM,GiskeC,
GrundmanH,HasmanH,HoldenM,HopkinsKL,IredellJ,Kahlmeter
G,KöserCU,MacGowanA,MeviusD,MulveyM,NaasT,PetoT,Rolain
J-M,SamuelsenØ,WoodfordN.2016.TheRoleofWholeGenome
Sequencing(WGS)inAntimicrobialSusceptibilityTestingofBacteria:
ReportfromtheEUCASTSubcommittee.ClinMicrobiolInfect.
256. ThakerMN,KalanL,WaglechnerN,EshaghiA,PatelSN,PoutanenS,
77
WilleyB.2015.Vancomycin-VariableEnterococciCanGiveRiseto
ConstitutiveResistanceduringAntibioticTherapy59:1405–1410.
257. SivertsenA,PedersenT,JaniceJ,Hegstad.2017.Vancomycinvariable
resistancemayoccurthroughcreationofanovelpromoterafterdeletions
bypolymeraseslippage,p.submitted.In27thEuropeanCongressof
ClinicalMicrobiologyandInfectiousDiseases.
258. ZarkanA,MacklyneH-R,TrumanAW,HeskethAR,HongH-J.2016.The
frontlineantibioticvancomycininducesazincstarvationresponsein
bacteriabybindingtoZn(II).SciRep6:19602.
259. SanMillanA,EscuderoJA,GiffordDR,MazelD,MacLeanRC.2016.
Multicopyplasmidspotentiatetheevolutionofantibioticresistancein
bacteria.NatEcolEvol1:10.
260. PorseA,SchønningK,MunckC,SommerMOA.2016.Survivaland
EvolutionofaLargeMultidrugResistancePlasmidinNewClinical
BacterialHosts.MolBiolEvol33:2860–2873.
261. HeS,HickmanAB,VaraniAM,SiguierP,ChandlerM,DekkerJP,Dyda
F.2015.InsertionSequenceIS26ReorganizesPlasmidsinClinically
IsolatedMultidrug-ResistantBacteriabyReplicativeTransposition.MBio
6:e00762.
262. HeS,ChandlerM,VaraniAM,HickmanAB,DekkerJP,DydaF.2016.
MechanismsofEvolutioninHigh-ConsequenceDrugResistancePlasmids.
MBio7:e01987-16.
263. StojanovM,MoreillonP,SakwinskaO.2015.Excisionofstaphylococcal
cassettechromosomemecinmethicillin-resistantStaphylococcusaureus
assessedbyquantitativePCR.BMCResNotes8:828.
264. MisiuraA,PigliYZ,Boyle-VavraS,DaumRS,BoocockMR,RicePA.
2013.RolesoftwolargeserinerecombinasesinmobilizingthemethicillinresistancecassetteSCCmec.MolMicrobiol88:1218–29.
265. HughesD.2003.Exploitinggenomics,geneticsandchemistrytocombat
antibioticresistance.NatRevGenet4:432–41.
78