Epidemiology of diabetic foot infections in a reference tertiary hospital in India

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Clinical

Microbiology

Epidemiology

of

diabetic

foot

infections

in

a

reference

tertiary

hospital

in

India

Sanjith

Saseedharan

,

Manisa

Sahu

_,

_Roonam

_Chaddha

_,

_Edwin

_Pathrose

_,

Arun

Bal

_,

_Pallavi

_Bhalekar

_,

_{Priyadharshini}

_Sekar

_,

_Padma

_Krishnan

a_{SLRahejaHospital(AFortisAssociate),IntensiveCareUnit,Mumbai,Maharashtra,India} b_{SLRahejaHospital(AFortisAssociate),DepartmentofMicrobiology,Mumbai,Maharashtra,India} c_{SLRahejaHospital(AFortisAssociate),DepartmentofDiabeticFoot,Mumbai,Maharashtra,India}

d_{UniversityofMadras,DrALMPGInstituteofBasicMedicalSciences,DepartmentofMicrobiology,Chennai,TamilNadu,India}

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Articlehistory:

Received27January2016 Accepted12September2017 Availableonline1November2017 AssociateEditor:AnaLuciaDarini

Keywords:

Diabeticfootinfection

blaNDM

MRSA

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Introduction:Thepresentstudyattemptstoexaminethemicrobialprofileandantibiotic susceptibilityofdiabeticfootinfectionsintheintensivecareunitofatertiaryreferralcentre fordiabeticfoot.Aspartofthestudy,wealsoattemptedtofindtheprevalenceofblaNDM-like

geneamongcarbapenem-resistantgramnegativeinfections.

Methodology:Aprospectivestudyof261patientswithdiabeticfootinfectionswasperformed duringtheperiodbetweenJanuary2014andJune2014.

Results:Atotalof289isolateswereobtainedfrom178tissuesamplesfrom261patients, 156(59.7%)malesand105(40.2%)females,withameanageof58years(−15years), hav-ingdiabeticfootinfection.Nogrowthwasseeninthirtyeight(17.6%)tissuesamples.Out ofthetotalsamples,44.3%weremonomicrobialand55.7%werepolymicrobial.Gram neg-ativepathogenswerepredominant(58.5%).Sevenofthetotalisolateswerefungal;0.7% showedpurefungalgrowthand1.7%weremixed,grownalongwithsomebacteria.The mostfrequentlyisolatedbacteriawereStaphylococcusaureus(26.9%),followedbyPseudomonas aeruginosa(20.9%).Ofthe58.5%gramnegativepathogens,16.5%wereEnterobacteriaceae resis-tanttocarbapenems.Amongtheseisolates,4(25%)werepositiveforblaNDM-likegene. Amongtherest,18.6%werecarbapenem-resistantPseudomonas,amongwhich4(36.3%)were

blaNDM.AmongtheStaphylococci,23.7%weremethicillin-resistantStaphylococcusaureus. Conclusions: Ourresultssupporttherecentviewthatgramnegativeorganisms,depending onthegeographicallocation,maybepredominantinDFIs.Thereisanincreasein multidrug-resistantpathogens,especiallycarbapenemresistanceandthisiscreepingrapidly.Weneed tobemorejudiciouswhileusingempiricantibiotics.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:sanjith@rahejahospital.com(S.Saseedharan).

https://doi.org/10.1016/j.bjm.2017.09.003

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Foot ulcers and other foot problems are a majorcause of morbidityand mortalityinpeople withDiabetesmellitus.1

Diabetic foot infections (DFIs) are the leading cause of hospitalizationfordiabeticpatientsworldwideandin devel-oping countries like India, it accounts for 20% of hospital admissions.2,3 _{DFI is a multifactorial process and three}

factors predispose to tissue damage, namely neuropathy, peripheral vascular disease, and susceptibility to infec-tion whenever there is a direct injury to the foot at risk.4–6 _{DFIs are usually polymicrobial, caused by aerobic}

gram positive cocci like Staphylococcus aureus, gram nega-tivebacilli(Escherichiacoli,Klebsiellapneumoniae,Pseudomonas aeruginosa), and anaerobes. Proper management of these infectionsneedsappropriateantibioticselection.7 _Empirical

treatment is based on the pathogens and the susceptibil-ity pattern seen in the community where the hospital is located.

Beta-lactam antibiotics are the most commonly used antibioticsforbacterialinfections.8_{However,theaccelerated}

emergenceofantibioticresistancetothesegroupsofdrugs amongtheprevalentpathogensisthemostseriousthreatto themanagementofsuchinfections,especiallycarbapenem resistance. These isolates are usually multidrug resistant, whichfurthercomplicatethescenario.

