RevBrasAnestesiol.2014;64(2):84---88
REVISTA
BRASILEIRA
DE
ANESTESIOLOGIA
OfficialPublicationoftheBrazilianSocietyofAnesthesiologywww.sba.com.br
SCIENTIFIC
ARTICLE
In
vitro
evaluation
of
antimicrobial
features
of
vasopressors
Habib
Bostan
a,
Yakup
Tomak
b,
Sengul
Alpay
Karaoglu
c,
Basar
Erdivanli
d,
Volkan
Hanci
e,∗aTheCouncilofForensicMedicine,MinistryofJustice,Istanbul,Turkey
bDepartmentofAnesthesiologyandReanimation,MedicalFacultyofSakaryaUniversity,Sakarya,Turkey cDepartmentofBiology,FacultyofArtsandSciencesofRizeUniversity,Rize,Turkey
dDepartmentofAnesthesiologyandReanimation,MedicalFacultyofRizeUniversity,Rize,Turkey
eC¸anakkaleOnsekizMartUniversity,MedicalFaculty,DepartmentofAnesthesiologyandReanimation,C¸anakkale,Turkey
Received5April2012;accepted28February2013 Availableonline11October2013
KEYWORDS
Antimicrobial activities;
Vasopressordrugs;
Drugcontamination
Abstract
Background: Drugsadministeredasintravenousinfusionmaybecontaminatedduringseveral stagesofproductionorpreparation.Howeverstudiesfocusingonantibacterialeffectsof vaso-pressordrugsareveryrare.Thisstudyinvestigatestheinvitroantimicrobialactivityofthe clinicallyusedformsofvasopressors.
Materialsandmethods: Invitroantimicrobialactivitiesofvasopressordrugsofdifferent con-centrations were investigated by using the micro dilution technique. Microorganisms used inthe test were Escherichia coliATCC25922, Yersiniapseudotuberculosis ATCC911, Pseu-domonasaeruginosaATCC10145,ListeriamonocytogenesATCC43251,Enterococcusfaecalis
ATCC 29212,Staphylococcus aureus ATCC25923, Bacilluscereus 702 Roma,Mycobacterium smegmatisATCC607, CandidaalbicansATCC60193,andSaccharomycescerevisiaeRSKK251. AntibacterialassayswereperformedinMueller-HintonbrothatpH 7.3andantifungalassays wereperformedinbufferedYeastNitrogenBaseatpH7.0.
Results:Twodifferentdopaminepreparationsshowed antimicrobialactivity. Nootherstudy drugshowedanyantimicrobialactivity.
Conclusions:Inouropinion,dopamine’santibacterialeffectsmaybeadvantageousfor inhib-itingthespreadofbacterialcontaminationduringthepreparationoftheinfusionsolutions. However,itisimportantthatstrictguidelines regardingtheneedfor sterileequipmentand deliverablesbeadheredtoduringallproceduresperformedintheintensivecareunits. ©2013SociedadeBrasileiradeAnestesiologia.PublishedbyElsevier EditoraLtda.Allrights reserved.
∗Correspondingauthorat:C¸anakkaleOnsekizMartUniversity,MedicalFaculty,DepartmentofAnesthesiologyandReanimation,Education
Hospital,CentralOperatingTheater,CumhuriyetMahallesi,SahilYolu,No:5,C¸anakkale,Turkey.
E-mail:vhanci@gmail.com(V.Hanci).
0104-0014/$–seefrontmatter©2013SociedadeBrasileiradeAnestesiologia.PublishedbyElsevierEditoraLtda.Allrightsreserved.
Introduction
Septic shock is the primary cause of death in critical care units. Shock states are primarily characterized by acute circulatory failure leading to tissue hypoperfusion, and potentially resultingin multi-organ failure. Observed hypotensioncanbetheconsequenceofthreemajor hemo-dynamicdisorders:hypovolemia,vascularfailure,andheart failure.1 When appropriate fluid administration fails to
restore adequate tissue perfusion and arterial pressure, vasopressors are usually necessary to increase mean sys-temicpressure,cardiacoutput,andoxygendelivery.2
In vitro studies focusing on catecholamine molecules demonstrated proliferation of bacteria.3---5 A portion of
catecholamines,whichareusedasvasopressor,are endoge-nouslyproducedinthebody.However,catecholaminesused asvasopressordrugsaresyntheticallyproducedandinfused forthetreatmentofcardiovascularfailurewhicharises dur-ing septic shock. Dopamine, dobutamine, adrenaline and noradrenaline aremost frequently used vasopressors pre-pared synthetically with supplemental chemicals having antioxidant and antimicrobial activity. Sodium metabisul-fite, N-acetylcysteine and disodium edetate are themost frequently used antioxidant and antimicrobials for this purpose in drugs commonly found in medical markets (Table1).
