h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Clinical
Microbiology
Epidemiology
of
diabetic
foot
infections
in
a
reference
tertiary
hospital
in
India
Sanjith
Saseedharan
a,∗,
Manisa
Sahu
b,
Roonam
Chaddha
a,
Edwin
Pathrose
a,
Arun
Bal
c,
Pallavi
Bhalekar
c,
Priyadharshini
Sekar
d,
Padma
Krishnan
daSLRahejaHospital(AFortisAssociate),IntensiveCareUnit,Mumbai,Maharashtra,India bSLRahejaHospital(AFortisAssociate),DepartmentofMicrobiology,Mumbai,Maharashtra,India cSLRahejaHospital(AFortisAssociate),DepartmentofDiabeticFoot,Mumbai,Maharashtra,India
dUniversityofMadras,DrALMPGInstituteofBasicMedicalSciences,DepartmentofMicrobiology,Chennai,TamilNadu,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27January2016 Accepted12September2017 Availableonline1November2017 AssociateEditor:AnaLuciaDarini
Keywords:
Diabeticfootinfection
blaNDM
MRSA
a
b
s
t
r
a
c
t
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:[email protected](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.8However,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.11Thereispaucity
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(30g) using KirbyBauerdiscdiffusionmethodandinterpretedaccording tothe CLSIguidelines.12 All theisolateswithaninhibition
zone diameter ofless than 18mmwere labelled as screen positive.
AlawncultureofE.coliATCC25922waspreparedonMHA plate.Asterilediscof6mmmoistenedwith20Lofsterile 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.17,18In
con-trast,grampositiveorganismspredominatethecausativesin thewesternpartoftheworld.19,20Whenlinelistingthe
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.,23revealedthatthereisahigh
preva-lence (27.9%)offungal infection indeeptissuesofdiabetic lowerextremitywoundswithCandidaparapsilosistoppingthe list. Fungalinfectionsaccountedfor9%ofthetotalisolates inastudybyBansalet al.24Candidatropicaliswasthemost
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%).26AstudyfromSouthIndiafoundthat
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-ginin2008.9NDMproducershavebeendescribedinstudies
fromvariouspartsoftheworld;although,manyreportsseem tooriginatefromtheIndiansubcontinent.11,30
However,thereispaucityofdatawithrespectto preva-lenceofNDMamongisolatesfromDFIsinIndia.Khanetal. havereportedNDMproducingEnterobacter cloacaeand Kleb-siellapneumoniaestrainsisolatedintwopatientswithdiabetic footulcersfromIndia.31Samantetal.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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