Antibacterial Activities and Possible Modes of Action of Acacia nilotica (L.)

16 Pages·2017·4.89 MB·English

molecules
Article
Antibacterial Activities and Possible Modes of
Action of Acacia nilotica (L.) Del. against
Multidrug-Resistant Escherichia coli and Salmonella
MuhammadBilalSadiq1,JoelTarning2,3,TayZarAyeCho1andAnilKumarAnal1,*
1 FoodEngineeringandBioprocessTechnology,AsianInstituteofTechnology,Bangkok12120,Thailand;
[email protected](M.B.S.);[email protected](T.Z.A.C.)
2 Mahidol-OxfordTropicalMedicineResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,
Bangkok10400,Thailand;[email protected]
3 CentreforTropicalMedicineandGlobalHealth,NuffieldDepartmentofClinicalMedicine,
UniversityofOxford,OxfordOX37FZ,UK
* Correspondence:[email protected]@gmail.com;Tel.:[phone];Fax:[phone]
AcademicEditor:PeterJ.Rutledge
Received:18November2016;Accepted:21December2016;Published:14January2017
Abstract: Medicinal plants are frequently used for the treatment of various infectious diseases.
The objective of this study was to evaluate the antibacterial activity and mode of action of
AcacianiloticaandtheantibiogrampatternsoffoodborneandclinicalstrainsofEscherichiacoliand
Salmonella. ThemechanismofactionofacaciaextractsagainstE.coliandSalmonellawaselucidated
byobservingmorphologicaldamagesincludingcellintegrityandcellmembranepermeability,as
wellaschangesincellstructuresandgrowthpatternsinkill-timeexperiments. Theclinicalisolates
of E. coli and Salmonella were found resistant to more of the tested antibiotics, compared to food
isolates. Minimum inhibitory concentration and minimum bactericidal concentration of acacia
leafextractswereintherangesof1.56–3.12mg/mLand3.12–6.25mg/mL,respectively,whereas
podsandbarkextractsshowedsomewhathighervaluesof3.12–6.25mg/mLand6.25–12.5mg/mL,
respectively,againstalltestedpathogens.Thereleaseofelectrolytesandessentialcellularconstituents
(proteinsandnucleicacids)indicatedthatacaciaextractsdamagedthecellularmembraneofthe
pathogens. Thesechangescorrespondedtosimultaneousreductioninthegrowthofviablebacteria.
ThisstudyindicatesthatA.niloticacanbeapotentialsourceofnewantimicrobials,effectiveagainst
antibiotic-resistantstrainsofpathogens.
Keywords:antibacterialactivity;antibiogram;kill-timeanalysis;SEM;bacterialmembranepermeability
1. Introduction
Thoughantibioticsprovidethemainbasisfortreatmentofbacterialinfections,emergingbacterial
resistance to commonly used antibiotics is a global concern. Repeated exposure and overuse
of antibiotics have led to an increasing rate of antibacterial resistance and the development of
multidrug-resistantstrainsofmicroorganisms[1]. Antimicrobialresistancemonitoringisessential
forprovidinginformationonthemagnitudeandtrendsofmicrobialresistanceinordertoplanand
monitortheeffectoftargetedinterventions[2].
SalmonellaandEscherichiacoliareincludedinthemainfood-bornepathogensresponsibleforfood
poisoningandsubsequententericinfections[3,4]. InSouthEastAsia,thereisanabsenceofofficial
Salmonellasurveillance,butitisestimatedthatupto22.8millioncasesofsalmonellosisoccurannually
with37,600deaths[5]. SalmonellaandE.coliisolatedfrompoultrymeatwerefoundresistanttovarious
commerciallyavailableantibiotics[6,7]. Foodcontaminatedwithdrug-resistantbacteriaisamajor
threattopublichealth[8].
Molecules2017,22,47;doi:10.3390/molecules22010047 www.mdpi.com/journal/molecules
Molecules2017,22,47 2of16
Thus, there is an urgent need of new antimicrobial agents to combat the rapid emergence of
bacterialresistance. However,therapidwidespreadresistancedevelopmentindicatesthatevennew
antimicrobialagentswillhavearelativelyshorttherapeuticlifespan[9]. Anunderappreciatedsource
of novel antimicrobial agents with potentially new mechanism of actions might be natural plant
sourcesandherbalproducts[10]. Plantscontainsecondarymetabolitesinadditiontomineralsand
primarymetabolitesthatareresponsibleforantioxidantandantibacterialeffects[11].
Theuseofmedicinalplantsasanalternativetherapyagainstvariousinfectiousdiseasesisage-long
practice. Thismightbebestexemplifiedinthefieldofantimalarialtherapy,wheretherecommended
first-line active drug compound (i.e., artemisinins) was originally derived from Artemisia annua.
Acacianilotica(L.)Del. isamedicinalplantbelongingtothefamilyMimosaceae. Theplantiswidely
distributedintropicalandsubtropicalregions. Ayurvedicmedicinepracticessuggesttheuseofleaves,
bark and pods of A. nilotica against cancer, cough, diarrhea, fever, small pox, piles and menstrual
problems[12]. Theplantisreportedtohaveantibacterialeffectsagainstpathogenicmicroorganisms
suchasMycobacteriumtuberculosis,Pseudomonasaeruginosa,EscherichiacoliandStaphylococcusaureus[13].