Considering several studies pointing catecholamine molecules’proliferatingeffectonbacteria,weinvestigated commercially prepared catecholamine products’ in vitro effectonproliferationofseveralyeastandbacterialstrains commonlyencounteredinsepticshock.
Materials
and
methods
MicroorganismsusedintestswereobtainedfromtheRefik SaydamHifzissihhaInstitute (Ankara,Turkey) andwere as follows: Escherichia coli ATCC 25922, Yersinia pseudotu-berculosisATCC911,PseudomonasauroginosaATCC10145, ListeriamonocytogenesATCC43251,Enterococcusfaecalis ATCC 29212, Staphylococcus aureus ATCC 25923, Bacillus cereus709ROMA,MycobacteriumsmegmatisATCC607, Can-dida albicans ATCC 60193 and Saccharomyces cerevisiae ATCC60193.
Antimicrobial effects of the drugs were tested quan-titatively in appropriate broth media using the double dilutionmethod,andtheminimuminhibitoryconcentration (MIC) values in g/mL were determined.6,7 Antibacterial assayswereperformedinMueller-Hintonbroth(MH)(Difco, Detroit,MI)atpH7.3andantifungalassayswereperformed inbufferedYeastNitrogenBase(YNB)(Difco,Detroit,MI)at pH7.0.Eachtesteddrugwaspreparedin0.1mLvolumesof sterile MHand YNBbroths in concentrations ranging from 5g/mLto5mg/mLformicrodilution.Onedrop(0.02mL) ofmicroorganism’ssuspension(approximately106 microor-ganismspermL)wasaddedtotheextract/brothdilutions. Afterincubationat35◦Cfor18---72h,themediawere exam-inedforgrowth.MICisdefinedasthelowestconcentration ofdrugshowingnogrowthofmicroorganism.Thedilutions without visible growth were used to determine minimum bactericidalconcentration(MBC)byspreading100Lofthe sampleacrossthesurfaceofdriedMHandYNBagarplates withsterileglassrods,andthenincubatingat35◦Cfor18h. MBCofeachextractisdefinedasthelowestconcentration
Table1 Studydrugsandingredients.
Catecholamine Ingredients Epinephrine In1mLAmpoule:
-Epinephrine0.5mg -Sodiumchloride8.5mg -Metabisulfite0.5mg -Waterforinjection Norepinephrine In4mLAmpoule:
-Norepinephrinebitartarate8mg(equivalentto4mgnorepinephrinebase) -Sodiummetabisulfite4mg
-Sodiumchloride34.35mg -Waterforinjection Dobutamine In20mLAmpoule:
-Dobutaminehydrochloride280mg(equivalentto250mgdobutaminebase) -Sodiummetabisulfite4.8mg
-Waterforinjection Dopamine In5mLAmpoule:
-Dopaminehydrochloride200mg -N-acetylcysteine2mg
-Disodiumedetate2mg -Waterforinjection Dopamine In5mLAmpoule:
86 H.Bostanetal.
that showed no growth of microorganism on agar plate.
Fluconazole, Ampicillin and Streptomycin were used as
standardantifungalandantibacterialdrugs,respectively. Ingredientsofstudydrugswidelyusedasvasopressorin medicalmarketarepresentedinTable1.
