Antimicrobial susceptibility of hospital acquired Stenotrophomonas maltophilia isolate biofilms

braz j infect dis 2 0 1 6;20(4):365–373

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Antimicrobial

susceptibility

of

hospital

acquired

Stenotrophomonas

maltophilia

isolate

biofilms

Erlin

Sun

,

Gehong

Liang

,

Lining

Wang

,

Wenjie

Wei

,

Mingde

Lei

,

Shiduo

Song

,

Ruifa

Han

_,

_Yubao

_Wang

_,

_Wei

_Qi

a_{TheSecondHospitalofTianjinMedicalUniversity,TianjinInstituteofUrology,Tianjin,China} b_{TheSecondHospitalofTianjinMedicalUniversity,DepartmentofInfectiousDisease,Tianjin,China} c_{UniversityofPennsylvania,DepartmentofAnatomyandCellBiology,Philadelphia,PA,UnitedStates}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8January2016

Accepted20April2016

Availableonline13May2016

Keywords: Stenotrophomonasmaltophilia Biofilm Antibioticresistance Nosocomial

a

b

s

t

r

a

c

t

Aims: WesoughttocharacterizetheantibioticsusceptibilityofstrainsofStenotrophomonas maltophiliaisolatedfromclinicalsamples,andtheroleofStenotrophomonasmaltophiliabiofilm

inantibioticresistance.

Methods:Fifty-oneclinicalStenotrophomonasmaltophiliaisolateswereobtainedfrompatients

withnosocomialinfectioninthesurgicalwardsandICUsofsixgeneralhospitalsinTianjin,

China.InvitromodelsofStenotrophomonasmaltophiliabiofilmswereestablishedand

con-firmedbyscanningelectronmicroscopyandfluorescencemicroscopywithsilverstaining.

Theminimalinhibitoryconcentrationsandbiofilminhibitoryconcentrationsofcommonly

usedantibioticsweredetermined.

Results:47of51strainswereresistanttothreeormoreantibiotics.42of51strainsformed

Stenotrophomonasmaltophiliabiofilmsinvitro.Stenotrophomonasmaltophiliabiofilmformation

greatlyreducedsensitivitytomosttestedantibiotics,butnottolevofloxacin.However,inthe

presenceoferythromycinscanningelectronmicroscopyrevealedthatlevofloxacininhibited

Stenotrophomonasmaltophiliabiofilmformation.FactorialANOVArevealedthaterythromycin

enhancedsusceptibilitytolevofloxacin,cefoperazone/sulbactam,andpiperacillin(p<0.05),

and an E modelrevealed thatlevofloxacin anderythromycin actedsynergistically in

biofilms,suggestingspecificuseofcombinedmacrolidetherapymayrepresentaneffective

treatmentforStenotrophomonasmaltophiliainfection.

Conclusions: Antibioticscouldactsynergisticallytocombattheprotectionconferredto

clini-calisolatesofStenotrophomonasmaltophiliabybiofilms.Macrolideantibioticsmaybeeffective

whereusedincombination.

©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthors.}

E-mailaddresses:irenesunsun@yahoo.com(E.Sun),qiweitianjin@yahoo.com(W.Qi).