A.niloticashowedhighantimicrobialpotentialagainstStaphylococcusaureus,E.coliandSalmonellatyphi
inacomparativeantimicrobialstudyamongacaciaspecies[14]. Ethanolandpetroleumetherextracts
ofA.niloticadisplayedantibacterialeffectsagainstS.aureus,E.coli,Proteusvulgaris,Proteusmirabilis,
Salmonella paratyphi and Klebsiella pneumoniae [15]. A number of researchers have investigated the
antimicrobialactivityofA.nilotica. However,themechanismofactionhasnotbeenstudiedindetail.
Thus,theobjectiveofthisstudywastoevaluatetheantimicrobialeffectsandpossibleantimicrobial
modesofactionofA.niloticaleaves,podsandbarkextractsagainstmultidrug-resistantstrainsofE.coli
andSalmonella.
2. ResultsandDiscussion
2.1. AntibiogramofSalmonellaandE.coli
The prevalence of antibacterial drug resistance is high among foodborne pathogens due to
extensive therapeutic and prophylactic use of antibiotics in animal farming. In South East Asia,
multidrug-resistant strains of E. coli and Salmonella are frequently isolated from food sources.
Thetreatmentofinfectionscausedbymultidrug-resistantbacteriaisbecominganincreasinglymore
difficulttaskandisofgreatfoodandpatientsafetyconcern[16,17].
Antibiogrampatternsofbacterialisolatesweredeterminedbythediscdiffusionassay,following
the guidelines of Clinical and Laboratory Standard Institute (CLSI). The results were interpreted
bymeasuringthediameterofinhibitionzone. TheantibacterialsusceptibilitytestingofE.coliand
Salmonella spp. obtained from clinical and food sources demonstrated that both clinical and food
sourceisolatesofE.coliandSalmonellawereresistanttovariousantibioticsasshowninTable1. E.coli
(E1)obtainedfromhumanclinicalisolateswasfoundtoberesistanttoampicillin(10µg),amoxicillin
(10µg), chloramphenicol(30µg), tetracycline(30µg), ciprofloxacin(5µg)andceftriaxone(30µg),
whereasclinicalisolateofSalmonellatyphimurium(S1)wasfoundtoberesistanttoampicillin(10µg),
amoxicillin (10 µg), tetracycline (30 µg) and streptomycin (10 µg). E. coli and Salmonella enterica
(E2andS2, respectively) isolated from poultry meat were found to be susceptible to all the tested
antibiotics,whereasE.coli(E3),isolatedfrombeefmeatwasresistanttoampicillin(10µg),amoxicillin
(10µg),chloramphenicol(30µg)andtetracycline(30µg);Salmonellatyphimurium(S3),isolatedfrom
poultrymeatwasresistanttotetracycline(30µg),andchloramphenicol(30µg);Salmonellaenteritidis
(S4),isolatedfrombeefmeatwasresistanttoampicillin(10µg),amoxicillin(10µg)andtetracycline
(30µg).
TheantibiogramoftestedpathogensdemonstratedthattheclinicalisolatesofE.coli(E1)and
Salmonella (S1) were highly resistant, which might be due to previous exposure of pathogens to
antibiotics and their ability to develop resistance upon repeated exposure in humans. Moreover,
both strains were found to be resistant to beta-lactam antibiotics including ampicillin (10 µg) and
Molecules2017,22,47 3of16
amoxicillin(10µg)indicatingtheabilitytoproducebeta-lactamase. Theseresultssupportaprevious
report,suggestingthatE.coliisolatedfromvariousmeatsampleswerefoundresistanttobeta-lactams
Molecules 2017, 22, 47 3 of 14
andtetracyclineantibiotics[18].
Table 1. Antibiogram of E. coli and Salmonella isolates.
Table1.AntibiogramofE.coliandSalmonellaisolates.
Bacteria Resistance Susceptible
BacterEia1 Amp,R Aesmisl,t aCnhcle, Gen, Tet, Cip, Ctx AmkS, Sutsrc, eSpxtti ble
E2 Amp, Aml, Chl, Gen, Amk, Str, Tet, Sxt, Cip, Ctx
E1 Amp,Aml,Chl,Gen,Tet,Cip,Ctx Amk,Str,Sxt
E3 Amp, Aml, Chl, Tet Gen, Amk, Str, Sxt, Cip, Ctx
E2 Amp,Aml,Chl,Gen,Amk,Str,Tet,Sxt,Cip,Ctx
E3 S1 Amp,A Ammpl,, AStmr,l T,Ceth l,Tet Chl, GGeenn,, AAmmkk,, SStxrt,, SCxitp,,C Ciptx, Ctx
S1 S2 Amp,Aml,Str,Tet AmpC, Ahlm,Gl,e Cnh,Al, mGekn,,S Axtm,Cki,p S,tCr,t Txet, Sxt, Cip, Ctx
S2 S3 Tet, Chl AmpA, Ammp,l,A Gmenl,, CAhml,kG, eSntr,,A Smxtk, ,CSitpr,, TCettx, Sxt,Cip,Ctx
S3 S4 Amp,T Aetm,Cl,h Tlet Gen, AAmmpk,, AStmr,l C,Gipe,n C,Atxm, Ckh,lS,t Sr,xSt xt,Cip,Ctx
S4 Amp,Aml,Tet Gen,Amk,Str,Cip,Ctx,Chl,Sxt
Amp = Ampicillin, Aml = Amoxicillin, Chl = Chloramphenicol, Gen = Gentamycin, Tet = Tetracycline,
ACmipp == CAimprpoicfliollxina,cAinm, lA=mAkm =o xAicmilliink,aCcihnl, =SCtrh =lo rSatmrepphtoenmicyocli,nG, eSnx=t G= eTnrtiammeytchino,pTreint/=SuTelftaramcyecthlinoex,aCzoiple=.
