olufunmisoolusolaolajuyigbeandanthonyjideafolayan · 2019. 7. 31. ·...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 769594 9 pageshttpdxdoiorg1011552013769594

Research ArticleEvaluation of Combination Effects of Ethanolic Extract ofZiziphus mucronata Willd subsp mucronata Willd andAntibiotics against Clinically Important Bacteria

Olufunmiso Olusola Olajuyigbe and Anthony Jide Afolayan

Department of Botany Phytomedicine Research Centre University of Fort Hare Alice 5700 South Africa

Correspondence should be addressed to Anthony Jide Afolayan aafolayanufhacza

Received 10 March 2013 Accepted 8 April 2013

Academic Editors H Chen A J Piantino Ferreira and G A Rocha

Copyright copy 2013 O O Olajuyigbe and A J Afolayan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A pragmatic approach to the treatment of infectious diseases withmulticausal agents and prevention of the development of resistantisolates is the combination of herbal remedies with the first-line antimicrobial agents to which most of them have become resistantThis study evaluated the interactions between the ethanolic bark extract of Ziziphus mucronata with known antimicrobial agentsin vitro In this study the results showed that varied zones of inhibitions (ZMEmdashchloramphenicol (17ndash42mm) ZMEmdashamoxicillin(17ndash35mm) ZMEmdashtetracycline (17ndash36mm) ZMEmdashciprofloxacin (20ndash41mm) ZMEmdashnalidixic acid (17ndash34mm) and ZMEmdashkanamycin (17ndash38mm)) were produced by the antibacterial combinations At the highest combined concentrations 12 isolates(ZMEmdashciprofloxacin) gt 10 isolates (ZMEmdashchloramphenicol) = (ZMEmdashkanamycin) gt 6 isolates (ZMEmdashamoxicillin) = (ZMEmdashnalidixic acid) and 5 isolates (ZMEmdashtetracycline) were inhibited with zones of inhibition greater than 20 plusmn 10mm Althoughthe agar diffusion assay suggested that the interactions between the ethanolic extract of Z mucronata and the antibiotics wereboth synergistic and additive in nature the fractional inhibitory concentration indices (FICI) showed that the interactions weresynergistic (5417) additive (2778) indifferent (1667) and antagonistic (139)While the fractional inhibitory concentrationindices (FICIs) for synergism ranged between 000391 and 05 that of additivity ranged between 0516 and 10 indifferences rangedbetween 1062 and 30 and antagonistic interactionwas 50The synergistic effects implied that the antibacterial combinations wouldbe more effective and useful in the treatment of multicausal and multidrug-resistant bacteria than a single monotherapy of eitherantibacterial agent

1 Introduction

Resistance of pathogens to antibiotics is an underappreciatedthreat to public health in nations around the globe [1] Itis a rapidly growing problem leading to an urgent needfor novel antimicrobial agents [2 3] While resistant bac-teria have become commonplace in healthcare institutionsinadequate empirical therapy resulting in increasedmortalityrate due to resistant Pseudomonas aeruginosa Staphylococcusaureus Klebsiella pneumoniae Escherichia coli Enterobacterspp and coagulase-negative staphylococci and enterococcihas been reported [4ndash6] With this increased incidence ofantimicrobial resistance and appearance of new infectiousagents many natural products have been investigated directly

for their antimicrobial activity and resistance modifyingability [7 8] While the natural products are known to playsignificant roles in the development of novel drugs andserved as leads for the treatment and prevention of diseases[9] plant-derived antimicrobials provide the much neededtherapeutics In phytomedicine research synergy assessmentbetweenmedicinal plants and commonly used antibiotics hasbecome a key area of interest because many diseases possessa multicausal agents and complex pathophysiology requiringtreatment with well-chosen drug combinations than with asingle-drug therapy

Subsequently a major strategy that could be employedin the treatment of new emerging infectious diseases and