1 _{Theseauthorscontributedequallytothispaper.}

http://dx.doi.org/10.1016/j.bjid.2016.04.002

1413-8670/©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

Introduction

Stenotrophomonas maltophilia (SMA) is an environmental

pathogen and opportunistic Gram-negative bacterium that

can infect immunocompromised patients or otherwise

healthypatientswhenintroducedbycontaminatedinvasive

medical devices.1 _{Dialysis technology, intubation, artificial}

implantsandotherwidelyemployedmedicalmaterialscan

be colonized by bacteria, and SMA has been observed to

form bacterial biofilms (BBF) on this equipment. In

surgi-caldepartments,device-relatedcontaminationbypotentially

pathogenicbacteriacanserveasasourceforcross-infection,2

and nosocomial SMA infections have received increased

attention in recent years.3–11 _{SMA bacteremia has been}

associatedwith mortality rates rangingfrom 14 to 69% in

immunocompromisedpatients.12–14

Treatment of SMA infection is complicated by its

nat-ural resistance to many antimicrobial drugs, including

carbapenems, and the rapid adaptation to the pulmonary

environment.15 _{SMAcanformBBFonhosttissues,}

dramat-ically enhancing the resistance of SMA to therapeutically

important antibiotics including aminoglycosides,

fluoro-quinolones, and tetracycline.16–20 _{Thus, biofilm formation}

represents an important mechanism of bacterial

antibi-oticresistance, and presents unique challenges in surgical

medicine, complicating therapeutic management of such

BBF.21,22

SMA biofilm formation was previously reported to be

associated with resistance to ceftazidime, cefepime,

ticar-cillin/clavulanic acid, piperacillin/tazobactam, aztreonam,

and gentamicin, but not to ciprofloxacin,

levoflox-acin, trimethoprim/sulfamethoxazole (TMP/SMX), or

meropenem.23 _{The fluoroquinolone moxifloxacin was}

reportedtointerferewithSMABBFformation24,25_{; however}

antibioticresistanceofclinicalisolateshasalsobeenwidely

reported,26_{mostlyinvolvingthestudyofstrainsisolatedfrom}

cysticfibrosis(CF)patients.

In this study we sought to investigate the

antibiotic-susceptibility of SMA strains isolated from invasive

infec-tions in non-CF patients. Using a methodology previously

reported27,28_{weestablishedaninvitromodelofSMABBF,and}

investigatedtheantibiotic-susceptibilityofSMAbiofilmsand

planktonicbacteria.Weassessedthecapacityofantibiotics,

appliedindividuallyandincombination,toreducegrowthand

biofilmformationofclinicalisolatesofSMA,inordertoguide

futureclinicaltreatmentofthesepatients.

Materials

and

methods

AntibioticsusceptibilityofSMAisolates

ClinicalSMAstrainswereobtainedfromhospitalizedpatients

with invasive infections that had originated from medical

manipulationinthesurgicalwardsandsurgicalICUsofsix

generalhospitalsinTianjin,China,between 2006and 2012

(Table1).TheMICsofSMAto12antibioticscommonlyused

forGram-negativebacilliweredeterminedbymicrobroth

dilu-tion,analyzedaccording tothe American National Clinical

and Laboratory Standards Institute(CLSI) guidelines.29 _The

following strainswere assessedinparallel forquality

con-trol:ATCC27853,ATCC25922andATCC25923,preservedinthe

InfectiousDiseaseInstituteoftheSecondHospitalofTianjin

MedicalUniversity,China.

InvitromodelofSMABBF

UsingamethodologypreviouslyreportedbyCeri27,28_we

estab-lished an in vitro model of SMA BBF, in a Mueller–Hinton

broth(MHB)-silicafilm system,aspreviouslydescribed.30,31

CryopreservedSMAwasrecoveredinsheepbloodagarplates

incubated aerobicallyovernight. A fresh singlecolony was

transferredtofreshMHBandincubatedfor8hat35◦_C,from

which a 200␮L suspensionof 0.5McFarland was prepared

andtransferredtoa12-wellflat-bottomplate,inwhich

ster-ilesilicafilm(1cm×1cm×1mm,L×W×T)and1.8mLMHB

wereco-culturedat35◦_{Cfor12and24h.Afterwashingthree}

timeswith0.9%sodiumchloridetoremoveplanktonic

bacte-ria, the BBF on the silica films was prepared. Theculture

mediumwas regarded asthenegativecontrol. Morphology

wasobservedbyscanningelectronmicroscopy(SEM)and

flu-orescencemicroscopy(FSM)asdescribedbelow.

Biofilmformationassessedusingfluorescencemicroscopy

withsilverstaining

Aspreviouslydescribed,32,33_{thebiofilmwasfixedin2.5%(v/v)}

glutaraldehydeinPBS(0.1M,pH7.4)for24h,thenimmersed

insaturatedcalciumchloridesolutionfor15min,andrinsed

withddH2Obetweeneachstep.Thefilmwasimmersedin5%

silvernitratesolutionfor15min,immediatelystainedwith1%

hydroquinonefor2min,thenrinsedwithddH2O.Thefilmwas

fixedin5%sodiumthiosulfatesolutionfor2min,thenrinsed

inddH2OandanalyzedbyFSM.

