Clinical and microbiological observational study on AmpC β-lactamase-producing Enterobacteriaceae in a hospital of Nepal

braz j infect dis.2013;17(2):256–259

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

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

Brief

communication

Clinical

and

microbiological

observational

study

on

AmpC

␤-lactamase-producing

Enterobacteriaceae

in

a

hospital

of

Nepal

Pankaj

Baral

_,

_Sanjiv

_Neupane

_,

_Basudha

_Shrestha

_,

_Kashi

_Ram

_Ghimire

_,

Bishnu

Prasad

Marasini

_,

_Binod

_Lekhak

a_{CentralDepartmentofMicrobiology,TribhuvanUniversity,Kirtipur,Kathmandu,Nepal}

b_{DepartmentofMicrobiology,FacultyofScience,MahidolUniversity,Bangkok,Thailand}

c_{DepartmentofPathology,KathmanduModelHospital,Kathmandu,Nepal}

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Articlehistory:

Received9August2012 Accepted24September2012 Availableonline27February2013

Keywords:

AmpC␤-lactamase Enterobacteriaceae Antibioticresistance

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LimitedinformationisavailableregardingAmpC␤-lactamase(ABL)-producing Enterobac-teriaceaecompared toextended-spectrum ␤-lactamase-producingenterobacteria.Since ABL-producingorganismsareoftenresistanttomultipleantimicrobialagents, therapeu-ticoptions againstthesepathogensare limited.Among 230clinicalEnterobacteriaceae isolates,64(27.8%)werefoundtoproduceABLinourstudy.Escherichiacoli(83.9%)wasa predominantpathogen,followedbyCitrobacterfreundii(5.2%).Asignificantproportionof ABL-producingisolates(81.3%)werefoundtobemultidrugresistantagainstcommonlyused antibiotics.Univariateanalysisshowedthatpriorhistoryoftakingantibiotics(oddsratio [OR],5.278;confidenceinterval[CI],2.838–9.817;p<0.001)andbeinginpatients(OR,4.587; CI,2.132–9.9;p<0.001)wereassociatedwithABLpositivity.Regularantimicrobialresistance surveillanceforABL-producingEnterobacteriaceaeiswarrantedforproperantimicrobial treatmentstrategyandpolicymakingduetoABL-positiveinfections.

©2013 ElsevierEditoraLtda.Allrightsreserved.

TheemergenceofAmpC␤-lactamase(ABL)-producing Entero-bacteriaceaeisnowamajorworldwideproblemduetotheir features of non-beta-lactam coresistance and high poten-tialto transferthe drugresistancefeatures horizontally to other Enterobacteriaceae members via their transmissible plasmid(s).1 _{In recent years, there are increasing reports} about the dissemination of ABL-producing Enterobacteria-ceae from hospital to community in different countries. However, little is known about the occurrence of ABL-producingEnterobacteriaceaeinNepal,althoughhighratesof

∗ _{Correspondingauthorat:CentralDepartmentofMicrobiology,TribhuvanUniversity,Kirtipur,Kathmandu,Nepal.}

E-mailaddress:pankaj.baral@gmail.com(P.Baral).

prevalencehavebeenreportedinneighbouringcountries,up to36.5%inIndia,2_{35.6%inPakistan}3_{and26.2%inChina.}4_We conductedaprospectivestudy inatertiarycarehospitalof Nepal,locatedatcapitalKathmandu,withtheaimto deter-minetheprevalence,etiologyandantimicrobialsusceptibility of ABL-producing Enterobacteriaceae (other than typhoidal salmonella), and the conjugational transferability of ABL-positive phenotypes in Escherichia coli (E. coli). Additionally, the present study also highlights the clinical characteris-ticsofpatientsinfectedwithABL-positiveEnterobacteriaceae

1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2012.09.012

brazj infect dis.2013;17(2):256–259

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and identified some of the risk factors for ABL-positive infections.