2 The Scientific World Journal

prevention of the development of resistant isolates is the com-bination of herbal remedies with the first-line antimicrobialagents to which most of them have become resistant WhileKamatou et al [10] showed that combination of antimicrobialagents had expressed significant interactionsWilliamson [11]reported that two or more compounds interact to producemutual enhancement amplification or potentiation of eachotherrsquos effects when combined Although these combina-tions could enhance the efficacy of the other antimicrobialagents and acted as alternative to treating infections causedby multidrug-resistant microorganisms having no effectivetherapy [12 13] the pharmacological effects of such mix-tures could have resulted from the diverse mechanisms ofaction resulting from the drug-herbal interactions Hencewhile natural products from plants are considered interestingalternatives for treatment of microbial infections [14 15]preventing the global increase of undesirable side effects ofcertain antibiotics and the emergence of previously uncom-mon infections [16 17] become imminent with the use of newcompounds which are not based on the existing syntheticantimicrobial agents [18]

The genus Ziziphus belongs to the Rhamnaceae familyThe members of the taxon are drought tolerant and veryresistant to heat [19] Ziziphus mucronata Willd subspmucronata Willd known as buffalo thorn is a small-to-medium-sized tree with a spreading canopy It is distributedthroughout summer rainfall areas of sub-Saharan Africaextending from South Africa northwards to Ethiopia andArabia In ethnomedicine the pastes of the roots and leavesare used to treat boils swollen glands wounds and soreswhilesteambaths from the bark are used to purify and improve skincomplexion [20] Its bark and roots are used for the treatmentof rheumatism gastrointestinal complaints and snake bites[21] In East Africa the roots are used for treating snakebites gonorrhea diarrhea and dysentery [22] Decoctionsof roots and leaves are used to ooze boils and treat soresand glandular swellings [23] In South Africa ethnobotanicalsurvey indicated that this plant is used for gastrointestinaldisorders including dysentery and diarrhoea [24] Unlikesome members of the Ziziphus genus there is a dearth ofscientific reports to indicate the pharmacological activitiesof this plant Hence this study was aimed at evaluatingthe combination effects of the ethanolic bark extract of Zmucronata and some first-line antibiotics to which microbeshave shown resistance against bacteria pathogens that areimplicated in clinical infections in order to determine theirpotential drug-herbal interactions

2 Materials and Methods

21 Collection of Plant Material The bark materials of Zizi-phus mucronata subsp mucronata were collected in August2010 from the plant growing within the University ofFort Hare campus in Alice South Africa The plant wasauthenticated in the Department of Botany and a voucherspecimen (OLAJ2010ZM01) was prepared and deposited inthe Griffin Herbarium of the University

22 Extract Preparation The bark sample was air-dried atroom temperature and pulverized using a milling machineThe extract of the bark material was prepared in accordancewith the description of Basri and Fan [25] About 100 g of thepulverized sample was extracted with 500mL of ethanol for48 h with shaking (Stuart Scientific Orbital Shaker UK) Theextract was filtered through Whatman no 1 filter paper andconcentrated under reduced pressure at 40∘C using a rotaryevaporator (Laborota 4000 efficient Heidolph Germany)The crude extract collected was allowed to dry at roomtemperature to a constant weight of 142 g The extract wasredissolved in absolute ethanol and made up to the requiredconcentrations for bioassay analysis using sterile deionizeddistilled water The reconstituted extract solution was steril-ized by filtering through 045120583mmembrane filter and testedfor sterility after membrane filtration by introducing 2mL ofthe extract into sterile nutrient broth before being incubatedat 37∘C for 24 h A sterile extract was indicated by the absenceof turbidity in the broth after the incubation period

23 Bacterial Strain The bacteria used in this study includedseven types of culture strainsmdashBacillus cereus ATCC 10702Enterococcus faecalis ATCC 29212 Escherichia coli ATCC8739 Klebsiella pneumoniae ATCC 10031 Proteus vulgarisATCC 6830 Pseudomonas aeruginosa ATCC 19582 and Ser-ratiamercescencesATCC9986 three environmental strainsmdashAcinetobacter calcoaceticus anitratus UP Bacillus subtilisKZN and S flexneriKZN and two clinical strainsmdashS aureusOK2a and S aureus OK

2b These organisms were obtainedfrom the Department of Biochemistry and MicrobiologyUniversity of Fort Hare Alice South AfricaThe antibacterialassayswere carried out usingMueller-Hinton IIAgar (Biolab)and broth The inocula of the test bacteria were preparedusing the colony suspension method [26] Colonies pickedfrom 24 h-old cultures grown on nutrient agar were used tomake suspensions of the test organisms in saline solution togive an optical density of approximately 01 at 600 nm Thesuspension was then diluted 1 100 by transferring 01mL ofthe bacterial suspension to 99mL of sterile nutrient brothbefore being used