Biofilmformationassessedusingscanningelectron

microscopy

Aspreviouslydescribed,25_{thesilicabiofilmwasfixedin2.5%}

(v/v)glutaraldehydeinPBS(0.1M,pH7.4)at4◦_Cfor2h,fixed

againwith1%osmicacidfor1h,thenrinsedwithPBS,

dehy-drated through a series of ethanol dilutions, then treated

withisoamylacetate.Thespecimenwasdriedinavacuum,

thencoatedwithplatinum–palladiumandanalyzedbySEMat

5–10kV.

Table1–SamplesfromwhichSMAwasisolated.

Tissues n(%)

Pus 7(13.7)

Intravascularcatheter 7(13.7) Postoperativeandburnwound 7(13.7) Bronchialsecretions/lavage 6(11.8) Urinarycatheter 6(11.8) Urine 5(9.8) Sputum 4(7.8) Bile 4(7.8) Blood 3(5.9) Asciticfluid 2(3.9)

brazj infect dis.2016;20(4):365–373

367

DeterminationoftheMIC

MICvaluesofthefollowingantibioticswerecalculatedwitha

brothmicrodilutionassayusingthetwofolddilutionmethod

accordingtotheCLSIguidelines34_{:ciprofloxacin (CIP),}

levo-floxacin(LEV),piperacillin(PIP),ceftazidime(CAZ),

cefopera-zone/sulbactam(SCF),erythromycin(ERY),sulfamethoxazole

(SXT), and gentamycin (GM) within the range of CIP was

256–0.125mg/L,or512–0.125mg/L.

DeterminationoftheBIC

After dilution of the fresh SMA to 3×1010_{CFU/mL, 100␮L}

oftheculturewastransferredtoeachwell ofaflat-bottom

96-wellmicrotiterplate.Aspreviouslydescribed,27,28,35,36_BBF

wereformedbyimmersingthepegsofamodifiedpolystyrene

microtiterlidintothisplate,whichwasthenincubatedat35◦_C

for24h.Peglidswereremoved,rinsedinPBS,thenplacedon

flat-bottommicrotiterplatescontainingantibiotics,and

incu-batedat35◦_{C.After24hpeglidswereremoved,rinsedthree}

times in PBS, then transferredonto flat-bottom microtiter

platescontaining100␮LofMHBperwell,andbiofilmswere

transferredfrompegstowellsbycentrifugationat805×gfor

20min.Theopticaldensitywasmeasuredat650nm(OD650)

onamicrotiterplatecolorimeterbeforeandafterincubation

at35◦_Cfor6h.

AdequatebiofilmgrowthwasdefinedasameanOD650

dif-ference(OD650at6hminustheOD650at0h)of≥0.05.BICsare

definedasthelowestconcentrationofantibioticinwhichthe

OD650was10%orlessofthemeanofthepositivecontrolwell

readings,representingatleasta1−log10growthdifference.

BBFcombinationsensitivitytestbycheckerboardmethod

ThesusceptibilityofbiofilmstoLEV,SCFandPIPaloneandin

combinationwithERYwasdeterminedbythecheckerboard

method.FivestrainsofSMA(numbered0020,0037,0040,0088,

0256)were selectedforthe followingtest. Silica filmswere

transferred toa 24-well flat-bottom microtiter plate. Then

2.0mLMHBwastransferredtothewell(blankcontrol),1.8mL

MHBwastransferredtoeachoftheotherwells.Accordingto

theMICvaluesofLEV,SCFandPIPagainsteachstrain,LEV,SCF

andPIPwasaddedtoyieldafinalconcentrationof1/2MIC,1

MICand2MIC,andERYwasaddedtoyieldafinal

concentra-tionof1/16MICand1/4MIC.Theplatewasincubatedat35◦_C

for24h,thenafterwashingwithsterilephysiologicalsaline,

the silica films were ultrasonicated at 60W for at 15min.

ThecolonyconcentrationwascalculatedfromtheOD650 by

theATCC27853standardcurve(Y=1.26514×10−8X+0.48028,

withalinear rangebetween1.5×108 _and9.6×103_CFU/mL,

p<0.01).Thedensityofcoloniesonthesilicafilmwasrecorded

fromthreewellsinparallel.