Ciprofloxacin,Amk=Amikacin,Str=Streptomycin,Sxt=Trimethoprin/Sulfamethoxazole.E1=E.coliand
E1 = E. coli and S1 = Salmonella typhimurium were isolated from clinical samples, E2 = E. coli,
S1=Salmonellatyphimuriumwereisolatedfromclinicalsamples,E2=E.coli,S2=SalmonellaentericaandS3=
SSa2l m= oSnaellmlaotnyeplhlai menutreiruimca wanedre Sis3o =la Steadlmforonmellap toyuplhtrimyumreiuamt, awnedreE 3is=olEat.ecdol ifraonmd Sp4ou=ltSrayl mmoenaeltl,a aenndte Eri3ti d=i sEw. ceorlei
iasnolda tSe4d =fr oSmalmbeoenfelmlae eanttsearmitipdliess .were isolated from beef meat samples.
2.22..2D. Deteectetciotinono foBf Beteat-aL-Lacatcatmams-s-a annddT Teetrtraaccyycclilinnee--RReessiissttaanntt GGeenneess
ThTehpe rperseesnecneceo foaf natnitbibioiotitcicr eressisisttaannccee ggeenneess eennccooddiinngg rreessiissttaannccee ttoo tthhee bbeetata-l-alactcatmam anadn dtettertarcayccylicnlein e
clacslassosf oafn atnibtiiboitoictiscsw wasasi ninvveesstitgigaateteddu ussiinngg ppoollyymmeerraassee cchhaaiinn rreeaaccttiioonn ((PPCCRR) )wwitihth ggenenomomici cDDNNAA, a,nadn d
amapmlpifiliefdiedp rpordoudcutcstsw wereerer erseosolvlvededb byyg geelle eleleccttrroopphhoorreessiiss ((FFiigguurree 11))..

FigFiugruere1 1.. E1E =1 E.= coEli .ancdol iS1a =n SdalmS1on=ellaS taylpmhoimneulrlaiumty wpheirmeu isroiulamtedw ferorem icsloinlaictaeld safmropmles,c Sli3n i=c Salalmsaomnepllale s,
S3ty=pShaimlmuorniuemlla wtyapsh iismoluartieudm frwomas pisooulalttreyd mfreoamt, apnodu lEtr3y =m Ee. acot,lia annddE S34= = ES.alcmoloinaenllda Sen4te=riStiadlims owneerllea iesnotleartietdid is
wefrroemis oblaeetef dmfreoamt sbameepflems.e aCtMsa mrepplreess.enCtsM breetap-rleascetanmts-rbeseitsat-alnatc tagmen-er easinsdta nTtAg eanned anTdB TrAepraensdenTt B
reptertersaecynctltienter-arceysicsltiannet- rgeesnisetsa. ntgenes.
E. coli (E1) obtained from human clinical isolates showed the presence of bla CMY and tet (A) genes,
whereas clinical isolate of Salmonella typhimurium (S1) showed the presence of bla CMY and tet (B). E. coli
(E3) isolated from beef meat showed the presence of bla CMY and tet (A); Salmonella typhimurium (S3)
Molecules2017,22,47 4of16
E.coli(E1)obtainedfromhumanclinicalisolatesshowedthepresenceofbla andtet(A)genes,
CMY
whereasclinicalisolateofSalmonellatyphimurium(S1)showedthepresenceofbla andtet(B).E.coli
CMY
(E3)isolatedfrombeefmeatshowedthepresenceofbla andtet(A);Salmonellatyphimurium(S3)
CMY
isolatedfrompoultrymeatandSalmonellaenteritidis(S4)isolatedfrombeefmeatshowedthepresence
oftet(B)andtet(A),respectively. E.coliandSalmonellaenterica(E2andS2,respectively)isolatedfrom
poultrymeatwerefoundnegativeforallthetestedresistantgenesandthisfindingwasinaccordance
withtheantibioticsusceptibilityresults.
2.3. AntimicrobialActivityofAcaciaExtracts
The antimicrobial activities of acacia extracts were investigated by the disc diffusion assay at
differentconcentrations(5and10mg/disc). Theresultswerereadafter24hofincubationat37◦C
bymeasuringthediameterofinhibitionzone(Table2). Allselectedpartsofacaciawerefoundtobe
effectiveagainsttheselectedpathogens. Thelowesttestedconcentration(5mg/disc)ofallextractsof
acaciainhibitedthegrowthofbothclinicalandfoodisolatesofE.coliandSalmonella. Similarresults
were previously reported by Kavitha et al. [19], who studied the antibacterial effects of A. nilotica
againstvariousclinicalbacterialisolates. Theleafextractsataconcentrationof10mg/discshowedthe
maximummeandiameterzoneofinhibitionof21.11±1.05mmand16.83±0.94mmagainstE.coli
andSalmonellastrains,respectively. Theleaveswerefoundmoreeffectiveininhibitingbacterialgrowth
ascomparedtopodsandbarkextracts. Theresultsdemonstratedthatallselectedpathogenswere
susceptibletoalltestedpartsoftheplant. ThisindicatedastrongantibacterialpotentialofA.nilotica
against the antibiotic-resistant pathogens tested here, arguably with a novel mechanism of action
than other tested antibiotics. The antibacterial results of the current study are in accordance with
literature,whereextractsofA.niloticawereeffectiveagainstclinicalbacterialisolatesofE.coliand
Salmonella[20,21].
