Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

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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

Effects

of

sub-minimum

inhibitory

concentrations

of

ciproﬂoxacin

on

bioﬁlm

formation

and

virulence

factors

of

Escherichia

coli

Guofeng

Dong

a

_,

_Jiahui

_Li

b

_,

_Lijiang

_Chen

b

_,

_Wenzi

_Bi

a

_,

_Xiaoxiao

_Zhang

b

_,

_Haiyang

_Liu

b

_,

Xiaoyang

Zhi

c

_,

_Tieli

_Zhou

b,∗

_,

_Jianming

_Cao

a,∗

a_Wenzhou_Medical_University,_School_of_Laboratory_Medicine_and_Life_Sciences,_Wenzhou,_China

b_The_First_Afﬁliated_Hospital_of_Wenzhou_Medical_University,_Department_of_Clinical_Laboratory,_Wenzhou,_China c_Yueqing_People’s_Hospital,_Department_of_Clinical_Laboratory,_Wenzhou,_China

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o

Articlehistory:

Received4September2018 Accepted16January2019 Availableonline21February2019

Keywords: Bioﬁlmformation Ciproﬂoxacin Escherichiacoli Sub-minimuminhibitory concentration Virulence

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Objective:Toevaluatetheinfluenceofsub-minimuminhibitoryconcentrations(MICs)of ciprofloxacin(CIP)onbiofilmformationandvirulencefactorsofEscherichiacoliclinical iso-lates.

Methods:Sub-MICsofCIPweredeterminedusinggrowthcurveexperiments.The biofilm-formingcapacityofE.coliclinicalisolatesandE.coliATCC25922treatedoruntreatedwith sub-MICsofCIPwasassessedusingacrystalvioletstainingassay.Thebiofilmstructureof E.coliisolatewasassessedwithscanningelectronmicroscopy(SEM).Theexpressionlevels ofthevirulencegenesfim,usp,andironandthebiofilmformationgenesofthepgaABCD locusweremeasuredusingquantificationRT-PCR(qRT-PCR)inE.coliisolatesandE.coli

ATCC25922.

Results:Basedonourresults,thesub-MICsofCIPwere1/4MICs.Sub-MICsofCIP signifi-cantlyinhibitedbiofilmformationofE.coliclinicalisolatesandE.coliATCC25922(p<0.01). SEManalysesindicatedthatthebiofilmstructureoftheE.colichangedsignificantlyafter treatmentwithsub-MICsofCIP.Expressionlevelsofthevirulencegenesfim,usp,andiron andthebiofilmformationgenesofthepgaABCDlocuswerealsosuppressed.

Conclusions: Theresultsrevealedthattreatmentwith sub-MICsofCIPfor24hinhibited

bioﬁlmformationandreducedtheexpressionofvirulencegenesandbioﬁlmformation

genesinE.coli.

∗ _{Corresponding}_authors.

E-mailaddresses:[email protected](T.Zhou),[email protected](J.Cao).

https://doi.org/10.1016/j.bjid.2019.01.004

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1. Introduction

Escherichia coliis acommon cause ofparenteral infections, includingurinarytractinfections,neonatalmeningitis,

Gram-negative bacteremia, and prostatitis.1,2 _The _occurrence _of

theseinfectionsismainlydependentontheimmunestatus

ofthe hostaswell asonbacterialfactors.2 _The

pathogeni-cityofE.coliismainlyrelatedtoitsvirulencefactors,which

are associated with host colonization and invasion, tissue

damage, stimulation ofinﬂammatory response, evasion of

immuneresponse,andbioﬁlmformation.Accordingto

pre-viousresearch,virulencefactorsincludegenesencodingiron acquisitionsystems(iron,iuc),adhesins(ﬁm,afaI,sfa,iha), tox-ins(cnf1,hlyA,set,astA,usp),andbioﬁlms(pgaABCD).3,4

Antibiotictherapyisofgreatimportancefortreating bac-terialinfections. Ciprofloxacin (CIP) isa second-generation fluoroquinoloneantibioticwidelyusedtotreatGram-negative andGram-positivebacterialinfections.CIPisafirst-line medi-cationforvariousinfections,5_and_is_the_most_commonly_used