Duringasix-monthperiodof2007,atotalof230 consec-utiveisolatesofEnterobacteriaceaefromthesame number ofnon-repeatpatientswerecollectedbyKathmanduModel HospitalLaboratoryfromdifferentclinicalspecimens, includ-ing urine, swabs and fluids. All suspected symptomatic patientswithculture-provendiagnosisofEnterobacteriaceae for the collected samples from the suspected anatomical sitesofinfections were considered to acquire Enterobacte-riaceae infections. Each of the included 230 patients was intervieweddirectlyusingastructuredquestionnaireto col-lectthedataaboutpatient’sdemographics,patient’scategory (outpatientorinpatient),priorantibioticexposure,historyof urinarytractinfection(UTI)andpresenceofanyunderlying illnesses(diabetesmellitus,malignancies,liverdiseaseand renal dysfunction) or comorbidities (recent surgical opera-tionsinthelastsixmonths,takingimmunosuppressant,using indwelling urinary catheter or other mechanical devices). Writteninformed consentswere obtainedfrom allpatients priortoinclusioninthestudy.Thisstudywasapprovedbythe EthicsandResearchCommitteeofKathmanduModel Hospi-tal.

All the received clinical samples were collected and processedbystandardmicrobiological methods.5 _Following collectionthesamplewastransferredtothelaboratory imme-diately and inoculated on blood agar and McConkey agar. Isolateswere identified basedupon colonialcharacteristics andconventional biochemicaltests.5 _{Antimicrobial} suscep-tibilitypatterns were determined bydiskdiffusion method usingcommercial antibiotic disks (Oxoid Ltd., Basingstoke, UK).6_{IdentificationofisolatesofEnterobactercloacae,}

Enterobac-teraerogenesandCitrobacterspp.werejudgedasABLproducers,

as they are known for chromosomal ABL producers and oftenproduceABLconstitutively.1,7 _{Thescreeningforother} presumptive ABL-producers in the possibility of presence ofextended-spectrum␤-lactamase(ESBL)was improvedby thecriteriaofcefoxitin(30␮g)insusceptibility,positivityfor ESBL screening and negative result forESBL confirmation. FortheassessmentofESBLproduction,double-disksynergy test using a disk containing of amoxicillin/clavulanic acid (20/10␮g)wasperformedusingpositiveandnegativecontrol strainsforESBLproduction.8_{Thephenotypicconfirmationof} ABL-positivewasdeterminedbyAmpCdisktest.9_TheAmpC diskcontaininga1:1mixtureofsalineand100× Tris–EDTA solutionwas madein-house accordingtoBlack et al.9 _The procedure ofAmpCdisk testing wasfollowed according to Blacket al.9 _{Inbrief,acefoxitindisk (30␮g)was placedon} thelawnofE.coliATCC25922onMueller-Hintonagarplate. Justbeforetheuse,AmpCdiskwasmoistenedwith20␮Lof salineandseveralcoloniesofatestorganismwere applied toadiskandthenplacedtoalmosttouchingacefoxitindisk ininoculatedsurfaceofagarwiththefaceofinoculateddisk incontactwiththeagarsurface.Afterincubationovernight, theABLproductionwasidentifiedbythepresenceofeither anindentationoraflatteningofthezoneofinhibition.We excludedtheisolatesofSalmonellaentericaserotypeTyphiand ParatyphiAbecause noneofthem were foundresistant to 2ndand3rdgenerationcephalosporinsatthetimeofstudy, and it wasfelt that including them could misestimatethe

prevalenceofABL-producingisolatesamong Enterobacteria-ceae.

The ABL-positive E. coliisolates which were susceptible for streptomycin were screened and selected for conjuga-tionstudytoevaluatethehorizontaltransferofABL-positive phenotypes.Evidenceofthesimilarsizeofintactplasmid(s) betweendonorsandtransconjugants,theresistantfeaturesto cefoxitin,cefatzidime(30␮g),cephotaxime(30␮g)and amox-icillin/clavulanic acid by transconjugants, and the positive result of AmpC disk test by transconjugants was judged as transferable ABL-positive phenotypes. Theplasmid was extractedandpurifiedusingcommercialkits(QiagenGmbH, Hilden, Germany) according tothe manufacturer’s instruc-tions. Conjugationwas doneas described byBaralet al.,10 using plasmid free E. coliHB 101 (F−S+_lac−_{) as the} recipi-entstrain.TransconjugantswereselectedonMacConkeyagar supplemented withstreptomycin (100␮g/mL) and cefoxitin (20␮g/mL).