2.4. MinimumInhibitoryConcentrationandMinimumBactericideConcentrationofAcacianiloticaExtracts
Minimuminhibitoryconcentration(MIC)valuesofacaciaextractsweredeterminedbybroth
macro-dilution method and minimum bactericide concentration (MBC) values were estimated by
sub-culturingallconcentrations(≥MIC)thathadnodetectablegrowth. MICandMBCofacacialeaves
wereintherangeof1.56–3.12mg/mLand3.12–6.25mg/mL,respectively,againstalltestedbacterial
strains(Table3). MICvaluesofleaveswerenotsignificantly(p>0.01)differentfrompodsandbark
extractsbutMBCvaluesofleavesweresignificantly(p<0.01)differentcomparedtopodsandbark.
TheMICvaluesofacacialeavesandbarkextractsagainstmultidrug-resistantE.coliwerelowerin
thecurrentstudythanpreviouslyreportedforE.coli, causingotitisinfection[22]. Theleavesand
barkextractsinthecurrentstudyalsoshowedlowerMICvalueagainstSalmonellatyphimuriumthan
previouslyreportedforSalmonellatyphi[21]. Thereporteddifferencesmightbeduetodifferentstrains
ofE.coliandSalmonella,and/ordifferentexperimentalprotocols.
Theantibacterialactivityofethanolextractsofleaves,podsandbarkofA.niloticaagainstdrug
sensitiveandmultidrug-resistantE.coliandSalmonellaspp. obtainedfromclinicalandfoodsources
wereexamined. AllpartsofplantwerefoundtobeeffectiveevenagainsttheclinicalisolatesofE.coli
andSalmonellatyphimuriumthatwereresistanttovariouscommerciallyavailableantibiotics. Therefore,
acaciacanbeanalternateapproachtotreatresistantpathogenseitherintheformofitspurifiedextract
orincombinationwithcommerciallyavailableantibiotics. Antibacterialactivitydeterminedbythe
disc diffusion method has certain limitations since this assay indicates only growth inhibition of
bacteriawithoutanyevidencethatthetestedextractsareeitherbacteriostaticorbactericidal. Therefore,
MIC and MBC values of extracts were determined to specify the dose and nature of the activity.
However, more research is needed to validate these findings in animal models and eventually in
humanclinicaltrials.
Molecules2017,22,47 5of16
Table2.AntibacterialeffectsofAcacianiloticaextractsagainstclinicalandfoodisolatesofE.coliandSalmonella.
Conc. ZoneofInhibition(mm)
Sample
(mg/disc) E1 E2 E3 MeanofE.coliStrains S1 S2 S3 S4 MeanofSalmonellaStrains
5 17.0±1.0 16.7±1.15 16.7±0.58 16.78±0.83a 12.3±0.58 11.3±1.53 11.3±2.08 11.7±1.15 11.67±1.30a
Leaves 10 21.7±0.6 21.0±1.0 20.7±1.53 21.11±1.05b 17.7±0.58 16.7±0.58 16.0±1.0 17.0±1.0 16.83±0.94b
5 9.3±0.58 8.3±0.58 7.7±0.58 8.44±0.88 8.3±0.58 8.7±1.15 7.7±1.15 8.3±0.58 8.25±0.86
Pods 10 19.0±1.0 18.3±0.58 15.3±0.58 17.56±1.81b 15.7±1.53 13.7±0.58 14.7±1.15 15.0±1.0 14.75±1.21b
5 8.7±0.58 9.0±1.0 8.3±0.58 8.67±0.71 5.3±4.61 8.7±0.58 8.7±1.15 8.0±1.0 7.67±2.53
Bark 10 13.0±1.0 12.3±0.58 11.3±1.53 12.22±1.20b 10.3±1.15 11.3±1.15 11.7±0.58 11.0±2.0 11.08±1.24b
Amikacin(Control) 30µg 22.4±1.51 23.8±1.73 22.5±1.1 22.8±1.43 23.4±2.14 23.5±1.53 22.0±1.0 23.0±1.0 22.97±1.24
Theresultswereexpressedasmean±S.Doftriplicates.Superscript“a”representsmeansthatarestatisticallydifferent(p<0.01)comparedtoallotherextractsatconcentrationof
5mg/discagainstE.coliandSalmonellastrains.Superscript“b”representsmeansthatarestatisticallydifferent(p<0.01)betweenallextractsatconcentrationof10mg/discagainst
E.coliandSalmonellastrains.E1=E.coliandS1=Salmonellatyphimuriumwereisolatedfromclinicalsamples,E2=E.coli,S2=SalmonellaentericaandS3=Salmonellatyphimuriumwere
isolatedfrompoultrymeat,andE3=E.coliandS4=Salmonellaenteritidiswereisolatedfrombeefmeatsamples.