ﬂuoroquinoloneforthetreatmentofurinarytractinfections, asitcanbereadilyabsorbedfromthegastrointestinaltract afteroraladministration.6 _Other _advantages_of_CIP _include

broad-spectrumcoverageandhighurinaryexcretionrates.6

Invivo,supra-inhibitoryconcentrationsofantibioticinevitably

becomesub-minimuminhibitoryconcentrations(MICs)once

insidethebody.7

Reports on the impacts of sub-MICs of antibacterial

drugsonbacteriahavebeencontradictory.In1988,Schadow

etal.8demonstratedthatsub-MICsofrifampincouldinduce

bioﬁlm formation by Staphylococcus epidermidis. However,

recentreportshaveshownthatsub-MICsofvarious

antimi-crobialagentshavebothinhibitoryandstimulatoryeffectson E.colibioﬁlmformationinhumaninfections.9,10_Therefore,_it

isofclinicalinteresttobroadenourcurrentknowledgeofthe effectsofsub-MICsofantimicrobialagentsonbioﬁlmsformed byE.colistrains.9_There_have_been_few_studies_on_the_inﬂuence

ofsub-MICsofCIPonE. colibioﬁlm formation.Inaddition, thereislimitedinformationandexperimentalevidenceofthe effectsofsub-MICsofantimicrobialsonthevirulenceofE.coli.

Therefore, the present study was designedto evaluatethe

effectsofsub-MICsofCIPonbioﬁlmformationandvirulence factorsofE.coli.

2. Materials

and

methods

2.1. Bacterialstrainsandsusceptibilitytesting

Five CIP-resistant E. coli clinical isolates (DC3233, DC3298, DC3316,DC3362,DC3365)andthreeCIP-sensitiveE.coli

clin-icalisolates(DC3220,DC3224, DC3242)were collected from

patients of the First Afﬁliated Hospital of Wenzhou

Medi-calUniversity,Wenzhou, Zhejiang Province, Chinain 2015.

Amongthem,DC3220,DC3298,DC3316,DC3362,DC3365and

DC3242 were isolated from different patients with urinary

tract infections, DC3224 was isolated from a patient with

pyelonephritis,andDC3233wasisolatedfromapatientwith

kidney stones. Identiﬁcation and antimicrobial

susceptibil-ity testing were conducted on all isolates using a VITEK

2system (bioMérieux, Marcy L’Etoile,France). The MICs of

CIPweredeterminedusingtheagardilutionmethod

recom-mended bythe Clinical and LaboratoryStandards Institute

(CLSI) guidelines.11_E._coli_ATCC₂₅₉₂₂ _served_as_the_control

strain.

2.2. Growthcurveexperiments

Thesub-MICofCIPforallfurtherexperimentswasselectedby creatingagrowthcurve.Brieﬂy,E.coliATCC25922wasgrownin thepresenceorabsenceof0,3/4,1/2,1/4MICofCIP.12_Samples

werecollectedatspeciﬁctimeintervalsandtheabsorbance

wasreadat600nm.

2.3. Effectofsub-MICsofCIPonbioﬁlmformation

The static bioﬁlm assay was performed as described

previously.7,13 Brieﬂy,eightE.coliclinicalisolatesandE.coli

ATCC25922weregrownovernightinLuria-Bertani(LB)broth

withorwithouttheirsub-MICofCIP.Theovernightcultureof cellswasthendiluted1:100offreshLBbrothwithorwithout sub-MICofCIP.Atotalof150␮Lofeachdilutionwasaddedtoa 96-wellpolystyrenemicrotiterplatewiththreereplicatesand incubatedat37◦Cfor24h.Afterincubation,planktoniccells

wereremovedfromthewells.Afterwashingthreetimeswith

sterilewater,thewellswerestainedwith0.1%crystalvioletfor

10minandtheunboundstainwaswashedthreetimeswith

sterilewater.Thiswasfollowedbyextractingthecell-bound

dyewith200␮L95%ethanolandmeasuringtheabsorbance

ofthesolutionat600nmusingamicroplatereader.Eachtest

wasrepeatedthreetimes.