Statisticalanalysis

CFUpercentimeterwasindicatedbymean±standard

devi-ation,andtheinteractionofcombinedtestingwasanalyzed

byfactorialexperimentdesignANOVA.Theimpactof

antibi-oticson biofilmgrowth was calculatedusing anE model

is based on the Bliss independence theory, described by

the equation Ii=(IA+IB)−(IA×IB), whereIi is the predicted

growth inhibitioncausedbythetheoretical combinationof

drugsAandB,andIAandIBrepresentthegrowthinhibition

causedbyeachdrugindividually.SinceI=1−E,whereE

rep-resentsgrowthinhibition,thefollowingequationisderived:

Ei=EA×EB. Ei represented the predicted growth inhibition

ofthetheoretical non-interactivecombination ofthedrugs

A and B, and EA and EB represent the growth inhibition

causedbyeach drugindividually.Interactionwasdescribed

asthedifference(E)betweenthepredictedand measured

growthinhibition(E=Epredicted−Emeasured).Statistically

sig-nificantinteractionsof<100%wereconsideredweak,thoseof

100–200%wereconsideredmoderate,andthose>200%were

consideredstrong.

Results

AntibioticsusceptibilityofclinicalSMAisolates

TheantibioticsusceptibilityofSMAisolateswasassessedand

intotal,47(92.1%)ofisolatedstrainswereresistanttothree

ormoreantibiotics.Themajorityoftestedstrainswere

resis-tanttocefotaxime(94.1%)amikacin(90.2%)cefritaxone(88.2%)

GM (82.4%) and CAZ(60.8%), and almost half ofall tested

strainswereresistanttocefoperazone(49.0%),PIP(45.1%)and

cefepime(45.1%).

ClinicalSMAisolatesformBBF

ThecapacityofclinicalisolatesofSMAstrain0314toform

biofilmsonthesurfaceofthesilicafilmsincubatedinMHB

wasassessedbyfluorescentmicroscopy.Maturebiofilmswere

formedby42ofthe51SMAisolates(82.35%).After24h,

sil-verstainingrevealedmaturebiofilmtobecomposedofblack

irregularsheets,scattereddotsandblackcottonymembranes,

and fine rod-shapedbacteria were observedatthe borders

(Fig.1).Noblackaggregationwasobservedonthesurfaceof

silicafilmculturedinMHBintheabsenceofSMA.These

find-ingswereconfirmedbySEM(Fig.2).Biofilmultrastructurewas

observedafter12h(Fig.2C),andmaturebiofilmwasobserved

at24h(Fig.2AandB).SMAwasobservedtobeclusteredon

the surfaceofthebiofilm,andSMA wasmostlyshortrods

ofabout1.5␮m×1.0␮m,partiallyorentirelyencompassedby

theextracellularmatrix(Fig.2AandC).Finebacillialternated

crosslotsofextracellularmucusfilaments,somecellswere

visibleinsplitphaseorundergoingapoptosis,andthe

bac-terialcommunitypresentonthebiofilmwasheterogeneous

(Fig.2B).Nobacteriawereobservedonthesurfaceofsilica

filmculturedinMHBintheabsenceofSMA(Fig.2D).

BBFformationenhancesSMAantibioticresistance

Theeffectofantibioticagentsonbiofilmformationwas

ana-lyzedbycomparingMICswithBIC.Theformationofbiofilms

enhancedtheresistanceofsomestrainstoalltested

antibi-oticsasidefromGMandERY,towhichallthetestedstrains

wereresistantevenintheabsenceofbiofilm,andLEV(Table2).

Afterbiofilmformation,21(50.0%)testedisolateswere

b r a z j i n f e c t d i s . 2 0 1 6; 2 0(4) :365–373

Table2–TheSusceptibilityof42SMAstrainsandtheirbiofilmsto8antibioticagents.