There is a recent emergence of the NDM metallo-beta-lactamase(MBL)encodinggenesamongdifferent enterobac-terialspecies and alsoin non-fermenterslike P. aeruginosa

andAcinetobacterbaumanniiinvariouspartsofworldincluding India.9–11 _{Infact,IndiaandPakistanarethemainreservoirs}

ofblaNDM-likecarryingEnterobacteriaceae.11_{Thereispaucity}

ofdata on MBL-producingorganisms carrying blaNDM-like

genefromdiabeticfootinfections.Takingthisintoaccount, westudiedthemicrobialprofileandsusceptibilitypatternof diabeticfootinfectionsinpatientswithType2Diabetes mel-litustoguideempirictherapyfordiabeticfootinfectionsin our hospitaland the occurrence ofblaNDM-like carbapen-emase gene among carbapenem-resistant gram negative pathogens.

Materials

and

methods

Studytype

Aprospectivestudywasperformedon261diabeticpatients withfoot ulcers over a periodofsixmonths from January 2014toJune2014.Thestudywasconductedatatertiarycare hospitalinMumbai,India.

Studypopulation

Allpatientswithtype2diabetes(irrespectiveofageandsex) who were hospitalized for surgical management of lower-extremity wounds from January 2014 till June 2014 were consideredforthestudy.Theirinformedconsentwasobtained anddemographicdetails,durationoflower-limblesion, dura-tionofdiabetes,andtypeofempirictherapyweredocumented

from their medical records. A deep tissue specimen was obtainedfromthewoundsduringsurgeryandsentfor bac-terialandfungalcultures.

Specimencollection

After surgical debridement ofthe slough and necrotic tis-sueoverthewoundintheoperationtheatre,thewoundwas washedthoroughlywithnormalsaline;adeeptissue speci-menofapproximately0.5×0.5cmwastakenfromthewound bed.Thespecimenwascollectedinasterilecontainersoaked withnormalsalineandwastransportedtoourmicrobiology laboratorywithoutdelayforfurtherprocessing.

Specimenprocessing

Partofthesteriledeeptissuespecimenwascrushedorground with a sterile mortar and pestle in the biosafety cabinet. Thecrushedspecimenwassubjected togramstainingand wasstreakedon5%sheepbloodagar(SBA),MacConkeyagar (MA), and Saboraud’s Dextrose agar (SDA) for fungal cul-ture. After inoculation, the SBA was kept in a candle jar and along with MA, it was kept in an incubator at 37◦C. Bacterial isolates and yeast-like fungus identification and susceptibility test was performed using VITEK 2 Compact automatedculturesystem(bioMeriux,France).CommercialI+ IE(imipenem+imipenem/EDTA)discfromHimedia,Mumbai, India wasusedforEDTAdisc synergytest. Allcarbapenem resistantEnterobacteriaceaeweretestedfor metallobetalacta-maseproduction.ModifiedHodgetest(MHT)wasperformed forallEnterobacteriaceaeisolatesresistanttocarbapenemsby Vitek2accordingtoCLSIguidelinesandEDTAdiscsynergytest wasdoneforthedetectionofmetallo-␤-lactamaseproduction forallgramnegativeisolates.ESBLproductionwasconfirmed byceftazidimeandceftazidime/clavulunicaciddiscsynergy testanddetectionofAmpC␤-lactamaseswasperformedas follows.

AmpCdetectionmethods

Theisolateswere screenedforpresumptiveAmpC produc-tion bytesting theirsusceptibilitytocefoxitin(30␮g) using KirbyBauerdiscdiffusionmethodandinterpretedaccording tothe CLSIguidelines.12 _{All theisolateswithaninhibition}

zone diameter ofless than 18mmwere labelled as screen positive.

AlawncultureofE.coliATCC25922waspreparedonMHA plate.Asterilediscof6mmmoistenedwith20␮Lofsterile salinewas keptand severalcoloniesoftestorganism were inoculatedonthis disc.Acefoxitindisc wasplacednextto thisdisc(almosttouching)ontheinoculatedplate.Theplates wereincubatedovernightat37◦C.Aflatteningorindentation ofthecefoxitininhibitionzoneinthevicinityofthediscwas consideredapositivetest.13

PCRtechniquefordetectionofblaNDM

TotalDNAsofthedifferentbacterialisolateswereextracted byalkalinelysisandPCRtodetectblaNDM-likegenewas per-formedasdescribedbyPoireletal.14

Table1–Infectionsanddepthofdiabeticfootlesions.

