1.
Introduction
Bronchialcolonizationbypathogensisarecognizedcauseof morbidityandmortalityinpatients withcystic fibrosis(CF). PseudomonasaeruginosaandStaphylococcusaureusaretwo ofthemainbronchialpathogensinCF.ChronicP.aeruginosa lungcolonizationisassociatedwithadeclineinlungfunction, increased morbidity, and a shorter life expectancy [1,2]. In 2013, 39.7% of the French CF patients had P. aeruginosa isolatedfromtheirsputum[3].Itisaubiquitous environmen-talbacteria,surviving instill water fora long time,and is recognizedasapotentialpipecontaminator[4].S.aureusis anothermajorpathogeninCF.Itsimpactonlungfunctionand itssurvivalismorecontroversialthanthatofP.aeruginosa. Colonizationbymulti-drugresistantS.aureusseemstohave an impact on lung function, exacerbation frequency, and survival[5,6].Recently,astudyhasshownthatthecoinfection byP.aeruginosaandS.aureuswasassociatedwithworsened lungfunction[7].S.aureushasbeenisolatedin62.1%ofthe sputumofFrenchCFpatients[3].
Inhealthychildren’snasopharyngealpassages,P.aeruginosa colonization is rare, not exceeding 5% [8,9], but S. aureus, whichis oneof thenasopharyngealcommensalbacteria,is detected in 27–49% of cultures [9,10]. In CF children, P. aeruginosanasopharyngealcarriageisup to14%[8],and S. aureuscarriageisalsosignificant(Oddsratio,2.4)[10].Since the oropharynx can serve as a reservoir for pathological microorganisms,itcouldleadtolowerairwaybacterial infec-tioninCFpatients[11].Thepatient’senvironmentis conside-red as a potential source of contamination and many recommendationshavebeenmadetoreducetheriskofthis potential pathogen, especiallyby increasingthe processof cleaning and disinfection. Hence, oral and dental hygiene must be considered. TheHaute Autorite´ de Sante´ (HAS) in France recommendsthefollowing:atleast twotooth brus-hings per day with a fluoride toothpaste. However, tooth-brushmaintenanceisnotspecified,resultinginavarietyof protocolsbeingused.Accordingtotheliterature, toothbrus-hes can be contaminated by microorganisms, which can originatefromtheoralcavity,theenvironment,thehands, aerosol contamination,contact withothertoothbrushes,or
fromstoragecontainers[12–14].Thereislimitedresearchon microorganisms,mostparticularlyS.aureusandP.aeruginosa toothbrushcontamination afteruse[13,15].Since toothbrus-hescanbeapotentialvectorforCFbronchialcontamination byS.aureusandP.aeruginosaandsincenodataexistonCF toothbrushbacterialcontamination,weconducteda descrip-tivepilotstudytoassesstheprevalenceoftwopathogens– S. aureus and P. aeruginosa – on the toothbrushes of CF children vs their siblings and healthy controls. Finally, we compared the toothbrush contamination profiles with the pulmonarycolonizationofCFchildren.
2.
Methods
2.1.
Study
design
and
patient
population
Participationinthestudy wasproposed toeach CFpatient agedfrom8to18years,andtheirparents,duringthe follow-up visitsat ToulouseChildren’s HospitalCF Centerfrom17 December2013to21February2014.CFpatientswereaskedto completea questionnaire aboutindividualtoothbrush care andotheroralcareissues,andtobringtheircurrentlyused toothbrush (<24h after the last use). CF diagnosis was confirmedby two sweat chloridetests>60 mEq/L and/or genetictestingpositivefortwoknownCF-causingmutations. Tocomparewiththegeneralpopulation, wealsoproposed thisstudytotheirhealthysiblings,whohadaknownnormal sweatchloridetest,andtohealthyvolunteers8–18yearsold, selectedfromtherelativesofthestaffworkingatthehospital orthebacteriologyinstitute.
For each CF patient,bacterial contamination of the tooth-brushwascomparedtothebacterialanalysisofthesputum sample performed on the same day, and to the sputum bacterial analysis of the preceding year. According to the EuroCareCFrecommendations,chronicP.aeruginosainfection was defined as 50% or more if these samples had been positive in the preceding 12 months [16]. If P. aeruginosa was isolated both on the CF patient’s toothbrush and in sputum, bacterial genotyping was conducted to compare the strains. For the sibling group, the toothbrush strain wascomparedwiththeirCFsibling’ssputumstrain. Conclusion.S.aureusandP.aeruginosacancolonizeCFpatients’
toothbrushes.Theimpactonpulmonarycolonizationremainsunk nown.Toothbrushdecontaminationmethodsneedtoconsiderthese bacteriainCFpatients.