Antibioticagent MIC BIC

Breakpoint R Strains(%) I Strains(%) S Strains(%) Range(␮g/ml) R Strains(%) I Strains(%) S Strains(%) Range(␮g/mL) Levofloxacin R≥8 S≤2 4(9.52) 3(7.14) 35(83.33) 0.125–32 21(50.00) 6(14.29) 15(35.71) 0.125–512 Ciprofloxacin R≥4 S≤1 16(38.10) 17(40.48) 9(21.43) 0.5–128 32(76.19) 9(21.43) 1(2.38) 0.5–1024 Ceftazidime R≥32 S≤8 26(61.90) 10(23.81) 6(14.29) 0.5–512 32(76.19) 2(4.76) 8(19.05) 0.5–1024 Cefoperazone/sulbactam R≥64 S≤16 16(38.10) 13(30.95) 13(30.95) 0.5–512 32(76.19) 1(2.38) 9(21.43) 0.5–1024 Piperacillin R≥128 S≤64 18(42.86) 0(0.00) 24(57.14) 0.5–512 33(78.57) 0(0.00) 9(21.43) 2–4096 Erythromycin R≥8 S≤0.5 42(100.00) 0(0.00) 0(0.00) 0.5–512 42(100.00) 0(0.00) 0(0.00) 0.5–1024 Sulfamethoxazole R≥152 S≤38 10(23.81) 8(19.05) 24(57.14) 2.375–608 33(78.57) 3(7.14) 6(14.29) 2.38–2432 Gentamycin R≥8 S≤4 41(97.62) 0(0.00) 1(2.38) 0.5–512 42(100.00) 0(0.00) 0(0.00) 0.5–1024

R,resistant;I,intermediatesusceptibility;S,susceptible.

SusceptibilityofthebacteriainBBFwasestimatedfromcriteriasetforplanktoniccells,becauseofnoidentifiedcriteriathatwereespeciallyapplicabletoBBF.MICs,weredeterminedbythemicrobroth two-folddilutionmethod.BICs,refertoapreviouslydevelopedtechnique(Hilletal.,35_{Moskowitzetal.,}36_Olsonetal.,27_Tomlinetal.28_{).Theresultsrepresentall42isolatesthatcouldformmature}

brazj infect dis.2016;20(4):365–373

369

Fig.1–InvitroSMABBFformationwasvisualizedbyfluorescencemicroscopy(×200).After24hSMAstrain0314culturedin

MHBformedBBFonsilicafilm.Blackirregularsheets,scatteredblackdots,andcotton-likemembraneswereobserved,and

finerod-shapedbacteriawereobservedattheborders(A).Noblackaggregationwasobservedonthesurfaceofsilicafilm

culturedinMHBintheabsenceofSMA(B).

Table3–Comparisonofantibioticsusceptibilityoftheplanktonicbacteriaandbiofilmsof42SMAstrains.

Antibioticagent MIC(␮g/ml) BIC(␮g/ml)

MIC50a MIC90b Range BIC50c BIC90d Range

Levofloxacin 0.25 2 0.125–32 4 64 0.125–512 Ciprofloxacin 2 8 0.5–128 8 256 0.5–1024 Ceftazidime 32 128 0.5–512 128 512 0.5–1024 Cefoperazone/sulbactam 16 64 0.5–512 128 1024 0.5–1024 Piperacillin 64 512 0.5–1024 256 >1024 0.5–1024 Erythromycin 32 256 0.5–1024 128 >1024 0.5–1024 Sulfamethoxazole 19 76 2.375–608 304 >2432 2.375–2432 Gentamycin 32 512 0.5–1024 256 >1024 0.5–1024

a _{MICinhibited50%oftheisolatestested.} b _{MICinhibited90%oftheisolatestested.} c _{BICinhibited50%oftheisolatestested.} d _{BICinhibited90%oftheisolatestested.}

toCAZ,32(76.19%)toSCF,and32(76.19%)toCIP.Theaverage

BIC90wasalsomuchhigherthantheMIC90(Table3).

CapacityofERYtoovercomeBBF-mediatedantibiotic

resistance

TheeffectsofcombiningERYwithotherantibioticswas

inves-tigatedinfiveSMAisolateswhichweredeterminedtohave

moderatesusceptibilitytoLEV,SCFandPIP.TheMICofLEV

inthechosenstrains(0020,0037,0040,0080and0256) was

between0.5and64mg/L,theMICofSCFwasbetween8and

32mg/LandtheMICofPIPwasbetween2and64mg/L.