Results
None of the study drugs containing norepinephrine, epinephrine and dobutamine showed any antimicrobial activity(Table2).Howeverstudydrugscontainingdopamine showed antimicrobial activity (Table 2), one of which showed no activity against yeast-like microorganisms. The solutions containing dopamine (125---1000g/mL), N-acetylcysteine and disodium edetate (1.25---10g/mL) showed bacteriostatic activity against Gram-positive and -negative microorganisms. Higher concentration solutions showed bactericidial activity against all microorganisms exceptY.pseudotuberculosis,which is capsular. Solutions ofdifferentconcentrationsofdopamine(125---500g/mL), N-acetylcysteine and disodium edetate (1.25---5g/mL) resulted in similar MIC and MBC values for bacterial strainslikeGram-positivebacillusL.monocytogenes, Gram-positivecoccus E. faecalis, Gram-negativeM. smegmatis, whichcontainmycolicacidintheircellwall.However,these valuesweremarkedlydifferentforeachsolution.Solutions containing dopamine (125---250g/mL), N-acetylcysteine and disodium edetate (1.25---2.5g/mL) showed bacte-riostatic activity against E. coli, P. aeruginosa and S. aureus. Solutions containing dopamine 2000g/mL, N-acetylcysteineanddisodiumedetate20g/mLshowed bac-tericidialactivity against the same microorganisms. Solu-tions containing dopamine 1000g/mL, N-acetylcysteine anddisodiumedetate10g/mLshowedbacteriostaticand bactericidalactivityagainstB.cereus.
Other drugscontainingdopamine (125---500g/mL)and sodium metabisulfite (62.5---125g/mL) showed bacteri-cidialactivityagainstallmicroorganismsusedinthetest.
Discussion
Inthis study,we havefound that twodifferentdopamine preparationsoutofalltestedshowedantimicrobialactivity. Drugsmanufactured forintravenous useshouldbe pre-pared and administered in sterile conditions. Infectious microorganismscanbeintroducedintothepatientthrough contaminated containers, rubber diaphragm, needles and infusion sets. Anesthetic agents and vasopressors may be contaminated by microorganisms during the preparation of an infusion. For this reason, the antimicrobial effects of anesthetic agents andvasopressors have been deemed important, and they have been investigated in previous studies.8Notably,propofolisknowntosupportthegrowthof
microorganisms.8Ontheotherhand,previousstudieshave
shownthatmorphinesulphate,thiopentalsodium,fentanyl citrate,dexmedetomidine andmidazolamallhave antimi-crobial effects.8---13 However, studies on the antimicrobial
effectsofvasopressorsdrugs,whicharecommonlyusedin intensivecareunits(ICU),areveryfew.
Studies demonstrating that catecholamines stimulate growth of microorganisms are increasing.4,5,14,15 Among
the causative factors are binding of catecholamines to transferrinandlactoferrin,enablingbacteriatoacquire nor-mally inaccessible ferric-iron14 and possible
␣-adrenergic specific response system of some bacteria to recognize catecholamines.15
On the other hand, additives having antioxidant and antimicrobial properties are commonly added to commercial vasopressor formulas to prevent bacterial contamination.16---19 However, studies investigating the
in vitro antimicrobial activity of clinically used commer-cial forms of vasopressors are very few. Most commonly usedadditivesinvasopressorsareN-acetylcysteineand diso-diumedetate,whichareknownaspotentantioxidantswith antimicrobialproperties.16---19
Thisstudyevaluatestheantimicrobialpropertiesofmost commonlyusedvasopressorsinthemedicalmarketsin dif-ferentconcentrationsbythemicro-dilutionmethod.
Vasopressordrugs areadministeredasinfusion through a preferablycentral, highcaliber veintoensure a steady state plasma concentration. We used the micro-dilution method to mimic different levelsof concentrations since catecholamines interact over a wide dose-response range andexhibitmultiplepotencies.
Ourstudyrevealedantimicrobialactivityofbothofthe dopamine preparates. No other study drug was able to inhibitmicroorganismal growthat anyconcentration.This findingcouldbeexplainedbythehighsodiummetabisulfite concentrationforoneoftheDopaminepreparatecompared tonorepinephrine,adrenalineanddobutaminepreparates. Sodiummetabisulfiteisanoxidizingagentactiveatlow pH. While all the study drugs have effective pH ranges between2.2and5.0,MHbrothhasapHvalueof7.3±0.1 andbuffered YNBusedinourstudy hasapH valueof 7.0 at25◦C(unbufferedmediumhasapHvalueof5.4±0.2at 25◦C).Therefore we donot thinkthat sodium metabisul-fite may exhibit any antimicrobial activity at the neutral pH. Sincehumanblood hasa slightly alkalinepH value of 7.35---7.45, andmost pathogenic bacteria prefera narrow pHrangeof6---8,11wethinkthatthisfindingisconcordant
withreal-lifeapplicationsofthedrugs.