2.4. Scanningelectronmicroscopy(SEM) characterizationofE.colibioﬁlms

Toobserve andcharacterize E.colibioﬁlms, anE.coliATCC

25922bioﬁlmwaspreparedinasix-wellmicrotiterplateas

describedpreviously.14_Each_well_of_the_treated_bioﬁlm_group

contained 1mLLB medium withsub-MIC of CIP and 1mL

overnightcultureofE.coliATCC25922.Controlwellscontained

1mLLBmediumand1mLovernightcultureofE.coliATCC

25922.After24hofcultivationat37◦C,thesampleswereﬁxed with2.5%glutaraldehyde.Ethanolwasthenusedforgradient

dehydrationofthesamples,followedbydryingatroom

tem-perature.Finally,theprocessedsamplesweresputter-coated

with gold and viewed using scanning electron microscopy

(SEM)attheLaboratoryofAnalyticalElectronMicroscopyat WenzhouMedicalUniversity.Thevoltagewassetat15kVand

sampleswereviewedatamagniﬁcationof2000×.

2.5. QuantitativereversetranscriptionPCR(qRT-PCR)

The effectsof sub-MICsof CIPon the expression levelsof

E.colivirulenceandbioﬁlmformationgeneswereevaluated

using quantitative reverse transcription PCR (qRT-PCR). For

RNAisolation,E.coliisolatesweregrowninfreshLBmedium withorwithoutsub-MICsofCIPat37◦Cfor24h.TotalRNA

was extracted from 3mLof culture using an RNeasyMini

Kit (Qiagen, Valencia, CA, USA)according to the

manufac-turer’sinstructions.TheextractedRNAsampleswerestored

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Table1–Primersusedforexpressionanalysisofvirulencegenesandbioﬁlm-forminggenesinE.coliisolates.

Function Genes Primersequence(5–3) Reference Ironacquisition systems iron AAGTCAAAGCAGGGGTTGCCCG 3 GACGCCGACATTAAGACGCAG iuc ATGAGAATCATTATTGACATAATTG 3 CTCACGGGTGAAAATATTTT Adhesins ﬁm GAGAAGAGGTTTGATTTAACTTATTG 3 AGAGCCGCTGTAGAACTGAGG afaI GCTGGGCAGCAAACTGATAACTCTC 3 CATCAAGCTGTTTGTTCGTCCGCCG sfa CTCCGGAGAACTGGGTGCATCTTAC 3 CGGAGGAGTAATTACAAACCTGGCA iha CTGGCGGAGGCTCTGAGATCA 3 TCCTTAAGCTCCCGCGGCTGA Toxins cnf1 AAGATGGAGTTTCCTATGCAGGAG 3 TGGAGTTTCCTATGCAGGAG hlyA AACAAGGATAAGCACTGTTCTGGCT 3 ACCATATAAGCGGTCATTCCCGTCA set GTGAACCTGCTGCCGATATC 3 ATTTGTGGATAAAAATGACG astA ATGCCATCAACACAGTATAT 3 GCGAGTGACGGCTTTGTAGT usp _{ACATTCACGGCAAGCCTCAG} 3 AGCGAGTTCCTGGTGAAAGC Bioﬁlm formation

pgaA TCTTGCGGCGTATATTGGTAGGT Thisstudy

CGACCCGACAATCACCAGTACG

pgaB CGACGAAATGCGGCAATAACAC Thisstudy

GCGGCGGCATATATTGTGGAAC

pgaC TCACCATCGGGATCAGCAAAT Thisstudy

GCAGCAGAATACCGGGAAAGA

pgaD GGGATCTTGCGGTTCACGTTC Thisstudy

AACGCACCAGGAAATCAGGGAC

Table2–Thecarryingofvirulencegenesandbioﬁlm-forminggenesofE.coli.