24 Antibiotics Used in This Study Antibiotic powders ofamoxicillin chloramphenicol ciprofloxacin tetracyclinehydrochloride kanamycin and nalidixic acid were usedStock antibiotic solutions were prepared and dilutions madeaccording to the CLSI (Clinical Laboratory StandardizationInstitute) method or the manufacturerrsquos recommendations[27 28]

25 Antibiotic Susceptibility Testing (Agar Diffusion Method)Each of the isolateswas standardized using colony suspensionmethod Each strainrsquos suspension was matched with 05McFarland standards to give a resultant concentration of 15times 106 cfumLThe antibacterial activity was determined usingthe well diffusion method according to the modified Kirby-Bauer diffusion technique [29] and the National Committeefor Clinical Laboratory Standard [30] by swabbing theMueller-Hinton agar (MHA) (Oxoid UK) plates with the

The Scientific World Journal 3

resultant saline suspension of each strain Wells were thenbored into the agar medium with heat sterilized 6mmcork borer The wells were filled with 100120583L of differentconcentrations prepared for the methanolic extract aloneantibiotics alone and their combinations taking care not toallow spillage of the solutions onto the surface of the agarTheplates were allowed to stand for at least 30min before beingincubated at 37∘C for 24 h [31] The determinations weredone in duplicate After 24 h of incubation the plates wereexamined for zones of inhibition [32] The diameter of thezones of inhibition produced by the extract alone antibioticalone and their combinationsweremeasured and interpretedusing the CLSI zone diameter interpretative standards [33]

26 Determination of Minimal Inhibitory Concentration(MIC) The minimum inhibitory concentrations (MICs) forthe extract and the antibiotics under study were determinedin duplicate by the macrobroth dilution method in Mueller-Hinton broth according to the standard methods of CLSI(Clinical Laboratory Standardization Institute) [27 28] Todetermine the MICs of each antibiotic 00019ndash500 120583gmLof each of the antibiotics and 0078ndash5mgmL of the extractwere prepared by two-fold serial dilutions in Mueller-Hintonbroth To determine their combinatorial effects combina-tions of different concentrations ranging from 025x MIC to8x MIC of each of the antibiotics and the extract were usedThe tubes were inoculated with 100120583L of each of the bacterialstrains Blank Mueller-Hinton broth was used as negativecontrol The bacteria-containing tubes were incubated at37∘C for 24 h Each combination assay was performed twotimes The MIC was taken as the lowest concentration ofthe extract and the antibiotics that showed no visible growthin the Mueller-Hinton broth after incubation at 37∘C for24 h [34 35]

27 Checkerboard Assay The antibacterial effects ofcombining ethanolic stem bark extract of Z mucronatawith antibiotics (chloramphenicol amoxicillin tetracyclineciprofloxacin nalidixic acid and kanamycin) were assessedusing a checkerboard method [36 37] The range of drugconcentration used in the checkerboard assay was such thatthe dilution range encompassed the MIC for each antibioticused in the analysis The fractional inhibitory concentration(FIC) was derived from the lowest concentrations of theextract and the antibiotics in combination permitting novisible growth of the test organisms in the Mueller-Hintonbroth after incubation at 37∘C for 24 h [38] FIC indices werecalculated using the formula FIC index = (MIC of extract incombinationMIC of extract alone) + (MIC of antibiotics incombinationMIC of antibiotics alone) In agreement withPetersen et al [37] G M Eliopoulos and C T Eliopoulos[39] Isenberg [40] and Prinsloo et al [41] synergy wasdefined as sum FIC le 05 additivity as 5 lt sum FIC le 1indifference as 1 lt sum FIC le 4 and antagonism as sum FIC gt 4

