prevalence antibiotic susceptibility and characterization of antibiotic resistant genes among carbapenem resistant gram negative bacilli and yeast in intestinal flora of cancer patients in north lebanon

Antibiotic resistance in cancer patientsJ. Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in south India.. Factors associated to preva

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Journal of Infection and Public Health

j o u r n a l h o m e p a g e :h t t p : / / w w w e l s e v i e r c o m / l o c a t e / j i p h

Prevalence, antibiotic susceptibility and characterization of antibiotic

resistant genes among carbapenem-resistant Gram-negative bacilli

and yeast in intestinal flora of cancer patients in North Lebanon

Rima Christophya, Marwan Osmana, Hassan Mallata, Marcel Achkarb, Azzam Ziedehc,

Walid Moukaddemc, Fouad Dabboussia, Monzer Hamzea,∗

a r t i c l e i n f o

Keywords:

Cancer

Yeasts

Carbapenemases

Colonization

a b s t r a c t

Theemergenceandspreadofcarbapenem-resistantbacteriaareasignificantclinicalandpublichealth concern.Theaimofthestudyistodeterminetheprevalenceofintestinalcarriageof carbapenem-resistantbacteriaandyeastsincancerpatientsunderchemotherapy.41stoolsamplescollectedfrom cancerpatientsinNinihospitalinTripoli,NorthLebanonhavebeenanalyzed.Afterisolatingyeastsand carbapenem-resistantbacteria,abiochemicalidentificationandantimicrobialsusceptibilityprofilewere determined.Themechanismofenzymaticcarbapenem-resistancewasdetectedbysearchingfor car-bapenemasesbybothHodgetestandPCRassays.Theassociationofseveralmechanismsofresistance wasalsosearched.46.3%(19/41)ofpatientswerecolonizedbyyeast.Candidaglabrata(6/19)wasthe majorspecies.Theprevalenceofcarbapenem-resistantbacteriawas24.4%(10/41)includingEscherichia coli(5/10),Enterobactercloacae(1/10),Enterobacteraerogenes(1/10)Edwardsiellahoshinae(1/10) Pan-toeaagglomerans(1/10)andPseudomonasstutzeri(1/10).PCRandsequencingoftheamplifiedfragments revealedthatPseudomonasstutzeri(1/1)carriedVIMgeneandEnterobacteraerogenes(1/1)andE.coli (1/5)carriedOXA-48gene.TheotherEnterobacteriaceaewereresistanttocarbapenemsbymechanisms otherthanacarbapenemaseincludinghyperproductionofcephalosporinase(4/10),extendedspectrum beta-lactamases(1/10)andbothcephalosporinaseandextendedspectrumbeta-lactamases(2/10).High prevalenceofintestinalcarriageofcarbapenem-resistantbacteriaandyeastsweredetectedincancer patientsunderchemotherapy.Inordertopreventthedevelopmentofendogenousinfectionandthe disseminationofantimicrobialresistance,animplementationofantibioticstewardshipprogramsand infectioncontrolmeasuresisrequiredinhospitalsparticularlyinthedepartmentofchemotherapy

©2017TheAuthors.PublishedbyElsevierLimited.ThisisanopenaccessarticleundertheCC

BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Introduction

Cancerpatientsareatincreasedriskofinfectioneitherbecause

theyhaveadeficiencyoftheimmunefunction[1]eitherbecause

ofchemotherapyorradiotherapywhichinduceneutropenia[2]

Theprophylacticantibioticuseisastandardpracticeinpatients

withneutropeniaafterchemotherapy[3].Disruptionoftheflora

ofthegastrointestinaltractisthencreated.Inaddition,treatment

withbroad-spectrumantibioticsincreasestheriskofcandidiasis

byincreasingtheconcentrationofCandidaspp.in the

testinal tract[4] The integrity of the mucousmembranes that can be damaged by the use of cytotoxic chemotherapy agents alsorepresenta riskfactorforthedevelopmentoffungal infec-tions in these patients [5] Potentially pathogenic bacteria are abletocolonize theintestinewhich leadstoa vicious circleof treatmentsandtheemergenceofnewbacteriaresistantto antibi-oticsincludingcarbapenem-resistantspecies[6].Carbapenemsare oftenused totreatinfections caused byenterobacteria produc-ingextended-spectrum␤-lactamases(ESBL)orcephalosporinase [7].Theydifferfromother␤-lactamsbytheirpost-antibioticeffect againstGram-negativebacteria[8].Unfortunately,theprevalence

of Enterobacteriaceae producing carbapenemases increasedover thelasttenyearswhich seriouslyleadstoatreatmentimpasse andachallengeinthetreatmentofnosocomialinfections[7].Two http://dx.doi.org/10.1016/j.jiph.2016.10.009