Table3.Minimuminhibitoryconcentration(MIC)andminimumbactericidalconcentration(MBC)ofA.niloticaextracts.
MinimumInhibitoryConcentration MinimumBactericideConcentration
MicrobialStrains Leaves Pods Bark Leaves Pods Bark
(mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)
E1 3.12 3.12 6.25 6.25 12.5 12.5
E2 3.12 6.25 6.25 3.12 12.5 12.5
E3 3.12 3.12 6.25 6.25 12.5 12.5
S1 1.56 3.12 3.12 3.12 6.25 6.25
S2 1.56 3.12 3.12 3.12 6.25 6.25
S3 1.56 3.12 3.12 3.12 6.25 12.5
S4 1.56 3.12 3.12 3.12 6.25 6.25
Theresultswereexpressedasmeanoftriplicates. E1=E.coliandS1=Salmonellatyphimuriumwereisolatedfromclinicalsamples,E2=E.coli,S2=SalmonellaentericaandS3=
Salmonellatyphimuriumwereisolatedfrompoultrymeat,andE3=E.coliandS4=Salmonellaenteritidiswereisolatedfrombeefmeatsamples.
Molecules2017,22,47 6of16
Molecules 2017, 22, 47 6 of 14
22..55.. KKiillll--TTiimmee AAnnaallyyssiiss
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stlhoewreerw garsoawstlho wase rcogmropwatrheda stoc otmhep caorendtrtool tthheatc oclnetarrollyt hinadticclaetaerdly thinadt iMcaItCed otfh laetaMveIsC, poofdlesa vanesd, pboardks
iannhdibbita trhkei nghroibwitthth oef gbraocwtetrhiao. Tfhbea clteearviae.s Tanhde lbeaarvke esxatnradctbsa artk ae cxotrnaccetnstarattaiocno nocf e2n ×t rMatIiCon koilfle2d× alMl thICe
bkailclteedriaalll ctehlelsb bayc ttehreia elncdel losf b2y4 thh.e endof24h.

FFiigguurree 22.. EEffffeeccttss ooff aaccaacciiaa lleeaavveess,, ppooddss aanndd bbaarrkk eexxttrraaccttss oonn tthhee vviiaabbiilliittyy ooff tteesstteedd EE.. ccoollii ((aa––cc)) aanndd
SSaallmmoonneellllaa ttyypphhiimmuurriiuumm ((dd––ff))..
The effects of leaves, pods and bark extracts on the viability of Salmonella typhimurium are shown in
Theeffectsofleaves,podsandbarkextractsontheviabilityofSalmonellatyphimuriumareshown
Figure 2. The MIC of all extracts inhibited the growth of bacteria over a period of 24 h as compared to
inFigure2. TheMICofallextractsinhibitedthegrowthofbacteriaoveraperiodof24hascompared
control, whereas the 2 × MIC showed a declined viability of test bacteria from 6.75 to 3.47 Log CFU/mL
to control, whereasthe 2 × MIC showeda declined viabilityof test bacteria from6.75 to 3.47Log
for leaves, 6.75 to 4.54 Log CFU/mL for pods and 6.65 to 3.70 Log CFU/mL for acacia bark.
CFU/mLforleaves,6.75to4.54LogCFU/mLforpodsand6.65to3.70LogCFU/mLforacaciabark.
The extracts of acacia showed variable kinetics against tested pathogens. The concentration-
The extracts of acacia showed variable kinetics against tested pathogens. The concentration-
dependent killing showed bacteriostatic and bactericidal effects of extracts. Test compounds were
dependentkillingshowedbacteriostaticandbactericidaleffectsofextracts. Testcompoundswere
considered bacteriostatic at the lowest concentration that reduced the original inoculum size by 0–3
consideredbacteriostaticatthelowestconcentrationthatreducedtheoriginalinoculumsizeby0–3Log
Log CFU/mL and bactericidal if inoculum size was reduced by >3 Log CFU/mL [23]. Complete
CFU/mLandbactericidalifinoculumsizewasreducedby>3LogCFU/mL[23].Completeelimination
elimination of E. coli was observed after 24 h of treatment with leaves and bark extracts at concentration
ofE.coliwasobservedafter24hoftreatmentwithleavesandbarkextractsatconcentrationof2×MIC.
