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ResearchAntibioticsusceptibilityofcoagulase-negativestaphylococciisolatedfromverylowbirthweightbabies:comprehensivecomparisonsofbacteriaatdifferentstagesofbiofilmformationYueQu1,AndrewJDaley2,TaghridSIstivan1,SuzanneMGarland2,3,4andMargaretADeighton*1AbstractBackground:Coagulase-negativestaphylococciaremajorcausesofbloodstreaminfectionsinverylowbirthweightbabiescaredforinNeonatalIntensiveCareUnits.
Thevirulenceofthesebacteriaismainlyduetotheirabilitytoformbiofilmsonindwellingmedicaldevices.
Biofilm-relatedinfectionsoftenfailtorespondtoantibioticchemotherapyguidedbyconventionalantibioticsusceptibilitytests.
Methods:Coagulase-negativestaphylococcalbloodcultureisolatesweregrownindifferentphasesrelevanttobiofilmformation:planktoniccellsatmid-logphase,planktoniccellsatstationaryphase,adherentmonolayersandmaturebiofilmsandtheirsusceptibilitiestoconventionalantibioticswereassessed.
Theeffectsofoxacillin,gentamicin,andvancomycinonpreformedbiofilms,atthehighestachievableserumconcentrationswereexamined.
Epifluorescencemicroscopyandconfocallaserscanningmicroscopyincombinationwithbacterialviabilitystainingandpolysaccharidestainingwereusedtoconfirmthestimulatoryeffectsofantibioticsonbiofilms.
Results:Mostcoagulase-negativestaphylococcalclinicalisolateswereresistanttopenicillinG(100%),gentamicin(83.
3%)andoxacillin(91.
7%)andsusceptibletovancomycin(100%),ciprofloxacin(100%),andrifampicin(79.
2%).
Bacteriagrownasadherentmonolayersshowedsimilarsusceptibilitiestotheirplanktoniccounterpartsatmid-logphase.
Isolatesinabiofilmgrowthmodeweremoreresistanttoantibioticsthanbothplanktonicculturesatmid-logphaseandadherentmonolayers;howevertheywereequallyresistantorlessresistantthanplanktoniccellsatstationaryphase.
Moreover,forsomecell-wallactiveantibiotics,concentrationshigherthanconventionalMICswererequiredtopreventtheestablishmentofplanktonicculturesfrombiofilms.
Finally,thebiofilm-growthoftwoS.
capitisisolatescouldbeenhancedbyoxacillinatthehighestachievableserumconcentration.
Conclusion:Weconcludethattheresistanceofcoagulase-negativestaphylococcitomultipleantibioticsinitiallyremainsimilarwhenthebacteriashiftfromaplanktonicgrowthmodeintoanearlyattachedmode,thenincreasesignificantlyastheadherentmodefurtherdevelops.
Furthermore,preformedbiofilmsofsomeCoNSareenhancedbyoxacillininadose-dependentmanner.
BackgroundCoagulase-negativestaphylococci(CoNS),predomi-nantlyStaphylococcusepidermidis,arethemostcommoncausativeagentsofneonatalsepsis[1-3],aconditionwhichhasbeenrelatedtosignificantmorbidityandmor-talityinneonatalintensivecareunits(NICUs)[2].
Thepresenceofacentralvenouscatheterinverylowbirthweight(VLBW)babies(48h),thechoiceiseitherflucloxacillinorvancomycinwithgentamicin.
Ciprofloxacinandrifampicinwerealsoeval-uatedinthisstudyastheefficacyoftheseantibioticsonCoNSbiofilmshasbeenreportedbyotherinvitrostud-ies.
PenicillinGwaspurchasedfromCSLBiotherapies,Parkville,AustraliaandallotherswereobtainedfromSigma-Aldrich,CastleHill,Australia.
EstablishmentofadherentmonolayersBacterialculturesofadherentmonolayerswereestab-lishedfollowingthemethodofMiyakeetal.
(1992),whichinvolvedtheadditionof50μLvolumesofbacterialTable1:Bacterialisolatesandgrowthmediausedforbiofilmformation.