Addi-tionofERYdidnotsignificantlyenhancetheefficacyofSCF

(p=0.06),but didsignificantlyenhanceLEVand PIPefficacy

(allp<0.05).FactorialANOVAanalysisrevealedthatthe

effi-cacyofSCF,LEV,and PIPwas significantlyimprovedinthe

Fig.2–UltrastructureofinvitroSMABBFvisualizedbyscanningelectronmicroscopy.After12hthebiofilmofSMAstrain

0314culturedinMHBonsilicafilmwasobserved(C),andwasmatureat24h(A,B).SMAbiofilmultrastructureincluded

clustersofproliferatingbacteria(A,C).SMAwasmostlyshortrodsofabout1.5␮m×1.0␮m,partiallyorentirely

encompassedbytheextracellularmatrix.Alargenumberoffinebacillialternatedacrossnumerousfilamentousstrands(B).

NobacteriawereobservedonthesurfaceofsilicafilmculturedinMHBintheabsenceofSMA(D).B×20,000,Bar=50␮m;A,

Table4–EffectofantibioticsaloneandincombinationontheSMAbiofilms.

Antibioticagent Colonydensity(104_CFU/cm2₎a

–

½

SCF NoERY 149.6±3.8 150.80±3.7 153.40±4.9 152.20±4.8 ERY1/16MIC 149.2±5.0 147.60±6.3 140.00±3.4 132.80±4.7 ERY¼MIC 150.2±4.0 141.00±5.4 132.20±3.3 130.00±4.5 PIP NoERY 148.4±5.6 146.20±3.4 153.20±3.7 148.20±5.5 ERY1/16MIC 147.4±4.8 144.60±5.5 143.80±5.4 133.20±5.2 ERY1/4MIC 150.6±5.8 143.40±5.9 140.40±4.6 130.80±3.7 LEV NoERY 153.0±4.1 131.80±4.5 117.40±5.1 108.20±4.9 ERY1/16MIC 149.2±5.4 120.40±3.6 89.80±4.4 80.60±3.9 ERY1/4MIC 151.2±4.3 96.00±7.5 75.60±5.8 66.20±5.5 LEV,levofloxacin;PIP,piperacillin;SCF,cefoperazone/sulbactam;ERY,erythromycin.

CombinationtestsallrevealedastatisticalinteractionbyfactorialexperimentdesignandANOVA,ofSCFwithERY(F=8.460,p=0.000),LEVwith ERY(F=20.825,p=0.000),andPIPwithERY(F=4.506,p=0.00)respectively.

a _{ResultswereexpressedasthemeanoffiveSMAisolates.}

presenceofERY(F=8.460,p=0.000;F=20.825,p=0.000;and

F=4.506,p=0.001,respectively;Table4,andone-wayANOVA,

p<0.05).ThesynergyofantibioticswasassessedbytheE

model, andthe combination ofERYandLEV was foundto

haveasynergisticeffectagainstfourofthefivestrainstested

(Table5).

SMA strain 0020 biofilm formation was also assessed

in the presence of SCF, LEV, PIP and/or ERY (Fig. 3). We

observedthatLEVdamagedBBFinthepresenceorabsence

ofERY(Fig.3AandB),butthecombinationofERYwithLEV

reducedthenumberofbacteriaanchoredtotheBBF,thinned

thepolysaccharidematrixandalteredbacterialmorphology.

Somespherical,irregularlyshaped,orcrackedbacteriawere

observed.Intheabsenceofantibiotics,theBBFwasmature

andbacterialcellswere packagedinalotofsticky

polysac-charidematrix.Bacterialgrowthwasstrongandsomebacteria

wereobservedtobeinthedivisionphase(Fig.3C).NeitherPIP

norSCF,inthepresenceorabsenceofERY,alteredthestateof

thebacteriaorbiofilm(Fig.3D).

Discussion

TreatmentofnosocomialSMA infectionsiscomplicated by

highratesofantibioticresistance.Although

pharmacokine-ticsand drug penetrationinfluence the clinical efficacy of

antibiotics against planktonic bacterial, resistance can be

characterized in in vitro susceptibility tests. In this study

wesoughttoinvestigatetheantibiotic-susceptibilityofSMA

strainsisolatedfrominvasiveinfectionsinnon-CFpatients.