Lege´notypagedessouchesdeP.aeruginosaamisene´videncela pre´sencechez2maladesd’unmeˆmeclonebacte´riensurlabrossea` dentetdanslesexpectorations,etunecorrespondancege´notypique entrel’expectorationd’unmaladeetlasoucheisole´esurlabrossea` dentdesonfre`re.
Conclusion.S.aureusouP.aeruginosapeuventcoloniserlabrosse a` dentsdepatientsmucoviscidosiques.L’impactdecephe´nome`ne surlacolonisationbronchiqueestinconnu.Lesme´thodesdede´s infectiondesbrossesa` dentsdoiventeˆtreteste´esspe´cifiquementsur cesgermesdanslamucoviscidose.
Writteninformed consentwas obtained fromeach patient andhisorherparentsbeforethecurrentlyusedtoothbrush wasprovided,regardlessoftype,design,durationofuse,or toothbrush care habits. The Toulouse University Hospital ResearchEthicsCommitteeapprovedthisstudy.
2.2.
Questionnaire
Aquestionnaire wasusedto obtaininformation fromeach childabouthometoothbrushcareandotheroralcareissues thatmayhaveinfluencedthebacterialcontaminationofthe toothbrush(datanotshown).
2.3.
Toothbrush
bacterial
isolation
and
identification
Usedtoothbrusheswererecoveredwithin24hafterthelast useandweretransportedinseparatesingle-usesealedplastic bags.Quantitativetoothbrushcultureswereperformedinthe FonderepharLaboratoryinToulouseandinthemicrobiology laboratoryatToulousePurpanUniversityHospitalFederative BiologyInstitute.
Thetoothbrushheadwas retrievedandsoakedin20mLof Tryptone-selandvortexedfor30stodislodgebacteria.Serial tenfolddilutionsweremadeand100-mLaliquotswere spread-plated onto blood agar and incubated under aerobic and anaerobicconditionsfor5–7daysat3618C.Onemilliliter of the initial solution was filtered on 0.45-mm membrane (Millipore),themembranewasdeposited,and100-mLaliquots werealsospread-platedontoCetrimideagarandChapman2 agar,for3–5daysunderaerobicincubationat3618C. Afterincubation,thetotalviablecountofeachdistinctcolony typewasdeterminedandallisolatespresentwithacolony countgreaterthan1colonyformingunit(CFU)/Petridishwere identified at thespecies level using morphological (Colony appearance,Gramstaining),respiratorytype,andbiochemical (oxydase,catalase,APIsystems)criteriaandagglutinationfor staphylococci(Staphyslide,Biome´rieux).S.aureusstrainswere alsocheckedformethicillinresistance.
Thetotal bacterial group(streptococci, etc.) contamination level wasexpressed asCFU/toothbrush.For specific patho-genssuchasS.aureusandP.aeruginosa,theCFU log/tooth-brushwascompletedbydetection/nodetectioncriteria.
2.4.
Bacterial
analysis
of
expectorated
sputum
Oneach visit,aninducedsputum sample wascollectedfor bacteriologicalanalysis,performedaccordingtothenational guidelines for CF sputum microbiology (in REMIC, Socie´te´ franc¸aisedemicrobiologieEd;2010,99–103),withadetection thresholdof102CFU/mLforS.aureusandP.aeruginosa. S. aureus was identified by means of positive DNAse and agglutination (ProlexW i2a, Perols, France) tests. P. aeruginosa was identified using Vitek2GN cards (BioMe´-rieux,Marcyl’E´toile,France).2.5.
Strain
genotyping
ThePFGEprocedurewas carriedoutusingtheBio-RAD pro-tocol.Inbrief,whole-cellDNAfromP.aeruginosaisolateswas digestedwithSpeI(for20hat378C)afterlysisbylysozyme andproteinaseK.Electrophoresisofenzyme-generated frag-mentswasperformedwithacontour-clampedelectricfield GenePathSystemapparatus(Bio-Rad)througha1%agarose gel.Migrationwasperformedfor20hat148C,withanelectric fieldof6V/cm.DNAbandingpatternswerevisualizedwith GelRedanddigitallyphotographed.DNAbandsizewas deter-minedfromtheLambdaLadderPFGMarker(BiolabsW
)(size range:50–1000kb).