Woundgradea _{No Percentage}

II(muscleonly) 88 40.7 III(tendonandcapsule) 106 49.1 IV(jointandbone) 22 10.2

Total 216 100

a _{UniversityofTexaswoundclassification.}

Table2–Distributionofsamplesaccordingthenumber

ofdifferentbacterialspeciesisolatedfromdeeptissue

(n=178).

Onebacterialspecies 79(44.3%)

Twobacterialspecies 73(41.0%)

Threebacterialspecies 21(11.7%)

Morethanthreebacterialspecies 5(3%)

Table3–Bacterialpathogensindiabeticfootinfection.

Nameofthepathogen Numberofpathogens

Staphylococcusaureus 76(26.9%) Pseudomonasaeruginosa 59(20.9%) Enterococcusfaecalis 36(12.7%) Escherichiacoli 34(12%) Klebsiellapneumoniae 27(9.5%) Citrobacterkoseri 10(3.5%) Proteusmirabilis 9(3.1%) Enterobactercloacae 8(2.8%) Citrobacterfreundii 5(1.7%) Streptococcuspyogenes 5(1.7%) Proteusvulgaris 4(1.4%) Enterobacteraerogenes 2(0.7%) Acinetobacterbaumannii 2(0.7%) Proteuspenerri 1(0.3%) Providenciastuartti 1(0.3%) Serratiamarcescens 1(0.3%) Acinetobacterlwoffii 1(0.3%) Stenotrophomonasmaltophilia 1(0.3%) Total 282

Results

Atotalof216diabeticpatientswithfootulcersadmittedtoour hospitalwerestudiedduringthe6monthsstudyperiodfrom January2014toJune2014.Outofthe261patients,156[59.7%] weremalesand105[40.2%]femalesofmeanage58years(±15 years)withaDFI.MajorityoftheDFIswereGradeIII(49.1%)

(Table1).

Atotalof289isolateswereobtainedfrom178tissue sam-ples.Nogrowthwasseeninthirtyeight(17.6%)tissuesamples. Distributionofthemicroorganismsintissuesamplesisshown

inTable2.Monomicrobialinfectionwasseenin44.3%cases.

Amongthebacterialisolates,atotalof117(41.5%)gram posi-tivepathogensand165(58.5%)gramnegativepathogenswere isolated.

Fromthe165gramnegativeisolates,102(61.8%)belonged tofamilyEnterobacteriaceae.AmongtheEnterobacteriaceae iso-lated,Escherichiacoliwasfoundtobemostcommonfollowed byKlebsiellapneumoniae(Table3).Sevenofthetotalisolates werefungal,ofwhich0.7%showedpurefungalgrowthand

46 51 80 100 100 100 25 39 68 60 30 46 30 100 100 100 20 40 40 20 0 20 40 60 80 100 120 Series1 Series2

Fig.1– SusceptibilitypatternofStaphylococcusaureus.

100 96 90 62 88 88 54 47 23 13 12 15 0 20 40 60 80 100 120

Fig.2–SusceptibilitypatternofE.coli.

92 68 87 51 75 75 52 43 ₄₁ 12 12 ₁₀ 0 10 20 30 40 50 60 70 80 90 100

Fig.3–SusceptibilitypatternofKlebsiellapneumoniae.

1.7%weremixed,grownalongwithsomebacteria.The fun-gal isolatescorresponded to Candidaalbicans (2), Candida tropicalis (2), Candidaspp. (1), Aspergillus fumigatus (1) and

Rhodotorulasp.

Piperacillin-tazobactamandcefoperazonesulbactamwere themostfrequentlyusedempiricantibiotics(68%)amongDFI patients. Theantibiotics usedwere ofbroadspectrum pri-marily becauseour hospitalisatertiary referral centrefor diabeticfootinfectionsandhence,wegetmanyadmissions fromsmallersetupsandnursinghomes.

Outof76isolates,18(23.7%)weremethicillinresistantS. aureus(MRSA).Saureus(MSSA+MRSA)strainswere100% sen-sitivetoVancomycin,Teicoplanin,andLinezolide(Fig.1).The next commongram positiveorganism wasEnterococcus fae-calisand 11%(4 out of36)were VRE(Vancomycinresistant Enterococcus)strains.

E. coliandK. pneumoniaewere themost frequent Entero-bacteriaceae isolated. Their susceptibility pattern is shown

in Figs. 2 and 3. Colistin was the most effective agent

85 ₈₁ 56 _{49 50 51} 68 _{50 49} 0 10 20 30 40 50 60 70 80 90

Fig.4–SusceptibilitypatternofP.aeruginosa.

andbeta-lactam/beta-lactamaseinhibitorcombinations.Two (7.4%)ofthe27 Klebsiellaisolateswereresistant tocolistin.