3 Results

In this study the bacterial isolates exhibited a varied degree ofsusceptibility to the extract alone antibiotics alone and their

combinations At the highest concentration (20mgmL) thezones of inhibition for the extract (ZME) ranged between17 and 27 plusmn 10mm At the highest concentration of eachof the antibiotics the zones of inhibition ranged between 15and 40 plusmn 10mm for chloramphenicol 18 and 42 plusmn 10mmfor amoxicillin 14 and 36 plusmn 10mm for tetracycline 17and 39 plusmn 10mm for ciprofloxacin 20 and 29 plusmn 10mmfor nalidixic acid and 23 and 36 plusmn 10mm for kanamycinThe agar diffusion assay showed that the isolates exhibitedvaried degree of concentration-dependent susceptibility toeach of the antibacterial combinations The antibacterialcombinations produced zones of inhibition ranging from17 to 42 plusmn 10mm for ZMEmdashchloramphenicol 17ndash35 plusmn10mm for ZMEmdashamoxicillin 17ndash36 plusmn 10mm for ZMEmdashtetracycline 20ndash41 plusmn 10mm for ZMEmdashciprofloxacin 17ndash34 plusmn 10mm for ZMEmdashnalidixic acid and 17ndash38 plusmn 1 0mmfor ZMEmdashkanamycin at their respective highest combinedconcentrationsThe inhibition zones from antibacterial com-binations were mostly prominent in size than those obtainedfrom either the extract or each of the respective antibioticsused alone With the exception of E coli ATCC 8739 and Bcereus ATCC 10702 being more susceptible to nalidixic acidalone S flexneri KZN showed the highest susceptibility tothe extract antibiotics and their combinations At the highestconcentrations combined 12 isolates (ZMEmdashciprofloxacin)gt10 isolates (ZMEmdashchloramphenicol) = (ZMEmdashkanamycin)gt 6 isolates (ZMEmdashamoxicillin) = (ZMEmdashnalidixic acid)and 5 isolates (ZMEmdashtetracycline) were inhibited by theantibacterial combinations with zones of inhibition greaterthan 20 plusmn 10mm (Tables 1(a)ndash1(c)) The agar diffusionassay therefore suggested that the interactions between theethanolic extract of Z mucronata and the antibiotics wereboth synergistic and additive in nature

Considering the susceptibility of the individualisolate to the extract and the respective antibiotics theirminimum inhibitory concentrations (MICs) ranged between0156mgmL and 0625mgmL for the extracts and between00048 120583gmL and 250120583gmL for all the antibioticsAccording to the MIC breakpoints of CLSI [33] thebacteria were classified as being susceptible intermediateand resistant based on their susceptibility to each testantibiotic Susceptibleintermediateresistant values for eachantibioticmdashchloramphenicol (le816ge32 120583gmL) amox-icillin (le816ge32 120583gmL) tetracycline (le48ge16 120583gmL)ciprofloxacin (le12ge4 120583gmL) nalidixic acid (le816ge32 120583gmL) and kanamycin (le1632ge64 120583gmL)mdashwereconsidered as the MIC breakpoints for the antibiotics bythe CLSI Though all the organisms were susceptible tochloramphenicol tetracycline and ciprofloxacin some ofthem were either resistant or intermediately susceptibleto amoxicillin nalidixic acid and kanamycin For theantibiotics the minimum inhibitory concentrations werein the ranges of 0977ndash1563120583gmL for chloramphenicol0977ndash250120583gmL for amoxicillin 00305ndash7813 120583gmL fortetracycline 00048ndash00781120583gmL for ciprofloxacin 1953ndash250120583gmL for nalidixic acid and 1953ndash125120583gmL forkanamycin (Table 2)The fractional inhibitory concentrationindices (FICI) showed that interactions between the


Table 1 Antibacterial effects of ethanolic extract of Z mucronata alone antibiotics alone and their combinations

(a)

Zones of inhibition (plusmn10mm) produced by the extract antibiotics and their combinations in vitroZME Chl ZME + Chl Amx ZME + Amx

5 10 20 625 125 250 5 + 626 10 + 125 20 + 250 625 125 250 5 + 625 10 + 125 20 + 250(mgmL) (120583gmL) (mgmL + 120583gmL) (120583gmL) (mgmL + 120583gmL)