Isolates iuc sfa cnf1 set1 iha astA usp hlyA afa ﬁm iron pgaA pgaB pgaC pgaD

DC3233 − − − − − − − − − − − − + − + DC3316 − − − − − − − − − − − − + − + DC3298 − − − − − − − − − − − − + − − DC3362 − − − − − − − − − + + + + + + DC3365 − − − − − − − − − + + + + + + DC3220 − − − − − − − − − + − − + − − DC3224 − − − − − − + − − + − + + − − DC3242 − − − − − − + − − + + + + + + ATCC25922 − − − − − − − − − + + + + + +

“+”:Theisolatecarrythegene. “−”:Theisolatedoesnotcarrythegene.

qRT-PCRanalysisusingacDNAsynthesiskit(TaKaRa,Tokyo, Japan)accordingtothemanufacturer’sinstructions.14,15_Gene

expressionlevelsweremeasuredwithqRT-PCRusinga7500

RT-PGEsystem(TOYOBO,Osaka,Japan)andSYBRGreen

RT-PCRKit(TOYOBO)withthespeciﬁcprimerslistedinTable1. Weselectedseveralisolatestoanalyzetheexpressionofﬁm

(DC3362,DC3365,DC3220,DC3224,DC3242,ATCC25922),usp

(DC3224,DC3242),iron(DC3362,DC3365,DC3242,ATCC25922), andpgaABCD(DC3362,DC3365, DC3242,ATCC25922)genes accordingtothegenecarryingconditions(Table2).The16S

rRNAgenewasusedasaninternalcontroltonormalizethe

data.Eachsamplewasmeasuredintriplicateandmean of

Ctvalueswereusedforanalysis.Geneexpressionlevelswere

calculatedusing2−Ctmethod[Ct(isolateswithsub-MIC ofCIP)=Ct(genesofisolateswithsub-MICofCIP)−Ct(16S rRNAgeneofisolateswithsub-MICofCIP);Ct(isolates with-outsub-MICofCIP)=Ct(genesofisolateswithoutsub-MICof CIP)−Ct(16SrRNAgeneofisolateswithoutsub-MICofCIP); −Ct=Ct(isolateswithoutsub-MICofCIP)−Ct(isolates withsub-MICofCIP)].14

2.6. Statisticalanalysis

The results ofthe OD were compared using the Student’s

t-test.Resultswithp-values<0.05wereconsideredtobe sta-tisticallysigniﬁcantinourstudy.

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1.5 1.0 0.5 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time(h) OD (600nm) Control 1/4MIC 1/2MIC 3/4MIC

Fig.1–ThegrowthcurveofEscherichiacoliATCC25922. Effectsofdifferentconcentrationsofciproﬂoxacin(0,3/4, 1/2,and1/4ofminimuminhibitoryconcentration)onthe growthofE.coliATCC25922.

3. Results

3.1. Susceptibilitytestingandgrowthcurves

TheMICsofCIPforDC3233,DC3298,DC3316,DC3362,DC3365,

DC3220, DC3224, DC3242 and ATCC 25922 were 32␮g/mL,

128␮g/mL, 64␮g/mL, 128␮g/mL, 128␮g/mL, 0.25␮g/mL,

1␮g/mL, 0.008␮g/mL, 0.015␮g/mL, respectively. According

to the growth curve,1/4 MIC ofCIP had negligible effects

on the growth ofE. coli ATCC 25922 and so we chosethis

concentrationforfurtherstudy(Fig.1).12

3.2. Effectsofsub-MICsofCIPonbioﬁlmformation

Toevaluatetheeffectsofsub-MICsofCIPonbiofilmformation, thebiofilm-formingcapacityofeightE.coliclinicalisolatesand E.coliATCC25922wasassessed.Theresultsdemonstratedthat sub-MICsofCIPsignificantlyinhibitedtheformationofE.coli biofilms(P<0.01)(Fig.2).

3.3. Scanningelectronmicroscopy(SEM)

Inadditiontomeasuringbioﬁlmformation,wealsoconducted

SEMexperimentstodeterminewhetherthestructureofthe

bioﬁlmschangedinresponsetoCIPtreatment. Theresults

revealedthatuntreatedE.coliATCC25922bioﬁlmsappeared

denseand clusteredinto amacrocolony.Incontrast,E. coli

ATCC 25922 bioﬁlmstreated with 1/4MIC ofCIP appeared

sparseanddispersed(Fig.3AandB).Theseresultswere

con-sistentwiththe quantitativebioﬁlm experiments, showing

thatboththebioﬁlmformationabilityandthestructureof

bioﬁlmswereaffectedby1/4MICofCIP.