Statistical analyses were performedusing SPSS (version 18.0,SPSSInc.,Chicago,IL)softwarepackage.Categorical vari-ableswereanalyzedbyChi-squaretestorFisher’sexacttest, asappropriate.Univariateanalysiswasperformedfor calcu-lationofoddsratio(OR)with95%confidenceinterval(CI)for potentialriskfactors.Ap-valueoflessthan0.05was consid-eredstatisticallysignificant.

Atotalof230Enterobacteriaceaeisolateswererecovered from1,503clinicalspecimensduring6-monthstudyperiod. Out of the total isolates, E. coli (83.9%) was the predom-inant pathogen, followed by Citrobacter freundii (5.2%) and

Enterobacterspp.(3%).Totally64(27.8%)ABL-producing

Entero-bacteriaceaewereobservedin230Enterobacteriaceaeisolates, amongthemE.coli(59.7%)wasthemostcommonorganism, followedbyC.freundii(18.8%).Themeanageofthepatients harbouringABL-producingEnterobacteriaceaewas49.3years (range2–92years).NoneoftheisolatesofProteusspp.and

Kleb-siellaspp.wereshowntoproduceABLinourstudy.Similar

toourstudy,highoccurrenceratesofABL-producing Entero-bacteriaceae were reported in other Asian countries.2,4,11 The majorityof ABL-producerswere found toexhibit high rates ofresistancetocommonlyused antibioticsasshown inTable1.Specifically, highrates ofresistanceobservedto fluoroquinolones(67.2%resistancetociprofloxacin,59.4%to norfloxacin,54.7%toofloxacin),aminoglycosides(48.4% resis-tance to gentamicin) and nitrofurantoin (20.3% resistance) are mostworrisome (Table1), because these drugscan be bought even by low-income class of Nepalese people and are easily available in different pharmacy counters across Nepal.TwoABL-producingC.freundiiwerefoundresistantto meropenem,whilenoneoftheisolateswasfoundresistant toimipenem.Itisnotuncommontofindcarbapenem resis-tance among ABL-producing isolates.12 _{Comparatively, rate} of meropenem insusceptibility(0.87% resistancein Entero-bacteriaceae) observed inour study is lower than a report fromIndia(22.16%resistanceamongGram-negativebacterial pathogens).13_{Asweexpected,asignificantproportionof} ABL-producingisolates(81.3%)showedresistanceto≥3different classesofantibiotics,definedasmultidrugresistant(MDR).

Out of 33 ABL-producing E. coli isolates, 72.7% (n=24) exhibited transferable ABL-positive phenotypesas revealed byconjugationstudy.ThemajorityofABL-producingdonor

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Table1–Antimicrobialresistance(%)byAmpC␤-lactamase(ABL)-positiveand-negativeEnterobacteriaceaeisolates.

Antibiotic ABL-positive(n=64) ABL-negative(n=166) p-value

Trimethoprim-sulfamethoxazole 78.1 28.9 <0.001 Nitrofurantoin 20.3 6.6 0.016 Ciprofloxacin 67.2 19.9 <0.001 Norfloxacin 59.4 16.3 <0.001 Ofloxacin 54.7 15.7 <0.001 Amikacin 18.8 1.8 <0.001 Chloramphenicol 20.3 6.6 0.002 Gentamicin 48.4 60.2 0.105 Meropenem 3.1 0 0.077 Imipenem 0 0