of 2 × MIC. This supported previous research that reported 99.9% reduction in the growth of Salmonella
Molecules2017,22,47 7of16
Molecules 2017, 22, 47 7 of 14
Thissupportedpreviousresearchthatreported99.9%reductioninthegrowthofSalmonellatyphiinan
Aty.pnhiil oitni caant iAm. en-ikloiltlicaas stiamye[-2k4i]l.l Tahsseapyo [d24s]w. Tehree apboldest owdeercer aebaslee ttoh edeincorecausleu mthes iizneofcruolmum6. 6si2zteo fr3o.4m7 L6.o6g2
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tthhaatt lleeaavveess, ,ppodosd sanadn bdarbka rekxtreaxcttrsa hctasveh aavne efafencte offne cthteo mnetmhebrmaneem pberramneeapbielirtmy eoaf bEi.l ictoyli oanfdE .Saclomlionaenldla
Styaplmhiomnuelrliaumty.p Ahilml pulraiunmt e.xtArallctpsl arenstueltxetdra icnt sinrcerseualsteedd rienlainticvree aesleecdtrricealal tcivonedeulecctitvriictyal ocfo bnadcutecrtiiavli tcyelolsf,
bwahcitcehri ianldcieclalste,dw ah liecahkaingde iocfa itnedtraacelellauklaarg iengorfeidniternatcse ellsupleacriainllgyr eeldeicetnrotslyetesps efrcoiamll ytheel ceecltlrso. lTyhtees lefarovmes
tohfe accealclsia. iTnhdeucleeadv emsaoxfimacuamci arienladtuivcee dpmermaxeimabuilmityr eolfa ti6v7e.2p5%er m±e a0b.8i2li%ty aonfd6 77.245.1%9%± ±0 .802.7%2%a nadt
7c4o.n1c9e%nt±rat0i.o7n2 %of a2t ×c oMnIcCen fotrra Eti.o cnoloi fan2d× SMalmIConfoelrlaE t.ycpohliimanudriuSmal,m reosnpelelcattivypehlyim, tuhraitu wm,arse mspoercet itvhealny ,ptohdast
wanads mbaorrke ethxatrnacptosd assa snhdowbanr kine xFtirgauctrse a3s. sMhoawinntaiinniFnigg uioren3 h.oMmaeinotsatainsiins gisi oinntehgormale otos ttahseis misaiinntteegnraanltcoe
tohfe enmearignyt esntaatnucse, soofluentee trrgaynssptaotruts, ,msoetlaubteoltirca rnesgpuolratt,iomne, tcaobnotlriocl roefg tuulragtoiorn p,rceosnsutrroel aonfdt umrgotoirlitpyr eosfs cuerlel,
athnedremfoorteil iaty sloifgchetl lc,htahnergeef oorfe tahes lsigtrhutccthuarnalg ienotefgthrietys toruf cthtuer acelliln mtegemritbyraonfeth ceacne lalfmfecetm mbreatanbeoclaisnma fafencdt
mleaedta btoo lcieslml daenadthl e[a2d5]t. ocelldeath[25].

Figure 3. Effects of acacia leaves, pods and bark extracts on the impermeability of cell membrane of
Figure3.Effectsofacacialeaves,podsandbarkextractsontheimpermeabilityofcellmembraneof
tested E. coli (a–c) and Salmonella typhimurium (d–f).
testedE.coli(a–c)andSalmonellatyphimurium(d–f).
The results in this study showed that bacterial cell membrane permeability changed with increasing
The results in this study showed that bacterial cell membrane permeability changed with
concentrations of extracts and incubation time period, which caused leakage of intracellular electrolytes.
increasingconcentrationsofextractsandincubationtimeperiod,whichcausedleakageofintracellular
Similar results were reported by Zhao et al. [26], showing an increase in electric conductivity of bacterial
electrolytes. Similar results were reported by Zhao et al. [26], showing an increase in electric
cells with increasing concentrations of sugarcane bagasse extracts. There was a modest change in
relative electrical conductivity of the control incubation during the first four hours. A rise in relative
Molecules2017,22,47 8of16
conductivityofbacterialcellswithincreasingconcentrationsofsugarcanebagasseextracts. Therewas
amodestchangeinrelativeelectricalconductivityofthecontrolincubationduringthefirstfourhours.
Ariseinrelativeelectricalconductivityafterfourhourswasfoundduetonormallysisandbacterial
deathresultinginincreasedrelativeelectricalconductivity.
2.7. IntegrityofBacterialCellMembrane
Theintegrityofthecellmembranewasdeterminedbytreatingthebacterialcellswithdifferent
concentrations of plant extracts (control, 1 × MIC and 2 × MIC) and measuring the released cell
constituents, including proteins and absorbance at 260 nm of the supernatant of tested bacteria.
TheeffectsofdifferentconcentrationsofeachextractontestedbacteriaareshowninTable4.Theresults
indicatedthattherewassignificantincrease(p<0.01)inthereleaseofcellularconstituentsandprotein
concentrationwithincreasingconcentrationsofacaciaextracts. Theseresultscorroborateprevious
reports,indicatingthatirreversibledamagemightoccurtobacterialmembranesaftertreatmentwith
plants,whichcouldleadtolossofessentialcellularcomponentssuchasproteinsandnucleicacids[27].
Theleavesshowedhigherproteinandnucleicacidreleasedcontentsascomparedtopodsandbarkat
concentrationsof1×MICand2×MIC.Theresultsindicatedthattestedextractscauseirreversible
damagetothecellmembrane,whichledtolossofcellularconstituentsandfinallytocelldeath.
Table4.Effectsofacacialeaves,podsandbarkextractsoncellconstituents’releaseoftestedE.coliand
Salmonellatyphimurium.
CellConstituentsRelease
E.coli Salmonellatyphimurium
Sample Conc.