E.coliisolateswere100%sensitivetocolistin.

AmongtheEnterobacteriaceae,sixteenisolates(16.8%)were resistant to carbapenems (Table 4). Nine of the 16 (56.2%) carbapenem resistant strains were MHT positive and 7 of the 16 isolates were positive for MBL detection by EDTA disk synergy test (EDS). All 16 isolates were studied for molecularcharacterizationforblaNDM-likegene.Fourofthe sevencarbapenem-resistantK.pneumoniaeandoneofthe3 carbapenem-resistantE.coliisolateswerepositivefor

blaNDM-likegene.AlltheisolatespositiveforblaNDMlikegenewere positive by EDS but only oneKlebsiella isolates harbouring

blaNDM-likegenewaspositivebyMHT(Table4).

Amongthegramnegativeisolates,63werenon-fermenters beingP. aeruginosa the mostfrequent(n=59[93.6%]).Eleven (18.6%) P. aeruginosa isolates were carbapenem resistant strains.Four(36.3%)ofthe11carbapenemresistantisolates werepositiveforblaNDM-likegene,all4werepositivebyEDS. Fifteenpercent(9/59)oftheP.aeruginosa,wereresistantto col-istin. Therewere isolatedtwoA. baumanniicasesand both werecarbapenemresistant,blaNDMpositive,andsusceptible onlytocolistinandtigecycline(Fig.4).

OutcomeofpatientswithDFI

Amputationoftheaffected partwasdonein120(55.5%)of thepatients,85(39.3%)hadmajordebridementdone,andthe remainder(5.2%)ofthepatientsunderwent minor debride-ment and medical management. Among patients carrying carbapenem-resistant gram negative pathogens, 2patients died.Inonecase,theisolatecorrespondedK.pneumoniaeand the other to A. baumannii. Both carbapenem resistant

iso-latesharbouredblaNDM-like.TheKlebsiellaisolatewascolistin resistantaswell.

Discussion

Currently,50.8millionIndianshavediabetes.Projections indi-cate that India will have the largest number of diabetic patients by the year 2030AD.15 _{Diabeticfoot infections are}

themostfrequentcomplicationrequiringhospitalization.3,16

Microbialprofilesofdiabeticfootinfectionsarewidelystudied anddifferindifferentregionsacrossIndiaandtheworld.In ourstudy,gramnegativebacteriaweremorecommonisolates inDFI,similartomanystudiesperformedinIndia.17,18_In

con-trast,grampositiveorganismspredominatethecausativesin thewesternpartoftheworld.19,20_{Whenlinelistingthe}

indi-vidualmicroorganisms,S.aureuswas ontop,followedbyP. aeruginosa,similartootherstudiesfromsameregion.21,22 _S.

aureuswasthemostfrequentpathogen(28%,42/150),followed byP.aeruginosa(24%,36/150)inthestudybyShanthietal.10

E.coliwasthemostcommonpathogenisolatedfollowedby

S. aureusinastudy from North India, but inother studies

P aeruginosa has been reported most frequently.3,17 _Fungal

pathogensandtheirroleinDFIislessfrequentlystudied.A studybyChellanetal.,23_{revealedthatthereisahigh}

preva-lence (27.9%)offungal infection indeeptissuesofdiabetic lowerextremitywoundswithCandidaparapsilosistoppingthe list. Fungalinfectionsaccountedfor9%ofthetotalisolates inastudybyBansalet al.24_{Candidatropicaliswasthemost}

commonfungalisolateintheirstudy.Incontrast,weobserved lowerprevalenceoffungalinfectionsinourstudy.

TherewereobservedlowerMRSAratescomparedtoother studiesfromIndiawhereincidenceofMRSAhasbeenreported ashigh as66%.25 _{Ourstudy alsorevealed thepredominant}

polymicrobial nature (55.7%) of infections. Monomicrobial infectionswereseenonlyin44.3%cases.Otherstudieshave revealed contrasting resultswithmonomicrobial infections beingmorepredominant.AstudybyTiwarietal.16 _showed

thatmonomicrobialinfectionscases(43.5%,27/62)weremore thanpolymicrobialinfections(35.5%,22/62).Inanotherstudy, equalnumberofpolymicrobialandmonomicrobialinfections werereported(44.4%).26_{AstudyfromSouthIndiafoundthat}

monomicrobialinfections(56%)weremorepredominantthan polymicrobialinfections(44%).27

Carbapenems are the mainstay in the treatment for multidrug-resistant gram negativebacteria; however, a

ris-Table4–Phenotypicdetectionofresistancemechanismsinbeta-lactamresistanceEnterobacteriaceae(n:95).