3.4. Quantiﬁcationofvirulenceandbioﬁlmformation geneexpressionwithqRT-PCR

Weinvestigatedtheexpressionofvirulenceandbioﬁlm

for-mationgenesineightE.coliclinicalisolatesandE.coliATCC

25922 incubated with sub-MICsof CIP.Thecarrying status

ofvirulenceandbioﬁlmformationgenesforeachisolateare showninTable2.Theresultsshowedthattheexpression

lev-elsofvirulenceand bioﬁlmformationgeneswere lower in

strainstreatedwithsub-MICsofCIPcomparedtountreated

0.20 0.15 0.10 0.05 0.00 DC3233 DC3298 DC3316 DC3362 DC3365 DC3220 DC3224 DC3242 ATCC25922 OD (600nm) Control 1/4MIC CIP

Fig.2–Bioﬁlm-formingcapabilitiesofEscherichiacoli

isolatesinthepresenceandabsenceofsub-minimum inhibitoryconcentration(MICs)ofciproﬂoxacin.“Control”,

E.coliisolatesandATCC25922wereculturedwithout1/4 minimuminhibitoryconcentrationofciprofloxacin.“1/4 MICCIP”E.coliisolatesandATCC25922wereculturedwith 1/4minimuminhibitoryconcentrationofciprofloxacin.The biofilm-formingcapabilitiesofE.coliisolatesincubated withciprofloxacinwereimpairedcomparedtountreated isolates.P<0.01.

strains (Figs. 4 and 5). This decrease in the expressionof

bioﬁlmformationgeneswasconsistentwithpreviousresults

demonstratingthatsub-MICsofCIPinhibitbioﬁlmformation. Wespeculatedthattheinhibitionofbioﬁlmformationcanbe attributedtotheinhibitionofgeneexpression.

4. Discussion

Extensive use ofantibioticsforthe treatment ofinfections

has led to the development of drug resistance in various

pathogens, including E. coli. Gupta et al.12 _proposed _that

theincreasingrateofresistancetoantibioticswarrants

fur-ther research into thediscovery of therapeuticagentsthat

target pathogen physiology rather than pathogen growth.

In addition to having bactericidal effects, someantibiotics couldalsoaffectthephysiologyofmicroorganismsbyaltering

gene expression at concentrations below the MIC values.

It has been shown that the sub-MIC of some antibiotics,

includingazithromycinandCIP,reducethepathogenicityof

Pseudomonas aeruginosabyinhibiting bioﬁlm formation and virulence.12,16 _The_present_study_was_designed_to_determine

whethersub-MICsofCIPaffectE.colivirulencebyinhibiting

bioﬁlmformation.

Bioﬁlmsarecommunitiesofmicroorganismsthatadhere

to each other on living or non-living surfaces within a

self-produced matrix of extracellularpolymeric substances

includingpolysaccharides,extracellularDNA,andproteins.17

Bioﬁlmshavebeenfoundtobeinvolvedinmultiple

micro-bial infectionsanditisestimatedthat80%ofall infections

are caused bybioﬁlm-forming bacteria and fungi.17 _In _the

present study,the resultsof crystal violet staining

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Fig.3–Effectofsub-minimuminhibitoryconcentration(MICs)ofciprofloxacinonbiofilmformationinEscherichiacoliATCC 25922.(A)BiofilmsofE.coliATCC25922treatedwithsub-MICofciprofloxacinappearsdispersedandsparse.(B)Biofilmsof

E.coliATCC25922culturedwithoutciproﬂoxacinappearsdense.

Fig.4–ExpressionratioofEscherichiacolivirulencegenesinthepresenceorabsenceofsub-minimuminhibitory concentration(MICs)ofciprofloxacin.(A)fimgene.(B)uspgene.(C)irongene.Sub-MICsofciprofloxacininhibitedthe expressionofvirulencegenes.

bysub-MICsofCIP.SEManalysesindicatedthatsub-MICsof

CIPinterferedwithbioﬁlmstructure.