WeestablishedaninvitromodelofSMABBF,andinvestigated

theantibiotic-susceptibilityofSMAbiofilmsand planktonic

bacteria.Fifty-oneclinicalisolatesofSMAwere takenfrom

patients infectedwith SMA through medical manipulation

and/or immunosuppressivetherapy in the surgical

depart-mentsofTianjinhospitals.

Inoursamplethevastmajorityofisolatedstrains(92.16%)

wereresistanttothreeormoreantibiotics.Thehighestrate

ofresistancewastocefotaxime(94.1%)andthelowest(only

13.7%)wastoLEV.TheintrinsicoracquiredresistanceofSMA

to antibioticshasbeen previouslyreported, and drastically

reducestheantibioticoptionsavailablefortreatment.22

BBF formation was also reported to enhance antibiotic

resistance.Bacterial adhesioninvolves developmentofpili,

changes in cell surface and hydrophobicity, and is crucial

toBBFformation,37,38 _{but themechanismofBBFformation}

varies by attachment substrate.38 _{We selected silicon film}

asanattachment matrixbecause ofitssuperiorresistance

tocorrosion,chemicalstabilityandnon-tackiness.A

major-ity ofSMAstrainsincludedinourstudyformedbiofilmsin

vitro, and maturebiofilms were observed within24h,

sug-gestingthattransmissionoftheseclinicalisolatesmayhave

beenfacilitatedbydevelopmentofbiofilmsonclinicaltools.

BBF structure was identified by silver staining

character-izedbySEMultrastructureanalysis.Theextracellularmatrix

around the bacteria appeared to be made up of secreted

exopolysaccharide.BBFformationwaspreviouslyreportedto

beassociatedwithS.maltophiliafimbriae1(SMF-1),whichis

composedoffimbrinsubunitsandsharessignificantsimilarity

withtheN-terminalaminoacidsequencesofseveralfimbrial

adhesinsofpathogenicEscherichiacolistrainsandtheCupA

fimbriaeinPseudomonasaeruginosa.39

In our sample, all clinical isolates were more resistant

to antibiotics in the BBF than in planktonic state. BBF

have been previously reported to confer antibiotic

resis-tance of up to 1000-fold,16–20 _{and thus BBF formation is}

thought tobeanimportantmechanism ofantibiotic

resis-tance by SMA. The BBF constitute a positively charged

polysaccharide matrix barrier to antibiotics with a

nega-tive charge, such as aminoglycosides.21 _{The extracellular}

polysaccharide matrixcanalsoabsorb highlevelsof

extra-cellularenzymes,suchas␤-lactamase.Wefoundthatmost

isolates were resistant to ␤-lactams, including third and

fourthgenerationcephalosporins,althoughresistanceto

cef-operazone/sulbactam was lower. SMA strains wrapped in

extracellularpolysaccharidematricesarelessabletoobtain

molecular oxygen and nutrients,andto eliminate

metabo-lites,whichcancausebacterialdormancyorretardedgrowth

rates,andlocalmetaboliteaccumulation.22_{Thus,inthisstudy,}

antimicrobialagentsthattargetbacterialgrowth,suchas

b r a z j i n f e c t d i s . 2 0 1 6; 2 0(4) :365–373

371

Table5–InvitrointeractionsofERYincombinationwithSCF,PIP,andLEVagainstBBF,andasindicatedbytheEmodel.

Strains Evaluemean(range)