The other oxidizing additive containedin tested drugs is N-acetylcysteine, which was shown to be a valuable mucolyticagent,capabletoaidinantimicrobialtreatment if combined with antibiotics. Examples where antimicro-bialactivity ofN-acetylcysteineisseen arelysisof gastric basal mucosallayer, which enables Helicobacterpylori to escapefromtheacidicgastricsecretions20 anddecreasing
formationofbiofilmsbyreducingproductionof extracellu-lar polysaccharide matrixandpromotingthe disruptionof maturebiofilm.21Bothoftheseactivitiesareaugmentedby
an acidicenvironment, andthe slightlyalkaline pH levels establishedinthestudybrothenvironmentsmayhave hin-deredN-acetylcysteine’santimicrobialactivity.However,as for metabisulfite, we conclude thata slightly alkaline pH levelismoreconcordantwithreal-lifeapplicationsofthese drugs.
Itisimportantthatstrictguidelinesregardingtheneed forsterileequipmentanddeliverablesbeadheredtoduring allproceduresperformedintheICU.Insomecircumstances, vasopressor drugs maybe contaminated with microorgan-ismsthat canthenleadtoinfections.8,9 Thus,the
features
of
vasopressors
87
Table2 AntimicrobialactivityofthecompoundsexpressedasMICvaluein100mLvolume.
Studydrugs Ingredient Concentration (g/mL)
MICvalues(100mL)
Ec Yp Pa Li Ef Sa Bc Ms Ca Sc
Norepinephrine 4mg/4mLAmpoule
Norepinephrine 1000 --- --- --- --- --- --- --- --- --- ---Sodiummetabisulfite 1000 --- --- --- --- --- --- --- --- --- ---Sodiumchloride 34.350 --- --- --- --- --- --- --- --- ---
---Adrenalin0.5mg/1mL Ampoule
Epinephrine 500 --- --- --- --- --- --- --- --- --- ---Sodiummetabisulfite 500 --- --- --- --- --- --- --- --- --- ---Sodiumchloride 8500 --- --- --- --- --- --- --- --- ---
---Dobutamine
250mg/20mLAmpoule
Dobutamine 12.500 --- --- --- --- --- --- --- --- --- ---Sodiummetabisulfite 240 --- --- --- --- --- --- --- --- ---
---Dopamin200mg/5mL Ampoule
Dopamine 40.000 125 125 250 250 125 125 1000 250 --- ---N-acetylcysteine 400 1.25 1.25 2.50 2.50 1.25 1.25 10 2.50 --- ---Disodiumedetate 400 1.25 1.25 2.50 2.50 1.25 1.25 10 2.50 ---
---Dopamine200mg/5mL Ampoule
Dopamine 40.000 500 500 500 250 250 500 250 250 500 125 Sodiummetabisulfite 10.000 125 125 125 62.5 62.5 125 62.5 62.5 125 62.5
Ampicillin 10 2 32 >128 2 2 2 <1
Streptomycin 10 4
Fluconazole 5 <8 <8
Bc,Bacilluscereus702Roma;Ca,CandidaalbicansATCC60193;Ec,EscherichiacoliATCC25922;Ef,EnterococcusfaecalisATCC29212;Li,ListeriamonocytogenesATCC43251;Ms, MycobacteriumsmegmatisATCC607;Pa,PseudomonasaeruginosaATCC10145;Sa,StaphylococcusaureusATCC25923;Sc,SaccharomycescerevisiaeRSKK251;Yp,Yersinia pseudotuber-culosisATCC911.
88 H.Bostanetal.
isofparamount importance.8,9Inouropinion, dopamine’s
antibacterialeffectsmaybesufficienttoinhibit contamina-tionduringthepreparationoftheinfusionsolutions.
Wehaveshownthatdopaminehasantibacterialeffects onsomemicroorganismsfrequently encounteredin hospi-talsettings.Wesuggestthatdopaminepreparationsshould be preferred in septic patients due to their antimicro-bial activity against several yeast and bacterial strains commonly encountered in septic shock. However, trans-lating such laboratory researches into recommendations requires delineation of the interactions between cate-cholamines, several molecules co-existing or co-secreted withthem,andmicroorganismsintheirexpected environ-ments.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
References
1.Levy B,CollinS, SennounN,et al. Vascular hyporesponsive-nesstovasopressorsinsepticshock: frombenchto bedside. IntensiveCareMed.2010;36:2019---29.