IthasbeenreportedthatthepgaABCDlocusofE.coli

pro-motesthe synthesisofpolysaccharideadhesins, whichare

requiredforbioﬁlmformation.4_We_found_that_the_expression

ofpgaABCDgeneswassuppressedbysub-MICsofCIP.Thiswas similartotheresultsofpreviousstudies,which foundthat

sub-MICsofCIPandceftazidimecouldinhibittheformation

ofbioﬁlmsofP.aeruginosabyinhibitingthequorum-sensing system.12,18_Based_on_our_results,_we_speculate_that_sub-MICs

ofCIP inhibitedbioﬁlmformationbyinhibitingthe

expres-sionofpgaABCD genes resultingin decreasedextracellular polysacchariderelease.

ThepathogenicityofE.coliisolatesisdueinparttotheir virulencefactors.3,19_Iron_is_an_important_element_for

bacte-rialsurvivalandforpromotingavarietyofcellularactivities intheorganism.Bacteriahavedevelopedaseveralstrategies foracquiringironduringinfection,includingtheexpressionof

ironacquisitionsystemsthatremoveironfromthe

environ-mentandconcentrateitinthebacterialcytosol.3_In_our_study,

theexpressionoftheirongene,whichisassociatedwithiron

acquisitionsystemswassuppressedbyCIP.

Theﬁmgenecluster,whichencodestype1ﬁmbriae,

medi-atesbacterialadhesiontoandinvasionofbladderepithelial

cells.3,20 _We_found_that_six_of_the_nine_E._coli_isolates_tested

carryingtheﬁmgenehaditsexpressionlevelsreducedafter CIPtreatment.Itwasalsopreviouslyreportedthatexpression oftheuspgeneincreasedE.coliinfectivityinamousemodel ofpyelonephritis,suggestingthatuspmayalsoplayarolein the pathogenesisofuropathogenic E.coli.3,21 _In_the _current

study,wefoundthattheexpressionofuspwassuppressed

byCIPtreatment.Basedontheseresults,wespeculatethat

sub-MICsofCIPreduceE.colivirulence.Theseresultsare con-sistentwithsomereportsbutarecontrarytootherresearches.

A previous study proposed that sub-MICsofamikacin and

CIP reduced bacterial virulence by interfering with

bacte-rialcellfunctions.22_Others_have_suggested_that_sub-MICs_of

CIP enhance Enterococcus faecium pathogenicity.23 _Since _we

observed somedifferences between individual isolates, we

speculatethattheeffectsofsub-MICsofantibacterialdrugson

thevirulenceofisolatesvary.Thespeciﬁcmechanismsneed

tobeexploredfurther.

Theresultsofourexperimentsshowedthatsub-MICsof

CIP reduced the virulence ofE. coli isolates. Limitationsof thisstudyincludetheabsenceofanimalexperimentsto ver-ifythereductioninE.colivirulenceandthelimitednumber ofisolates.Wewillcontinuethisresearchtoovercomethese

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Fig.5–Effectofsub-minimuminhibitoryconcentration(MICs)ofciprofloxacinonexpressionratioofE.colibiofilm formationgenes.(A)pgaAgene.(B)pgaBgene.(C)pgaCgene.(D)pgaDgene.Theexpressionlevelsofbiofilmformation genesweresuppressedbysub-MICsofciprofloxacin.

thataffectbioﬁlmformation,virulencegeneexpression,and bacterialcolonization.

Inconclusion,wefoundthatbiofilmformationand expres-sionofthevirulencefactorsgenespgaABCD,iron,fimandusp weresignificantlyinhibitedbysub-MICsofCIP.Theseresults

suggestthatCIPmaybeagoodtreatmentoptionfor

bioﬁlm-associatedbacterialinfections.Becauseantibioticsareoften

presentatsub-MICsinthebody,thiscouldprovidea

theo-reticalbasisfortheclinicaluseofdrugsinthetreatmentof E.coli-relatedinfections.Thisphenomenondeserves contin-uedattentionofclinicians.

5. Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgment

ThisworkwassupportedbytheZhejiangProvincialProgram

fortheCultivationofHigh-LevelInnovativeHealthTalents[no. (2012)241].

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