A 17 18 20 23 27 30 20 25 28 20 23 26 0 14 17B 17 18 20 20 25 30 22 26 29 14 16 21 0 13 16C 17 18 20 23 27 30 23 25 27 16 19 21 0 13 16D 15 16 18 21 27 30 23 25 29 15 18 20 13 15 18E 14 15 17 22 25 29 22 26 28 30 32 34 28 31 34F 16 17 19 22 26 30 20 26 29 0 15 18 13 16 18G 16 17 18 22 27 31 19 22 25 13 16 18 0 12 16H 15 17 19 23 25 27 20 23 26 28 30 32 28 30 33I 14 15 18 0 0 15 13 15 17 30 34 37 28 31 34J 15 17 18 23 25 27 0 15 17 30 33 35 27 31 33K 15 16 18 20 24 27 23 25 28 27 31 35 25 28 30L 23 25 27 33 37 40 38 40 42 31 36 42 29 33 35

(b)

Zones of inhibition (plusmn10mm) produced by the extract antibiotics and their combinations in vitroZME Tet ZME + Tet Cip ZME + Cip


A 17 18 20 0 15 18 0 15 17 27 30 35 20 22 25B 17 18 20 0 0 14 0 15 16 18 21 24 18 20 22C 17 18 20 0 0 15 13 15 17 13 15 19 21 24 26D 15 16 18 0 13 16 0 14 16 19 20 22 18 22 25E 14 15 17 25 28 32 20 22 25 23 26 32 15 17 20F 16 17 19 0 0 15 13 15 17 13 15 17 20 23 27G 16 17 18 0 15 17 13 15 19 18 21 24 20 23 25H 15 17 19 25 27 30 22 25 27 17 20 23 26 29 31I 14 15 18 0 13 16 0 15 17 17 21 24 15 18 22J 15 17 18 25 27 30 15 18 20 13 15 17 16 19 23K 15 16 18 25 28 31 20 23 25 25 27 30 15 16 20L 23 25 27 29 32 36 30 33 36 30 35 39 33 37 41

(c)

Zones of inhibition produced by the extract antibiotics and their combinations in vitroZME Nal ZME + Nal Kan ZME + Kan


A 17 18 20 21 26 29 15 16 18 26 29 32 24 26 29B 17 18 20 21 25 29 13 15 17 24 25 28 23 24 28C 17 18 20 19 22 26 15 17 20 25 27 29 23 25 28D 15 16 18 20 23 25 13 15 17 24 27 30 24 26 28E 14 15 17 16 20 25 23 24 26 19 21 23 17 19 22


(c) Continued



F 16 17 19 18 21 25 14 16 17 25 26 28 22 24 27G 16 17 18 20 22 26 14 16 18 25 26 30 23 25 28H 15 17 19 17 20 23 20 21 25 20 23 25 18 20 23I 14 15 18 17 21 25 13 16 18 21 24 27 21 23 25J 15 17 18 15 17 20 25 28 30 19 20 23 13 15 19K 15 16 18 23 26 27 21 24 26 22 24 25 0 14 17L 23 25 27 22 24 28 25 28 34 30 32 36 32 25 38

Key A E coli ATCC 8739 B B cereus ATCC 10702 C B subtilis KZN D P aeruginosa ATCC 19582 E S marcescens ATCC 9986 F A calcoaceticusanitratus UP G K pneumoniae ATCC 10031 H P vulgaris ATCC 6830 I E faecalis ATCC 29212 J S aureus OK2a K S aureus OK2b L S flexneriKZN

extract and the antibiotics were synergistic (5417)additive (2778) indifferent (1667) and antagonistic(139) Although the fractional inhibitory concentrationof the extract was between 00031 and 20 and that of theantibiotics was between 000391 and 20 the fractionalinhibitory concentration indices (FICIs) for the antibacterialcombinations ranged between 000391 and 50 While thefractional inhibitory concentration indices (FICIs) forsynergism ranged between 000391 and 05 that of additivityranged between 0516 and 10 indifferences ranged between1062 and 30 and antagonistic interaction was 50 (Table 3)