SCF PIP LEV SCF PIP LEV SCF PIP LEV SCF PIP LEV

0020 8.97(−0.64to16.62) 9.43(2.05to15.78) 17.38(−0.85to29.65) 54.44(5) 56.55(6) 105.13(5) −0.64(1) 0(0) −0.85(1) NI NI SYN 0037 15.37(4.13to22.26) 4.18(−2.04to8.96) 27.29(12.17to38.71) 92.32(6) 27.09(5) 163.72(6) 0(0) −2.04(1) 0(0) NI NI SYN 0040 10.05(1.34to18.15) 3.54(−10.83to15.75) 19.33(8.55to28.94) 60.29(6) 34.88(4) 115.96(6) 0(0) −13.62(2) 0(0) NI NI SYN 0088 7.64(−0.68to13.68) 7.10(2.04to13.33) 12.77(0.10to20.21) 46.10(6) 42.57(60 76.63(6) 0(0) 0(0) 0(0) NI NI NI 0256 9.01(5.39to12.84) 12.57(8.06to17.99) 17.37(6.12to25.99) 54.06(6) 75.44(6) 104.21(6) 0(0) 0(0) 0(0) NI NI SYN NI,nointeraction;SYN,synergism;ANT,antagonism;n,numberofinteractions.

Fig.3–ImpactofantibioticagentsonultrastructureofinvitroBBFvisualizedbyscanningelectronmicroscopy.SMAstrain

0020BBFformedacompletematurestructureinwhichthebacterialwerepackagedwithdensematrix(C).Inthepresence

oferythromycin(B)orerythromycinandlevofloxacin(A)theBBFmatrixbecamethin,feweradheredbacteriawereobserved

andsomeexhibitedsphericalorirregularform.Theblackarrowindicatesabacterialcellundergoingdivisionphase.Inthe

presenceoferythromycinandpiperacillintheBBFremainedsimilartothatseenintheblankcontrol(D).D×20,000;

Bar=100␮m;A,B,C×5000;Bar=50␮m.

tolocalmetaboliteaccumulation,the microenvironmentof

bacterialivingdeepintissuesisacidic,whichinactivatesmost

ofthetherapeuticdrugsthatoptimallyactinneutral

condi-tions.

SMAgeneexpressionprofileshavebeenreportedtochange

significantlyonBBFformation,presentingarangeof

mecha-nismsfordevelopmentofantibioticresistance.40

Development of BBF also enables evasion of the host

immune response. The glycocalyx shell protects bacteria

fromphagocytosisandsecretedquorum-sensingfactor

N-(3-oxododecanoyl)-l-homoserinelactone(HSL)andC4-HSLcan

inhibitleukocyteproliferationandcytokinesecretion.41

Pro-tectionfromthehostimmuneresponseprovidesaprotected

nicheinwhichSMAcanevolveresistancetopreviously

effec-tiveantimicrobials.

In this study, formation of biofilm conferred resistance

to all tested antimicrobials aside from LEV, according to

CLSI break points. LEV alone inhibits mature BBF in a

concentration-dependentmanner, whichindicates thatthe

invivomaximumpermissibledosageforBBFshouldbe

inves-tigated. Complete elimination ofbiofilm infection remains

challenging, but inhibiting bacterial adherence or

destroy-ingthematrixmayproveusefultactics,incombinationwith

activatedhostimmunity.Macrolideshavebeenreported to

assistotherantibioticsinbactericidaleffectsonP.aeruginosa

BBF,byinhibitingsynthesisofGDP-mannosedehydrogenase,

the maincomponent ofbiofilms.42,43 _{Moreover, macrolides}

caninhibitneutrophilaccumulation,andpromoteimproved

CD4+_/CD8+_Tcellratios.44,45_{InthisstudyERY,a14-membered}

ring macrolide,actedsynergistically withLEV,SCFand PIP,

even whereSMA strains were resistant to ERYalone. ERY

markedly enhanced efficacy of LEV and was observed to

reducebacterialadhesiontobiofilmsandunderminedbiofilm

architecture.WeselectedLEV,SCFandPIPtotestin

combina-tionwithERYbasedontheirBICvalue,butothercombinations

ofantimicrobialsmayalsoshowclinicaleffectiveness.

There-fore,furthercombinationsofantimicrobialsshouldbetested

infuture.

Insummary,wefoundthatantibioticscouldact

synergis-ticallytocombattheprotectionconferredtoclinicalisolates

ofSMAbyBBF.Ourfindingssuggestthatmacrolideantibiotics

maybeeffectivewhereusedincombination.Howeverfurther

specificinvivostudieswillberequiredtoconfirmwhetherthis

approachcantreatnosocomialSMAinfectionintheclinic.

Funding

The study was supported bythe National Natural Science

FoundationofChina(GrantNo.:81402095).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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