2.PóvoaP, CarneiroAH. Adrenergic support inseptic shock: a criticalreview.HospPract(Minneap).2010;38:62---73.
3.Lyte M.Theroleof catecholaminesinGram-negative sepsis. MedHypotheses.1992;37:255---8.
4.LyteM,ErnstS.CatecholamineinducedgrowthofGram nega-tivebacteria.LifeSci.1992;50:203---12.
5.NealCP,FreestonePP,MaggsAF,HaighRD,WilliamsPH,LyteM. CatecholamineinotropesasgrowthfactorsforStaphylococcus epidermidisandothercoagulase-negativestaphylococci.FEMS MicrobiolLett.2001;194:163---9.
6.NationalCommitteeforClinicalLaboratoryStandard.Methods for determiningbactericidalactivity ofantimicrobialagents; approvedguideline.NCCLSdocumentM26-A;1999.
7.WoodsGL,Brown-ElliottBA,DesmondEP,etal.Susceptibility testingofmycobacteria,nocardiae,andotheraerobic actino-mycetes;approvedstandard.NCCLSdocumentM24-A,vol.23; 2003.
8.HanciV,CömertF,AyogluH,KulahC,YurtluS,TuranIO. Evalua-tionoftheantimicrobialeffectsofatracurium,rocuroniumand
mivacurium.Antimicrobialeffects ofmusclerelaxants.Drugs TherStud.2011;1:e2.
9.Ayoglu H, Kulah C, Turan I. Antimicrobial effects of two anaestheticagents:dexmedetomidineandmidazolam.Anaesth IntensiveCare.2008;36:681---4.
10.Graystone S, Wells MF, Farrell DJ. Do intensive care drug infusions support microbial growth? Anaesth Intensive Care. 1997;25:640---2.
11.CrowtherJ,HrazdilJ,JollyDT,GalbraithJC,GreacenM,Grace M.Growth of microorganisms in propofol,thiopental,and a 1:1mixtureofpropofolandthiopental.AnesthAnalg.1996;82: 475---8.
12.SosisMB,BravermanB,VillaflorE.Propofol,butnot thiopen-tal,supports the growth ofCandida albicans. Anesth Analg. 1995;81:132---4.
13.Keles¸GT,KurutepeS,TokD,etal.Comparisonofantimicrobial effectsofdexmedetomidineandetomidate-lipurowiththoseof propofolandmidazolam.EurJAnaesthesiol.2006;23:1037---40.
14.SandriniSM,ShergillR,WoodwardJ,etal.Elucidationofthe mechanismbywhich catecholaminestress hormonesliberate ironfromtheinnateimmunedefenseproteinstransferrinand lactoferrin.JBacteriol.2010;192:587---94.
15.FreestonePPE,HaighRD,LyteM.Blockadeof catecholamine-induced growth by adrenergic and dopaminergic receptor antagonistsinEscherichiacoli O157:H7,Salmonella enterica andYersiniaenterocoliticaBMCMicrobiol.2007;7:8.
16.ErcanS, OztürkN, Celik-Ozenci C,Gungor NE, Yargicoglu P. Sodiummetabisulfiteinduceslipidperoxidationandapoptosis inratgastrictissue.ToxicolIndHealth.2010;26:425---31.
17.Baker MT,Dehring DJ, Gregerson MS. Sulfite supportedlipid peroxidationin propofol emulsions.Anesthesiology. 2002;97: 1162---7.
18.Olofsson AC, Hermansson M, Elwing H. N-acetyl-l-cysteine affects growth,extracellularpolysaccharide production, and bacterial biofilm formation on solid surfaces. Appl Environ Microbiol.2003;69:4814---22.
19.Mansouri MD, Darouiche RO. In vitro antimicrobial activity ofN-acetylcysteineagainstbacteriacolonisingcentralvenous catheters.IntJAntimicrobAgents.2007;29:471---83.
20.HuynhHQ,CouperRT,TranCD,Moore L,KelsoR,ButlerRN. N-acetylcysteine, a novel treatment for Helicobacter pylori infection.DigDisSci.2004;49:1853---61.