4 Discussion

The currently observed rapid increase in consumption ofherbal remedies worldwide was stirred by several factorsincluding the notion that all herbal products are safe andeffective [42 43] However over the past decade severalnews-catching episodes in developed communities relatedlife-threatening adverse effects to taking herbal products ortraditional medicines [44 45] Pak et al [46] and Saad et al[47] reported that adulteration inappropriate formulationor lack of understanding of plant and drug interactionshave led to these adverse reactions that are sometimes life-threatening or lethal to patients Today while prescribing thepractice of specific class of antibiotics to certain organismshas played critical roles in the development of resistanceagainst that antibiotic [48 49] combining antibiotics towhich microbial resistances have been known globally withmedicinal plants unraveling and understanding antimicro-bial resistance would help to minimize the emergence ofmultidrug-resistant organisms [50]

Understanding natural products as a proven templatefor the development of new scaffolds of drugs [51 52] thepropelling force behind the current trends in phytochemicalresearches involving herbal-drug interactions is the discoveryof new biologically active compounds for medicinal usesWhile the success of natural products in drug discovery has

been credited to their high chemical density the effect ofevolutionary pressure to create biologically active moleculesand the structural similarity of protein targets across manyspecies [53] the synergy between the ethanolic extract ofZ mucronata and the antibiotics demonstrated that thereare explorable phytochemicals in the plant that acted syner-gistically with each of the antibiotics to produce significantantibacterial effects at their supposed target sites Thesephytochemicals combining with the antibiotics could haveinhibited different stages of some biochemical pathways inthe isolates In both groups of bacteria the extract could haveincreased the permeability of the outer membrane barriersby interacting with cell membrane andor lipopolysaccharidelayer to allow the antibiotics to gain access to cytoplasmictargets [54 55] While the synergy indicated a broaderspectrum of activity and a decreased risk of emergence ofresistant strains [56] it could shorten the total durationof therapy and decrease drug related toxicities by allowingthe use of lower doses Hence identifying isolating andevaluating the promising bioactive phytochemicals in theplant extracts become essential [57]

Consequently in agreement with previous studies indi-cating diverse interactions between medicinal plants anddifferent antibiotics [8 58ndash60] this study showed that thecombination of ethanolic extract of Z mucronata with theantibiotics was more synergistic than being indifferent orantagonistic The antibacterial combinations resulted in syn-ergy that strongly inhibited the growth of the bacterial iso-lates Although the indiscriminate use of antimicrobial agentsin the treatment of bacterial infections has led to the emer-gence of resistant strains and a great loss of clinical efficacy ofpreviously effective first-line antimicrobials resulting in theshifting of antimicrobial treatment regimen to second-line orthird-line antimicrobial agents that are often more expensivewith many side effects [61] the synergistic interaction ofthe extract of Z mucronata and the antibiotics could be apowerful tool in preventing or suppressing the emergenceof resistant strains decreasing dose-related toxicity attaining


Table 2 Susceptibility of the bacterial isolates to Ziziphus mucronata extract and the antibiotic

Minimum Inhibitory concentrations of extracts and the different antibiotics used in combinationZME Chloramphenicol Amoxicillin Tetracycline Ciprofloxacin Nalidixic acid Kanamycin

(mgmL) (120583gmL)E coli ATCC 8739 0156 1563 (I) 1563 (S) 0977 (S) 00048 (S) 3906 (S) 3125 (I)B cereus ATCC 10702 0156 391 (S) 7813 (S) 00305 (S) 00781 (S) 1563 (I) 125 (R)B subtilis KZN 0313 391 (S) 625 (R) 0977 (S) 00195 (S) 7813 (S) 3906 (S)P aeruginosa ATCC 19582 0313 391 (S) 3906 (S) 0488 (S) 0156 (S) 7813 (S) 3125 (I)S marcescens ATCC 9986 0625 098 (S) 3125 (I) 1563 (S) 00781 (S) 1953 (S) 1953 (S)A calcoaceticus anitratus 0313 781 (S) 250 (R) 049 (S) 00195 (S) 3125 (S) 1563 (S)K pneumoniae ATCC 10031 0156 195 (S) 0977 (S) 0488 (S) 0039 (S) 3906 (S) 1563 (S)P vulgaris ATCC 6830 0313 781 (S) 250 (R) 7813 (I) 00195 (S) 1953 (S) 3125 (I)E faecalis ATCC 29212 0313 195 (S) 3906 (S) 3906 (S) 0313 (S) 3125 (I) 12500 (R)S aureus OK2a 0156 781 (S) 125 (R) 0977 (S) 00781 (S) 3125 (I) 7813 (S)S aureus OK2b 0313 781 (S) 7813 (S) 0197 (S) 00195 (S) 6250 (R) 3125 (I)S flexneri KZN 0313 7813 (S) 250 (R) 0197 (S) 0039 (S) 250 (R) 15625 (S)