2.6.
Statistical
analysis
Concerningstatisticalanalysis,weconstitutedthreegroups: groupA(CFpatients),groupB(healthysiblings),andgroupC (healthyvolunteers).Appropriatestatisticalanalysiswas per-formed using SPSS software. Categorical variables were comparedusingaChi2test.Thedegreeoflineardependence betweentwo quantitative variables was studiedusing the Pearson’scorrelationcoefficient.
The relation between a quantitative variable and a dichotomous categorical variable was calculated using Student t-tests for independent samples, whereas the relationwithmorethantwo-modalitycategoricalvariables was investigated with analysis of variance (Anova). A 95% confidenceinterval was selected for statistical signi-ficance.
3.
Results
3.1.
Patient
population
Fifty-seven children were included in this study and were dividedintothreegroups:
groupA:27CFpatientsoutofthe29CFfamiliestowhom the study was proposed. Their median age was 14 years ( 2.7)andsexratio1.1.Ofthesepatients,26wereableto performroutinespirometry,median FEV1 was92% (range: 39–148%) of the predicted level, the nutritional status (estimatedbytheratioofactualweighttoidealweightat thesameage)was98% (range,74–135%) ofthepredicted level.Overthepastyear,17ofgroupACFpatientshadatleast one P. aeruginosa-positive culture (eight of them having chronicP.aeruginosainfection),23hadatleastoneS. aureus-positive culture,and 13 children had at least one positive culture of each. The presence of methicillin-resistant S. aureus (MRSA) in respiratory cultures from three CF patientsshouldbenoted;
groupB: 15 healthy siblings,whose median age andsex ratiowere,respectively,12.6years(3)and1.5;
group C: 17 healthy volunteers: their median age was 13.2years(2.3)andtheirsexratiowas1.1.
There was no significant difference in sex or median age between children with CF and the two other groups (P>0.05). According to the answers of thecorresponding questionnaires, the toothbrush duration of use differed greatly forthe57 toothbrushes collected:minimum 2 days andmaximum1year.Therewasasignificantdifferenceinthe meanvalue(P=0.042)betweengroups,whichwaslowerin groupAwith42daysofuse,comparedto91daysingroupC. There was no significant difference in tooth brushing fre-quency, time, autonomy, or toothbrush storage. Themean valueoftoothbrushingfrequencywas1.8/day(range:0.5–4). Everychildusedtoothpasteandrinsedhisbrushthoroughly withtapwaterafterwards.
3.2.
Toothbrush
bacterial
contamination
3.2.1. Oralflorabacteria
Microbiologicalanalysisoftoothbrushesonbloodagar iden-tifiedalargenumberofbacterialspecies,whichcorresponded to the main oral flora genera (Streptococcus, Actinomyces) sometimeswiththedetectionofanaerobicstrainsbelonging toFusobacterium,Peptostreptococcus,Porphyromonas,or Pre-votellasp.Therewasnosignificantdifferenceinthese bac-teria’sdistributionamongthethreegroups(P>0.05). The mean CFU value was 5.8105/mL, with a minimum survivalcountunderthethresholdofdetectionanda maxi-mum of 6.2106CFU/mL (fig. 1). There was no significant differenceamongthethreegroups(P>0.05);however,group A tended to harbor a lower count(2.5105CFU/mL) than groupC (1.3106CFU/mL). Toothbrushcontamination with oralbacteriasignificantlyincreasedwithtoothbrushduration ofuse(P<0.01).
3.2.2. S.aureus
S. aureus contamination was observed in ten out of the 57toothbrushes(17%)withnosignificantdifferencebetween the three groups (P>0.05); however, S. aureus detection frequencytended tobehigheringroupA(6/27,22%),than groupB(2/15,13%)orgroupC(2/15,13%)(fig.2).TheS.aureus bacterial count was low, ranging from 1 to 60 CFU/mL. S. aureusdetectionfrequencywaslowerastoothbrush dura-tionofuseincreased(P=0.029)andastheoralflorabacterial countincreased(CFU/mL)(P=0.005).