ESBL AmpC CRE MHT EDS

Klebsiellapneumoniae(n=27) 9(33.3%) – 7(25.9%) 5(1-NDM+ve) 4(NDM+ve)

Escherichiacoli(n=34) 21(61.7%) 2(5.8%) 3(8.8%) 2 1(NDM+ve)

Enterobactercloacae(n=8) 4(50%) – 2(25%) – – Enterobacteraerogenes(n=2) 1(50%) – 1(50%) 1 0 Citrobacterkoseri(n=10) 3(30%) – – – – Citrobacterfreundii(n=5) 2(40%) – – – – Proteusmirabilis(n=9) 1(11%) - 3(33%) 1 2 95 41(43.1%) 2(2.1%) 16(16.8%) 9 7

ingnumberofcarbapenemases(andthus,resistance)tothis group of drugs is increasingly being reported from differ-entpartsoftheworld.28 _{Metallo-beta-lactamasehavebeen}

describedpreviouslyinP.aeruginosaisolatesfromdiabeticfoot infections.29NDMisthelatestinthearmamentariumof car-bapenemases.NDM1(NewDelhimetallo-beta-lactamase)was firstidentifiedinisolatesfromaSwedishpatientofIndian ori-ginin2008.9_{NDMproducershavebeendescribedinstudies}

fromvariouspartsoftheworld;although,manyreportsseem tooriginatefromtheIndiansubcontinent.11,30

However,thereispaucityofdatawithrespectto preva-lenceofNDMamongisolatesfromDFIsinIndia.Khanetal. havereportedNDMproducingEnterobacter cloacaeand Kleb-siellapneumoniaestrainsisolatedintwopatientswithdiabetic footulcersfromIndia.31_{Samantetal.have,forthefirsttime,}

describedProvidenciarettgeristrainsharbouringblaNDM-like

in4patients withdiabetic foot ulcers from India.32 _Inour

study,wefoundthepresenceofblaNDM-likeinnonfermenters likePseudomonasandAcinetobacteraswellasinE.coliandK. pneumoniae.

With the current local microbiological and antibiogram data, cefoperazone/sulbactam or piperacillin/tazobactam withclindamycinwasthesuggestedastheempiricantibiotic ofchoiceforDFIwithescalationtocarbapenem(meropenem) with teicoplanin depending upon the culture sensitivity report.

Duetoresourceconstrainswelookedonlyforthe

blaNDM-likegenes.However,itsworthlookingintotheprevalenceof other carbapenemase genes likeKPC, IMP,VIM etc.In one shortprojectforCREscreeninginourhospitalwefoundan increasedprevalenceofKPClikegenesinK.pneumoniae iso-latedfromstoolsamples.(unpublisheddata)

Conclusion

Tothebestofourknowledge,thisstudyhasdealtwith per-haps the largest data in the worldrelated to diabetic foot infectionsforstudyingthepathogenspectrumalongwithan attempttostudy the prevalenceofblaNDM-like gene. This studygoesontoindicatethatinatertiarydiabeticfoot refer-ralcentre,polymicrobialgramnegativeinfectionsare more predominantinDFIs.Thestudyhasalsodemonstratedthe emergenceofdifficult-to-treatgramnegativesasalmostone fifthofthegramnegatives.Therefore,theauthorsbelieveitis nowhighlyrecommendedtohavestrictantimicrobial stew-ardshipprogrammes andrestrict unnecessaryuseofbroad spectrumantibioticstocurbthemenaceofantibiotic resis-tance.

Brief

summary

Alreadyknown:

Gramnegativebacteriaare morecommonthan gram posi-tivebacteriainDFIcasesinIndia.Monomicrobialinfections aremorepredominantthanpolymicrobialinfectionsinmost studies.NDMprevalenceinDFIcaseshasbeendemonstrated byfewstudies.13

Whatthisstudyadds:

Wehandledalargerdatapoolforcarbapenem-resistant bacte-riainDFIcasesthanmoststudies.Polymicrobialinfections (54%)werefoundtobemorepredominantthan monomicro-bialinfections(44.3%)inDFIcases.LowerrateofMRSAamong grampositivebacteria.FoundthepresenceofblaNDM-likein nonfermenterslikePseudomonasandAcinetobacteraswellas inE.coliandpneumoniae.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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