IsolateSpeciesStatusGrowthmediumforbiofilmformationaTSBTSB+1%glucoseTSB+4%NaClicaAicaCicaD1S.
warneriInvasive+---2S.
haemolyticusInvasive----3S.
epidermidisInvasive++++4S.
epidermidisInvasive+---5S.
epidermidisInvasive++++6S.
capitisInvasive+++-7S.
epidermidisInvasive++++8aS.
capitisInvasive+++-8bS.
capitisInvasive+++-9S.
capitisInvasive+++-10S.
epidermidisInvasive+---11S.
epidermidisInvasive++++12S.
epidermidisContaminantw---13S.
epidermidisContaminant++++15S.
capitisContaminantw++-16S.
capitisContaminant+++-17S.
capitisContaminant+++-18S.
capitisContaminant+++-19S.
epidermidisContaminant----20S.
epidermidisContaminant++++21S.
epidermidisContaminant++++22S.
capitisContaminantw+++23S.
epidermidisContaminant++++24S.
epidermidisContaminant++++RP62AS.
epidermidisReference+SP2S.
hominisReference-aSelectionofgrowthmediumwasbasedontheproductionofthehighestbiofilmdensityforeachisolate.
Theamountofbiofilmwasindicatedas:"+",strong(OD600≥0.
24);"w",weak(0.
12≤OD6002*log2inMICorMBCforoxacillin,vancomycin,ciprofloxacinandrifampicin,andanincreaseof>3*log2forMICorMBCforpenicillinandgentamicin,wereconsideredsignificant[38].
Experi-mentstargetingtheeffectsofantibioticsonpreformedbiofilmswererepeatedatleastthreetimesintriplicate.
Onewayanalysisofvariance(ANOVA)orthenon-para-metricMann-Whitneytestwasusedfortwo-setcompar-isonsandap-valueof10241024>102432≤132Biofilm(0.
9-1.
9)*109>1024e>102464>102464>102416>1024d0.
25>10240.
0040.
015Isolate3LogPlanktonic(2.
9-8.
3)*1058160.
250.
250.
250.
25240.
120.
250.
0080.
015Monolayer(5.
0-11.
0)*10532640.
250.
50.
120.
5280.
250.
50.
0020.
004StatPlanktonic(0.
6-2.
2)*109>102432>1024>10241024256Biofilm(0.
3-1.
3)*109>1024>102416160.
25216160.
2520.
54Isolate8aLogPlanktonic(2.
6-5.
5)*105128128323288120.
250.
250.
0080.
03Monolayer(6.
4-11.
1)*105>128>12832646464180.
120.
50.
0040.
015StatPlanktonic(4.
4-4.
9)*108>1024>1024>1024>102410242Biofilm(0.
6-1.
8)*1081024>1024256>1024256>10248>10240.
12>10240.
0044Isolate9LogPlanktonic(3.
1-5.
2)*10532643232816120.
250.
250.
0150.
03Monolayer(4.
6-10.
8)*105>128>12832>1281632280.
250.
50.
0040.
015StatPlanktonic(0.
7-0.
8)*109>1024>1024>1024>1024>1024256Biofilm(1.
1-1.
7)*109>1024>102464>1024>128>10248>10240.
25>10240.
0084Isolate11LogPlanktonic(2.
7-8.
5)*1058864>1283264110.
060.
12>128>128Monolayer(3.
2-17.
8)*1053264>128>1283264120.
060.
12>128>128StatPlanktonic(0.
8-1.
6)*109>1024>1024>1024>10245121024Biofilm(0.
2-0.
6)*109>1024>1024256>1024256>10248>10240.
251664>1024αBiofilm-positiveisolates(OD600>0.
24).
bMICsforCoNSatstationaryphasearenotprovidedasthevaluescouldnotbedeterminedbystandardmethods.
cValuesunderlinedindicateasignificantincreaseintheMICsorMBCsbetweenlog-planktonicandadherentmonolayermodesofgrowth.
dValuesinitalicindicateasignificantchangeintheMBCsbetweenstationary-planktonicandbiofilmmodesofgrowth.
eValuesinboldindicateasignificantincreaseinMICsorMBCsbetweenlog-planktonic/adherentmonolayerandbiofilmmodesofgrowthQuetal.
AnnalsofClinicalMicrobiologyandAntimicrobials2010,9:16http://www.
ann-clinmicrob.
com/content/9/1/16Page8of12adherentmonolayers,however,itisalsolikelythatthedifferencewasduetothedifferentkineticsofbiofilmfor-mationbetweenP.
aeruginosaandStaphylococcusspp.
[19].
ConventionalMICshavebeenusedtoguidethetreat-mentofbiofilm-relatedinfectionsatthefebrilestage,basedontheassumptionthatbiofilm-releasedcellsaresimilarintheirsusceptibilitiestocellsintheplanktonicphase[23].