isolatesharboredmultipleplasmidsmostcommonlyofsize

32.5kb and 38kb, whereas plasmid of size32.5kb was the

mostcommonplasmidamongtheABL-producing

transcon-jugants.ABL positivitywiththe MDR pattern (amoxicillin-,

ciprofloxacin-, cefixime-, trimethoprim-sulfamethoxazole-,

ofloxacin-, and norfloxacin-resistance) was found most

commonly (78%) to be transferred to recipient strain by

conjugation. Thehigh prevalencerate oftransferable

ABL-positivephenotypesobservedinourstudymayindicate an

increasedtransmissionrateofdrugresistantplasmidsthat

haveacquiredgenesforAmpCenzymes amongpathogenic

Enterobacteriaceae isolates, which can consequently make

thebacteriawhichareknownforlackingorpoorly

express-ingchromosomalAmpC␤-lactamasegene,suchasinE.coli

and Klebsiella pneumoniae.1 _{The clinical characteristics of} 230patientsinfectedwithEnterobacteriaceaeare shownin Table2.Univariateanalysisrevealedthatprioruseof antibi-oticinthelastsixmonths(OR,5.278;CI,2.838–9.817;p<0.001) andbeing inpatient(OR,4.587;CI,2.132–9.9;p<0.001)were associatedwithABL-positiveinfections(Table2).Itisnot sur-prisingtomentionthatinNepalthereisextensivepracticeof irrationalantibioticuse,especiallybyself-medicationbecause drugsare easilyaccessibleinthepharmacycounters with-outprescription,andthismaycreatetheselectionpressure toemergeABL-positiveMDRpathogensasweobservedinthe

presentstudy.However,furthermolecularstudyinvolvingthe isolatesfromdifferentregionsofNepalisneededtoconfirm suchsituationinNepal.ThehighfrequencyofABL-producing Enterobacteriaceae infections (57.6%)amongthe inpatients impliestheextensivedisseminationofABL-producing bacte-riainhospitalenvironments (Table2).Anincreasedrateof occurrenceofABL-producers(63.4%)wasalsoobservedamong nosocomialisolatesofE. coliandK. pneumoniaeinarecent reportfromIndia.14_{Itmayindirectlysuggestthatthe} contrib-utorsthatledtotheemergenceandspreadofABL-producing Enterobacteriaceaeamongthehospitalizedpatientsofboth countrieshaveacommonmechanism.

Inconclusion,weobservedahighprevalencerateof ABL-producingEnterobacteriaceaeamongNepalesepatientsand the majority ofthem showed increased rates ofantibiotic resistance.Toourknowledge,thisisamongthefirstreports to describe about clinical and microbiological characteris-ticsofABL-producingEnterobacteriaceae,anddocumenting plasmid-mediated transfer of ABL-positive phenotypes of

E. coli from clinical isolates of Nepal. Our study not only stronglysuggeststhedisseminationofABL-producing Entero-bacteriaceaeinourlocality,butalsoamongthepatientsofall geographicallocationsofNepal, becausemanypeoplefrom outside of Kathmandu regularly visit our hospital seeking forbettertreatmentfacility.Regularnationwideantimicrobial

Table2–UnivariateanalysisofAmpC␤-lactamase(ABL)positivityamongpathogenicEnterobacteriaceaeisolates. Potentialriskfactors Totalisolatesn=230(%) ABL-positiveisolatesn=64(%) OR(CI95%) p-value Ageofpatient ≤30years 100(43.5) 23(23) 0.648(0.358–1.175) 0.152 31–60years 74(32.2) 23(31.1) 1.265(0.689–2.323) 0.448 >60years 56(24.3) 18(32.1) 1.318(0.685–2.536) 0.407 Gender Male 54(23.5) 18(33.3) 1.413(0.732–2.729) 0.302 Female 176(76.5) 46(26.1)

Presenceofanyunderlyingillnesses and/orcomorbidities

85(37) 30(35.3) 1.781(0.989–3.205) 0.053

PriorhistoryofUTI(lastoneyear) 90(34.3) 26(28.9) 1.09(0.605–1.965) 0.773

Prioruseofantibiotic(lastsixmonth) 74(32.2) 38(51.4) 5.278(2.838–9.817) <0.001

ForcaseswithUTI 202(87.8) 54(26.7) 0.657(0.285–1.511) 0.32

Categoryofpatient

Inpatient 33(14.3) 19(57.6) 4.587(2.132–9.9) <0.001

Outpatient 197(85.7) 45(22.8)

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resistancesurveillanceincorporatingthedetectionofABLsis warrantedinordertorestrictthepossibleburdenassociated withABL-positiveEnterobacteriaceaeinfections.

Conflict

of

interest

Allauthorsdeclaretohavenoconflictofinterest.

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