Protein CellConstituents Protein CellConstituents
(µg/mL) (OD260nm) (µg/mL) (OD260nm)
2×MIC 62.70±4.20 0.42±0.007 48.26±5.25 0.4±0.006
Acacialeaves 1×MIC 32.33±4.00 0.34±0.007 25.67±4.84 0.31±0.001
Control 8.63±2.31 0.086±0.013 10.11±2.22 0.09±0.004
2×MIC 30.85±2.80 0.31±0.029 18.63±2.31 0.27±0.01
Acaciapods 1×MIC 17.52±2.31 0.23±0.036 12.33±2.22 0.164±0.025
Control 6.78±2.94 0.102±0.006 5.67±2.94 0.1±0.017
2×MIC 39.74±4.62 0.30±0.013 31.96±3.90 0.29±0.006
Acaciabark 1×MIC 22.33±2.94 0.18±0.016 18.67±1.64 0.14±0.009
Control 5.67±2.22 0.088±0.011 7.18±1.65 0.1±0.01
Theresultswereexpressedasmean±S.D.oftriplicates.MIC=minimuminhibitoryconcentration.
Themacromoleculesofmicrobialcells,includingnucleicacidsandproteins,whichconstitutekey
structuralcomponents,werereleasedfromthetestedbacteriaaftertreatmentwithleaves,podsand
barkextractsofacacia. Themeasurementsofspecificleakagemarkersaftertreatmentwithextracts,
includingnucleicacidsandprotein,isanindicatorofbacterialcellmembraneintegrityincomparison
tounexposedcells[28]. Theresultsfromthisstudyindicatedrapidlossofproteinsandnucleicacids
fromtreatedpathogens,duetoirreversibledamagetothecytoplasmicmembranes. A.niloticaisrich
inphenoliccompounds[29]andphenolicacidscancauseirreversiblechangestocellmembrane[30].
Thus integrity of cell membrane was concluded as an important factor to inhibit the pathogenic
bacterialgrowth. Furtherresearchisneededtofindthetargetdamagesitesonbacteriacellstomake
surethateitherantimicrobialeffectwasfromdamagetolipopolysaccharideormembraneproteins
incellwall.
2.8. ScanningElectronMicroscopeObservations
Toinvestigatetheantibacterialmodeofaction,itisessentialtoevaluatechangesinbacterialcell
membranepermeability,integrity,morphologyandsurfacecharacteristics[27]. Thephysiologicaland
morphologicalchangesinE.coli(Figure4)andSalmonellatyphimurium(Figure5)wereobservedby
Molecules2017,22,47 9of16
scanningelectronmicroscope(SEM)aftertreatmentwithacaciaextracts. Resultsshowedadirectly
destructiveeffectofacaciaextractsontestedpathogens. Thetreatedbacterialcellsshowedobvious
Mmoolercpulheso 2l0o1g7,i c2a2,l 4c7h angesascomparedtountreatedcells. Mostoftreatedbacterialcellsbecamep9i totfe 1d4,
deformedandbroken. Theseobservationssupportedtheresultsofcellpermeabilityandintegrity
aaMssosslaeacyyu,,l eaas nn20dd1 7ii,n n2d2d,i i4cc7aa tteedd tthhaatt eexxttrraaccttss ooff AA.. nniilloottiiccaa hhaadd mmaajjoorr eeffffeeccttss oonn tthhee cceellll wwaallll aanndd ccyyttooppllaa9ss momf i1icc4
mmeemmbbrraannee ooff bbaacctteerriiaa..
assay, and indicated that extracts of A. nilotica had major effects on the cell wall and cytoplasmic
membrane of bacteria.

Figure 4. Scanning electron microscope imaging of untreated bacterial cells of (A) E. coli (clinical isolates);
Figure 4. Scanning electron microscope imaging of untreated bacterial cells of (A) E. coli
treated bacterial cells of E. coli (clinical isolate) with (B) leaves; (C) pods; (D) bark extracts of acacia.
(clinicalisolates);treatedbacterialcellsofE.coli(clinicalisolate)with(B)leaves;(C)pods;(D)bark
Figure 4. Scanning electron microscope imaging of untreated bacterial cells of (A) E. coli (clinical isolates);
extractsofacacia.
treated bacterial cells of E. coli (clinical isolate) with (B) leaves; (C) pods; (D) bark extracts of acacia.

Figure 5. Scanning electron microscope imaging of untreated bacterial cells of (A) Salmonella typhimurium

(clinical isolates); treated bacterial cells of Salmonella typhimurium (clinical isolate) with (B) leaves;
Figure 5. Scanning electron microscope imaging of untreated bacterial cells of (A) Salmonella typhimurium
(FCig) uproed5s.; (SDca)n bnairnkg eexletrcatrcotns omf iacrcoascciao.p eimagingofuntreatedbacterialcellsof(A)Salmonellatyphimurium
(clinical isolates); treated bacterial cells of Salmonella typhimurium (clinical isolate) with (B) leaves;
(clinicalisolates);treatedbacterialcellsofSalmonellatyphimurium(clinicalisolate)with(B)leaves;(C)pods;
(C) pods; (D) bark extracts of acacia.