3.2.3. P.aeruginosa
P. aeruginosawasdetectedinsixofthe57toothbrushes(10%), with no significant difference between the three groups (p>0.05); however,P.aeruginosa detectionfrequency ten-dedtobehigheringroupA(4/27,15%)thaningroupB(2/15, 13%)orgroupC(0/15)(fig.3).TheP.aeruginosabacterialcount waslow,rangingfrom1to80CFU/mL.P.aeruginosadetection frequencywaslowerastheoralflorabacterialcount increa-sed(CFU/mL)(P=0.007).
3.2.4. ContaminationwithS.aureusandP.aeruginosa
Among the 57 toothbrushes, three were simultaneously contaminated by S. aureus and P. aeruginosa: two in groupAandoneingroupB.
Figure1. Oralflorabacterialspeciesidentifiedonbloodagar(CFU/mL). GroupA:CFpatients;groupB:healthysiblingsofCFpatients;groupC: healthyvolunteers.
Figure2. PercentageoftoothbrusheswithpositiveS.aureusculture. GroupA:CFpatients;groupB:healthysiblingsofCFpatients;groupC: healthyvolunteers.
3.3.
Bacteriological
sputum
analysis
and
correlation
with
toothbrush
colonization
Wedidfoundnoconcordancebetweensputumcultureresults anddatafromthepatients’toothbrushes(P>0.05),withthe resultofthesample collectedthesame dayastheir tooth-brush,theretrospectivesamplescollectedinthepreceding12 months, or the pulmonary colonization status (chronic or intermittent)(P>0.05).NoMRSAwasisolatedon toothbrus-hes,eveninthecaseofthethreeCFpatientsknowntohave MRSAintheirsputumcultures.P.aeruginosawasfoundon thetoothbrush of a single CF patientwho had never had P. aeruginosa isolated inhis sputum.For twopatients,the P. aeruginosa toothbrush clone was compared with P. aeruginosaisolated inthesputumcollectedonthesame day.Forthethirdpatientthetoothbrushclonewascompared tothelastpositivesputumsampleforP.aeruginosa. IngroupA,toothbrushandsputumP.aeruginosagenotyping confirmedthepresenceofthesamecloneinbothsamplesfor twoofthethreeCFpatientsconsidered(tableI).Ofthetwo identicalclones,oneP.aeruginosawasfromthesameday’s sputumandtheotherwasfromaprevioussample.IngroupB, one of the two strains of P. aeruginosa isolated fromthe siblings’ toothbrushes was considered genetically identical to the strain isolated in his CF sibling’s sputum (table II).
Observed genotypes varied from one patient to another, confirmingnoprevalenceofanyspecificgenotype,regardless ofthegroupconsidered.
4.
Discussion
ThisstudyisthefirstdocumentationofCFpatients’ tooth-brushcontaminationbybronchialpathogenssuchasS.aureus andP.aeruginosa.Thispilotstudyfoundaprevalenceof15% forP.aeruginosaonCFpatients’toothbrushes and22%for S. aureus.Inthe healthyadults, bacterial contamination of toothbrushes is known to occur soon after initial use [12], increaseswithrepeateduse,andcanpersistfrom1to5days
[12,17–19].Asdescribedintheliterature,weobservedoralflora
colonizationoftoothbrushesincreasingwiththedurationof use(p<0.01):nosignificantdifferenceinthecontamination levelwasnotedbetweenthethreegroupseventhoughwe surprisinglynotedalowermeanvalueintheCFpatientgroup. ConcerningS. aureusand P. aeruginosacontamination, the threegroupswereequallyconcerned,eventhoughwenoteda higherrateofcontamination onCFpatients’toothbrushes. Thisdifferencewasnotsignificant,possiblyduetothesmall sizeofthepopulationincluded.Variationsinpathogen detec-tionmaystemfrombacterialgrowthcompetitionon tooth-brushesbetweenoralflora,S.aureusandP.aeruginosa.Total bacterialcounts increasedwith theduration of use of the toothbrusheswithaconcomitantdecreasein S.aureusand P. aeruginosa detection. Repetitive antibiotic therapy (or occasionalantibiotic use fornon-CF groups),or chronic CF bronchial colonization could also have directly influenced survivalofbronchialpathogenbacteriaontoothbrushes.
15% 13% 0% 0% 5% 10% 15% 20% 25% A (n = 27) B (n = 15) C (n = 15) Group
Figure3.PercentageoftoothbrusheswithpositiveP.aeruginosaculture. GroupA:CFpatients;groupB:healthysiblingsofCFpatients;groupC: healthyvolunteers.