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

mechanismsof resistanceare described, theacquisition of

car-bapenemases genesthat encode enzymescapableof degrading

carbapenemsoroverexpressionofß-lactamasesthathaveavery

lowaffinityforthecarbapenemssuchascephalosporinaseandESBL

traitalongwithoutermembraneporinlossorhyper-expressionof

effluxpumps[9,10]

Ontheotherhand,theadequateantibiotictherapyisakeyissue

inmultidrugresistantbacteriamanagement.Severalstudieshave

shownthatcancerpatientsinfectedwithresistantbacteriamore

oftenreceiveinadequateinitialempiricalantibiotictherapy,which

mayimpairoutcomes,increasemortalityandprolong

hospitaliza-tion[11].Therefore,theaimofthisstudywastodeterminethe

prevalenceofcarriageofcarbapenem-resistantbacteriaandyeasts

intheintestinalfloraofcancerpatientsunderchemotherapy

Samplesanddatacollection

41cancerpatientswereincludedinthis study,28were

suf-feringfromsolidtumorsand13withmalignantblooddisorders

ThesepatientsaretreatedatNinihospitalinTripoli,NorthLebanon

Astandardquestionnairewascompletedinordertoobtain

infor-mationincludingtheage,gender,typeofcancer,dateofdisease

diagnosis,surgicaloperationsinrelationtocancerand

microbio-logicalhistory.41stoolsampleswerecollectedbetweenFebruary

andJuly2014andtransportedquicklytothelaboratoryina

ther-mostaticallycontrolledcontainer

Yeastisolationandantifungalsusceptibility

IsolationofyeastswasmadebycultureofstoolonSabouraud

agar (Pronadisa®—Spain) supplemented with chloramphenicol

(0.5g/l)followedbyincubation for24hat37◦C.The

identifica-tionwasconductedbytheuseofAuxacolorTM2(BioRad®—France)

Thedeterminationofthesensitivityofdifferentisolatesto

antifun-galswasmadeusingFungitestTM (BioRad®—France)accordingto

themanufacturer’srecommendedprocedures

Isolationofcarbapenem-resistantbacteria

Isolationafterenrichmentmethod

An amount of stool in the volume of a pea is

intro-duced in a volume of 5ml of enrichment Tryptic Soy Broth

(Scharlau®—Spain) supplemented by an antifungal Nystatin

(2500IU/l)(Medistan®—Lebanon)andadiscofertapenemof10␮g

(BioRad®—France)andthenincubatedfor24hat37◦C.10␮lofthis

suspensionisthenspreadonMacConkeyagar(Liofilchem®—Italy)

followedbyincubationfor24hat37◦C[12]

Directisolationmethod

Anamountofstoolinthevolumeofapeaisintroducedina

volumeof5mlofsteriledistilledwater.10␮lofthissuspension

isinoculatedontoMacConkeyagarsupplementedbyertapenem

(Invanz®—Canada)withaconcentrationof1mg/landwithNystatin

(2500IU/L)andthenincubatedfor24hat37◦C

Biochemicalidentificationandresistancepattern

The identificationof Enterobacteriaceae and oxidase-positive

Gram-negativebacilli wascarriedoutthroughtheuseof RapID

ONE (Remel®—USA) and RapID NF (Remel®—USA), respectively

The susceptibility to antibiotics of strains resistant to at least

one carbapenem was performed by the disk diffusion

accord-ing to “Comité de l’Antibiogramme de la Société Franc¸aise de

Table 1

TTACTGCCCGTTGACGCCC

GAGCACTTCTTTTGTGATGGC

ATGCTGGCCTTGGGGAACG

CGAATGCGCAGCACCAG

CCTCTCAATGGTGTGGGT

ACGAATTCGAGCATCACCAG

AACTAYCCAATAYRTAAC

CTGACAGTTACCAATGCTTA

TGGGTRAARTARGTSACCAGA

TTAGCGTTGCCAGTGCTC

AGTGTGTTTAGAATGGTGATC

TAACTTGACCGACAGAGG

GGGYSGCTTAGATAGTGCTGAT

Microbiologie—EuropeanCommitteeonAntimicrobial Susceptibil-ity(CASFM—EUCAST)recommendations