3. Ma(Dte)rbiaalr kanexdtr Mactestohfoadcasc ia.
3. Material and Methods
3.1. Preparation of Plant Extracts and Microbial Sample Collection and Identification
3.1. PLreeapvareast,i opno dofs P alnandt bEaxrtkra ocft st haned A M. nicirlootbiicaal pSalamnpt l(ew Ciolldle) cwtioenre a ncodl Iledcentetdif icfraotimon L ahore, Pakistan. The
plant was authenticated and voucher specimen (Voucher number S6 HbGCS with Reference number
Leaves, pods and bark of the A. nilotica plant (wild) were collected from Lahore, Pakistan. The
7998) was deposited to Botany Society Government College of Science, Lahore, Pakistan. The selected
plant was authenticated and voucher specimen (Voucher number S6 HbGCS with Reference number
plant parts were washed thoroughly under running tap water to remove the surface dirt, followed
7998) was deposited to Botany Society Government College of Science, Lahore, Pakistan. The selected
by rinsing with sterilized distilled water. The plant samples were dried under shade in open air for
plant parts were washed thoroughly under running tap water to remove the surface dirt, followed
by rinsing with sterilized distilled water. The plant samples were dried under shade in open air for
Molecules2017,22,47 10of16
3. MaterialandMethods
3.1. PreparationofPlantExtractsandMicrobialSampleCollectionandIdentification
Leaves, pods and bark of the A. nilotica plant (wild) were collected from Lahore, Pakistan.
The plant was authenticated and voucher specimen (Voucher number S6 HbGCS with Reference
number 7998) was deposited to Botany Society Government College of Science, Lahore, Pakistan.
Theselectedplantpartswerewashedthoroughlyunderrunningtapwatertoremovethesurfacedirt,
followedbyrinsingwithsterilizeddistilledwater. Theplantsamplesweredriedundershadeinopen
airfor48h. Thedriedsamplesweregroundedbymeansofamechanicalgrinder(PhilipsCo. Ltd.,
Shanghai,China)andfinallyintofinelydividedpowderbypestleandmortar.
The extraction from these dried parts of A. nilotica was conducted following the method as
describedbyAdwanetal.[31],withslightmodifications. Powderedplantsamples(30g)wereplaced
in250mLofethanol(80%,v/v)inconicalflasksandplacedonshakingincubator(Gallenkamp,UK)at
200rpmfor48hat25◦C.Theextractswerefilteredandconcentratedbymeansofarotaryevaporator
(Büchi rotavapor R-144, Flawil, Switzerland) followed by lyophilization for 24 h in a freeze dryer
(Scanvac Cool Safe 55-4, Scanvac, Denmark). The freeze dried extracts were stored at 4 ◦C until
furtheruse.
E.coli,Salmonellaentericasubsp. enterica,SalmonellaenteritidisandSalmonellatyphimuriumwere
isolatedfrombeefandpoultrymeatsamplesandidentifiedbybiochemicalandimmunologicaltesting
intheBioprocessTechnologylaboratoryattheAsianInstituteofTechnology,Bangkok,Thailand. Other
human clinical isolates, namely E. coli and Salmonella typhimurium were acquired from the clinical
laboratoryofThammasatHospital,Pathumthani,Thailand.
3.2. AntibiogramofSalmonellaspp. andE.coli
Antibacterial susceptibility patterns of Salmonella spp. and E. coli isolates were determined
by using the disc diffusion assay, following the guidelines of CLSI, M100-S23 [32]. The following
commonly used antibiotics were tested: ampicillin (10 µg), amoxicillin (10 µg), chloramphenicol
(30 µg), gentamicin (10 µg), amikacin (30 µg), streptomycin (10 µg), tetracycline (30 µg),
trimethoprim–sulfamethoxazole(25µg),ciprofloxacin(5µg),andceftriaxone(30µg)(Oxoid,UK).
Thepre-incubated24hculturesofE.coliandSalmonellawereadjustedto0.5McFarlandstandardtoget
108CFU/mL.Thebacterialsuspensionswerespreadbyusingthesterilizedcottonswabonthesurface
ofMueller-HintonAgar(MHA)(Himedia,India)plates. Antibioticdiscsweredispensedbyusing
sterilizedforcepsonthesurfaceofagarmediumandgentlypressed. Theplateswerethenincubated
for24hat37◦Candresultsweremeasuredasdiameterofinhibitionzoneintriplicates.
3.3. DetectionofBeta-Lactam-andTetracycline-ResistantGenes
E.coliandSalmonellaisolatesweresub-culturedovernightinnutrientbrothandgenomicDNA
wasextractedbyusinggenomicDNApurificationkit(Insta-maxgenematrixBio-Rad,Hercules,CA,
USA)accordingtomanufacturer’sinstructions. Thepresenceofgenesassociatedwithresistanceto
tetracycline, tet (A), tet(B) and beta lactams (bla , bla ) were determined by PCR and set of
SHV CMY
primersusedforeachgenewereacquiredfromSigmaAldrich,Singapore(Table5). PCRreactions
wereperformedinatotalvolumeof25µLincluding,14µLPCRmastermix(TagPCRmastermixkit,
Bio-Rad,Hercules,CA,USA),9µLofDNA(50–200ng/µL)and1µLofeachreverseandforward
primer(10µM).PCRreactionswerecarriedoutbyusingDNAthermos-cycler(Bio-Rad)asfollows:
initialdenaturationfor30sat95◦Cfollowedby30cycles,for30sat95◦C,annealingfor30s(65.2,57.2,
61.1and60.3◦Cforbla ,bla ,tet(A)andtet(B),respectively)and1minat68◦C,followedby
CMY SHV
afinalextensionstepof5minat68◦C.Amplifiedsamplesweresubjectedtogelelectrophoresisby
usingQIAxceladvancesystem(QIAGENInc.,Valencia,CA,USA)andresultswereinterpretedby
QIAxcelscreenGel1.4.0.