TableI
P. aeruginosagenotyping:bacterialstraincomparisonina singlesubject:expectoratedsputumvstoothbrushculture.
Subject Group Sample Ribotypeof
P. aeruginosa
Subject2 A Expectoratedsputum a
Toothbrushculture b
Subject14 A Expectoratedsputum c
Toothbrushculture c
Subject5 A Expectoratedsputum d
Toothbrushculture d
GroupA:CFpatients.
TableII
P. aeruginosagenotyping:bacterialstraincomparisonamongsiblingpairs:patientexpectoratedsputumvssiblingtoothbrushculture.
Siblings Subject Group Sample RibotypeofP.aeruginosa
Couple1 Subject11 A Expectoratedsputum e
Subject31 B Toothbrushculture f
Couple2 Subject15 A Expectoratedsputum g
Subject37 B Toothbrushculture g
Theactualimpactoftoothbrushcontaminationonbronchial primarycolonizationbyS.aureusorP.aeruginosaseemstobe very difficultto assess, becausebronchial colonization can occurfromdifferentsourcesandishighlydependentonthe child’s pulmonary status. Indeed, nebulizers, toilets, and washbasins have been described as potent sources of P. aeruginosaaerosolization [20]. However, it is very inter-estingtonotethatthesameP.aeruginosaclonewas identi-fiedontoothbrushandsputuminthreecases:twoCFpatients and surprisingly in thesputum of one CF patient and his brother’stoothbrush.Noclonalprevalencewasnoted.Atthis time, considering the environmental variability of P. aeruginosa, noconclusion asto theoriginof toothbrush contamination(thepatient’ssputumortheenvironment)or therepercussiononthepatient’slungcolonization(director indirectcolonization)canbedrawn.Inarecentreportusing multilocussequencetyping,Kiddetal.[21]foundthatevenif several CF strains were frequentlyencountered in multiple ecological settings, the most frequently encountered CF strains wereconfinedto CFpatientswith anon-clonal epi-demic structure.It was earlier demonstrated [22] that the microbialcommunitiesthatchronicallyinfecttheairwaysof patients with CF vary little over a year despite antibiotic perturbation.The presentspeciestended to varyfromone subject toanother, withless variationwithin eachpatient, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. Thus, thepotentialroleofexternalreservoirsinmaintaining patho-gensinCFpatientsmustbeconsidered.Withthedescription of various genotypes between CF patients but similarity between strains isolated from sputum and toothbrushes, ourobservations emphasize thattoothbrushes may consti-tuteanotherpathogenreservoiramongallthosedescribedin theliterature.
UnrelatedCF patientsmay acquireP. aeruginosafromthe environmentorbycross-infection,forexampleduring noso-comial outbreaks. The efficacy of measures to prevent nosocomialacquisitionofP.aeruginosawasearlier demons-trated [23–25]. Currently, CF patients should follow the generalrecommendationsofthehealthauthorities includ-ingtheCentersforDiseaseControlandPrevention, suggest-ing that patients not to share toothbrushes, rinse their toothbrushthoroughly withtapwaterfollowingbrushing, not routinely cover the toothbrush or store it in closed containers,allowingittoair-dry,andrenewthetoothbrush every3or 4months,sooner ifthebristlesappearwornor splayed.
CFpatientsaresupposedtousetheirtoothbrushesatleast twiceaday,whichare,asdemonstratedinthepresentstudy, potentially contaminated with bronchial pathogens. Many different toothbrush maintenance methods are available
[14,15,18,26–29],includingcleaningand/or
disinfection/steri-lization,whichstillneedtobetestedonbronchialpathogens suchasP.aeruginosa.Otherstudiesareneededtodetermine
theactualimpactoforal-dentalhygienehabitsonbronchial colonizationeven if the ubiquityof P. aeruginosacertainly limitsconclusions.Simpleoral-dentalhygiene recommenda-tionsshouldbediscussed,suchasmorefrequenttoothbrush renewal(oncepermonth)and/orafterP.aeruginosaprimary colonizationtreatment.
5.
Conclusion
Toothbrushhygiene mightbeconsideredasoneofthe rea-sonableactionstobetakenbyparentstoavoidcontactwith environmentalP.aeruginosa.
Disclosure
of
interest
Theauthorsdeclarethattheyhavenocompetinginterest.
Acknowledgments
OurthanksareextendedtoSamandNathalieAssaffortheirhelpful revision.
Financial support: Microbiological analysis was granted by Pierre FabreLaboratories.
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