For thedetectionof a cephalosporinase,Müller–Hintonagar withcloxacillin(0.25g/l)wasused.Discsofertapenem,imipenem, meropenem, amoxicillin/clavulanic acid, cefoxitin, ceftazidime, cefotaxime, cefepime and aztreonam were used This test was repeatedonMüller–Hintonagarwithoutcloxacillin After incu-bationfor 24hat 37◦C, thediameter of inhibition zoneswere compared betweenbothMüller–Hinton agar,withandwithout cloxacillin.Forthedetectionofacarbapenemase,theHodgetest wasperformed[13].Thesameprocedurewasrepeatedfor Pseu-domonasstrainsbyusingimipenem(10␮g)insteadofertapenem DetectionofcarbapenemasesandESBLgenes

BacterialDNAwasextractedbyGenEluteTMBacterialGenomic DNA Kit (Sigma–Aldrich®—USA) The Protocol followed is the oneproposedbythemanufacturer Thegeneamplification was carried out by the thermal cycler (MycyclerTM Thermal cycler, Biorad—France)usingtheprimersdescribedinTable1

Results

46.3% (19/41) of patients were infected withyeast Candida glabrata(C.glabrata)(6/19)wasthemajorspeciesfollowedby Can-didaalbicans(2/19),Candidainconspicua(2/19),Candidatropicalis (1/19),Candidaparapsilosis(1/19)and7strainswerenon identifi-able.Table2expressesthevariousantifungalsusceptibilityprofile Theprevalenceofcolonizationofbacteriaresistantto carbapen-emsincancerpatientsunderchemotherapyinNorthLebanonwas

IsolatedstrainswereEscherichiacoli(5/10), Enterobactercloacae

(1/10),Enterobacteraerogenes(1/10),Edwardsiellahoshinae(1/10),

Pantoeaagglomerans(1/10)andPseudomonasstutzeri(1/10).Note

thatonly2strainsofcarbapenem-resistantbacteriaweredetected

bybothmethodsused.Table3expressesthepercentageof

sensi-tivitytoantibioticsfor10carbapenem-resistantstrains

Moreover,outof10strainsresistanttocarbapenems,3were

positiveforHodgetest.2(E.aerogenesandE.coli)ofthemharbored

OXA-48geneandthethirdone(P.stutzeri)harboredVIMgene

InadditiontoOXA-48,E.colialsocontainCTX-Mgene.Theother

carbapenem-resistantstrainshadanoverproduced

cephalospori-naseand/orESBL(Table4)

Discussion

Carbapenem-resistantbacteria areassociated withincreased

morbidity and mortality and are capable of silently colonizing

the digestive tract The present study shows a high

preva-lenceofcarbapenem-resistantbacteriaintestinalcarriage(24.4%)

in cancerpatients under chemotherapy in Nini hospital, North

Lebanon.Recentstudyshowedahighincidenceand prevalence

of carbapenem-resistantEnterobacteriaceae (CRE) intestinal

car-riageinaMexicantertiarycarehospital,particularlyinhematologic

malignancypatients[21].Inaddition,Tischendorfetal.[22]

sug-gestedanoverall16.5%riskofinfectionwithCREamongstpatients

colonizedwithCRE

RegardingthedetectionofGramnegativebacilliresistantto

car-bapenems,resultshaveshownthattheisolationmethodproposed

bytheCentersforDiseaseControl(CDC)failedtodetect4strains

detectedbythemethodofdirectisolationonMacConkeyagar

sup-plementedbyertapenem.Severalauthorsfounddifferencesinthe

detectionofthecolonizationofcarbapenemasesproducingstrains

betweenthemethodsused[23–25].Themajormechanismof

resis-tancedetectedin ourstudywasoverexpressionof ß-lactamase

(cephalosporinaseorESBL)withaproblemofimpermeability

Ourrecentstudy,alsoperformedinNinihospital,including2767

Enterobacteriaceaeisolatesduringtheperiod2008–2012showed

thatamong7enterobacterialresistantspeciestocarbapenem,88%

producedOXA-48carbapenemasewiththepredominanceofE.coli

(73%)[26].Inaddition,thisstudyshowedanincreaseintherateof

CREcolonizationwhichwas0.4%between2008and2010and1.6%

in2012[26].Anotherstudyinthesamehospitalfrom2006to2013

showedthat46%ofP.aeruginosastrainswithdecreasedsensitivity

toimipenemwereharboringblaVIM-2gene[27]