Reporteddifferencesbetweentheconven-tionalMICsandbiofilmMICswereattributedtolackofstandardizationofinitialinoculaandtothepresenceofsmallcolonyvariants[23].
However,inourstudy,wefoundthatMICsofcellwallactiveantibioticswerefre-quentlyhigherforbiofilmgrownbacteriathanplanktoniccultures.
ThisisconsistentwithrecentstudiesbyMoskowitzetal.
(2004)andMelchioretal.
(2006),whoreportedthatbiofilmMICsofβ-lactamantibiotics,butnototherantibiotics,weremuchhigherthantheconven-tionalMICsforP.
aeruginosaandS.
aureusrespectively[21,24].
Theseresultsarenotsurprisinggiventhatcellwallactiveantibioticsmainlyaffectrapidlygrowingbac-teria.
Biofilm-releasedcellsarelikelytobelessactivethandividingplanktoniccellsatmid-logphase,probablybecausetheyhaverecentlyundergoneaswitchfromabiofilmmodeofgrowthtoafree-livingmode,similartocellsatlag-phasegrowth,andrequireachangeingeneexpressiontoadapttothenewenvironment.
TheMBCsofpenicillinG,gentamicin,oxacillin,andvancomycinforCoNSgrowninabiofilmmodeweregen-erally>1024μg/ml,whichiswellbeyondthehighestachievableserumconcentrations.
Althoughsomeoftheseantibioticsatthehighestachievableserumconcen-trationswereeffectiveagainstbacteriagrownplanktoni-callytomid-logphase,theywereinadequatetokillTable3:Antibioticsusceptibilityoffourbiofilm-negativeisolatesagrownindifferentmodes.
IsolateandmodeofgrowthInitialbacterialdensity(CFU/ml)Penicillin(μg/ml)Gentamicin(μg/ml)Oxacillin(μg/ml)Vancomycin(μg/ml)Ciprofloxacin(μg/ml)Rifampicin(μg/ml)MICMBCMICMBCMICMBCMICMBCMICMBCMICMBCSP2LogPlanktonic(1.
2-4.
1)*1050.
50.
5880.
060.
06120.
120.
120.
0080.
03Monolayer(2.
6-4.
7)*10528c16160.
120.
120.
510.
120.
120.
0040.
008StatPlanktonic(3.
5-9.
5)*107b>10241024d2420.
008Biofilm(1.
0-3.
2)*107128>1024e8320.
122240.
1220.
0020.
008Isolate15LogPlanktonic(2.
2-5.
5)*10512812832641632440.
250.
250.
0150.
03Monolayer(5.
2-16.
4)*105>128>12832641664480.
120.
250.
0040.
008StatPlanktonic(3.
7-7.
9)*108>1024>1024>1024>1024>102464Biofilm(0.
7-0.
9)*108>1024>1024256>1024256>102416>10240.
255120.
0040.
008Isolate19LogPlanktonic(2.
9-7.
4)*105>128>128646428220.
250.
250.
0080.
015Monolayer(3.
2-14.
6)*105>128>12864>12828280.
120.
250.
0020.
008StatPlanktonic(0.
7-1.
5)*109>1024>1024>1024>102410242Biofilm(0.
1-0.
2)*1091024>102464>102464>10244>10240.
2540.
0080.
06Isolate22LogPlanktonic(2.
8-5.
6)*105>128>1286464816440.
120.
250.
0150.
03Monolayer(4.
8-16.
3)*105>128>12832>1283232280.
120.
250.
0040.
008StatPlanktonic(0.
5-0.
7)*109>1024>1024>1024>1024>1024256Biofilm0.
2*1091024>1024>128>1024>128>102416>10240.
2510.
0080.
03aBiofilm-negativeisolatesincludedbiofilm-weakproducer(0.
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Received:5February2010Accepted:27May2010Published:27May2010Thisarticleisavailablefrom:http://www.
ann-clinmicrob.
com/content/9/1/162010Quetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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doi:10.
1186/1476-0711-9-16Citethisarticleas:Quetal.
,Antibioticsusceptibilityofcoagulase-negativestaphylococciisolatedfromverylowbirthweightbabies:comprehensivecomparisonsofbacteriaatdifferentstagesofbiofilmformationAnnalsofClinicalMicrobiologyandAntimicrobials2010,9:16