Ontheotherhand,astudyconductedintheUnitedStatesin

2013onthecolonizationofcarbapenemsresistantstrainsof18

patientswithmalignantblooddiseaseshasshownthatthe

major-ityofisolatedstrainswereKlebsiellapneumoniae(14strains)andE

cloacae(3strains).Themechanismofresistancewasrelatedtothe

productionofKPCandCTX-M-15enzymesincombination with

outer-membraneporinloss[28].Furthermore,athirdstudy

per-formedin2014in Colombiaon45cancerpatientsshowedthat

KPCgenewasmorefrequentininfections(82.3%)comparingto

colonization(17.7%)[29]

Withregardtothescreeningforyeast,ourstudyshowedthat

C.glabratawasthemajorspecieswhichisin agreementwitha

studyconductedinGermanyin1999on116patientswithacute

leukemia[30].C glabratawasreportedaspredominantspecies

(51%),followedbyC.albicans(18%)andC.krusei(4%)[30].Areview

of37reportsincluding1591casesofsystemicCandidainfection

amongoncologypatientsshowedtheimportanceofnon-albicans

Candida.46%ofcaseswereduetonon-albicansCandidasuchas

C.tropicalis(25%),C.glabrata(8%),C.parapsilosis(7%),andC.krusei

(4%)[31].Moreover,severalstudieswereconductedinLebanon Table

Table 3

E coli E coli E coli E coli E coli E aerogenes E cloacae E hoshinae P agglomerans Pseudomonas stutzeri

S, sensitive; I, intermediate; R, resistant; nd, not determined.

AMP, ampicillin (10 ␮g); AMC, amoxicillin/clavulanic acid (20/10 ␮g); TIC, ticarcillin (75 ␮g); TCC, ticarcillin/clavulanic acid (75/10 ␮g); PIP, piperacillin (30 ␮g); PPT, piperacillin/tazobactam (30/4 ␮g); ATM, aztreonam (30 ␮g); IMP, imipenem (10 ␮g); MEM, meroprnem (10 ␮g); ERT, ertapenem (10 ␮g); CEF, cefalotin (30 ␮g); CXM, cefuroxim (30 ␮g); FOX, cefoxitin (30 ␮g); CFM, cefixim (5 ␮g); CTX, cefotaxime (5 ␮g); FEP, cefepime (30 ␮g); CAZ, ceftazidime (30 ␮g); GMN, gentamicin (10 ␮g); TMN, tobramycin (10 ␮g); AKN, amikacin (30 ␮g); NET, netilmicin (10 ␮g); PI, pipemidic acid (20 ␮g); OFX, ofloxacin (5 ␮g); CIP, ciprofloxacin (5 ␮g); SXT, trimetho-prim/sulfamethoxazole (1,25/23,75 ␮g); CS, colistin (50 ␮g); TGC, tigecyclin (15 ␮g); FSF, fosfomycin (200 ␮g).

Phenotypic and genotypic test for carbapenems resistant Gram negative bacilli.

Strains Hodge test Cephalosporinase inhibition Carbapenemases genes ESBL genes

KPC IMI NMC-A SME GES IMP VIM NDM-1 OXA-48

nd, not determined.

albi-cansstrains,12%wereresistanttoitraconazole,6%toketoconazole,

7.7%tovoriconazole,1.7%toamphotericinB,and5%tofluconazole

AnotherinvestigationconductedbyBitaretal.[33]hasshowna

sig-nificantandrapidlevelofincreasedresistancetoamphotericinB

(37.6%),eventhoughitsuseislimitedinLebanonduetoitstoxicity

Conclusion

Inconclusion,ourstudyhasallowedustoreportahigh

preva-lenceofintestinalcarriageofcarbapenem-resistantbacteriaand

yeastsin cancerpatientsunder chemotherapy.Otherwise,

can-cerpatientsareathighriskfordevelopingendogenousinfections

relatedtothecompromisedhostdefensesandthesequelaeof

treat-ment.Thisdrawsattentiontotheurgentneedtoimproveantibiotic

useandtosupportimplementationofantibioticstewardship pro-gramsandinfectioncontrolmeasuresinhospitalsparticularlyin the departmentof chemotherapy These resultsalso argue the importance need to carry out screening tests for carbapenem-resistantbacteriabeforeeachadmissionofacancerpatient

Nonedeclared

Conceived and designed the experiments: Monzer Hamze, FouadDabboussiandHassanMallat.Performedtheexperiments and wrote the paper: Rima Christophy and Marwan Osman

Mokad-dem

Acknowledgements

WewouldliketothankTahaAbdou,HusamKhaled,Mariam

Yehya,ImaneDarwichandFarahCharrouffortheirexcellent

tech-nical assistance.Thisstudy wasfinanced byDoctoral Schoolof

ScienceandTechnology,LebaneseUniversity,Tripoli,Lebanon

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