Table 3 Effects of combining ethanol extract of Ziziphus mucronata subsp mucronata with antimicrobial agents against selected bacterialstrains

Effects of the combined ethanol extract and different antibacterial agents on the tested bacterial isolatesA B C D E F G H I J K L

FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125

Chloramphenicol FICAs 0125 0125 05 025 025 1 2 025 0125 025 025 05FICIs 0375 0375 075 05 0375 3 3 0375 0375 0375 05 0625Rem Syn Syn Add Syn Syn Ind Ind Syn Syn Syn Syn AddFICE 025 1 025 0125 0125 0125 05 0125 0031 025 0031 0031

Amoxicillin FICAs 025 1 0125 0125 0125 0003906 025 025 025 025 0125 05FICIs 05 2 0375 025 025 0129 075 0375 0281 05 0156 0531Rem Syn Ind Syn Syn Syn Syn Add Syn Syn Syn Syn AddFICE 0125 0062 0125 0062 025 025 1 0125 05 025 0062 0125

Tetracycline FICAs 05 0125 025 1 025 0016 1 025 025 05 0125 05FICIs 0625 0187 0375 1062 075 0344 2 0375 075 075 0187 0625Rem Add Syn Syn Ind Add Syn Ind Syn Add Add Syn AddFICE 025 025 0125 025 1 1 05 0062 0125 025 0125 025

Ciprofloxacin FICAs 0125 025 0125 025 1 1 0125 0031 0125 025 025 0125FICIs 0375 05 025 05 2 2 0625 0094 025 05 0375 0375Rem Syn Syn Syn Syn Ind Ind Add Syn Syn Syn Syn SynFICE 025 2 1 02496 0125 0125 05 025 0125 05 025 1

Nalidixic acid FICAs 0125 1 2 05 2 05 05 05 05 05 05 4FICIs 0375 3 3 075 2125 0625 1 075 0625 1 075 5Rem Syn Ind Ind Add Ind Add Add Add Add Add Add AntFICE 025 025 05 05 0125 0125 025 0125 05 1 025 0062

Kanamycin FICAs 0125 0063 025 0125 05 00625 0125 025 001563 05 0125 0031FICIs 0375 0313 075 0625 0625 0188 0375 0375 0516 15 0375 0094Rem Syn Syn Add Add Add Syn Syn Syn Add Ind Syn Syn

Key A E coliATCC 8739 B B cereusATCC 10702 C B subtilisKZN D P aeruginosaATCC 19582 E S marcescensATCC 9986 FA calcoaceticus anitratusUP GK pneumoniaeATCC 10031 H P vulgarisATCC 6830 I E faecalisATCC 29212 J S aureusOK2a K S aureusOK2b L S flexneriKZN Rem remarksSyn synergy Ant antagonisms add additivity FICE FIC of extract FICAs FIC of antibiotics FICIs FIC indices


a broad spectrum of activity [62] and selecting appropriateantimicrobial therapy [63 64]The synergistic effects of thesecombinations would therefore be useful in the treatment ofmulticausal and multidrug-resistant bacteria [65ndash67]

5 Conclusions

Resistance to antibiotics is a ubiquitous and relentless clinicalproblem compounded by a dearth of new therapeutic agentsThe retreat of the pharmaceutical industries from researchand development of new antibiotic has exacerbated thechallenge of widespread resistance and signals a criticalneed for innovation Although antimicrobial combinationsare commonly used in medicine to broaden antimicrobialspectrum and generate synergism it should be promotedand encouraged as a strategy for reducing the emergence ofantibiotic-resistant strains This study showed that antibacte-rial combination of extract of Z mucronatawith the differentantibiotics was more of synergy and would be effective inthe treatment of microbial infections in which multidrug-resistant bacteria are involved The active compounds inZiziphus mucronata if isolated may be used as a thera-peutic drug candidate for controlling microbial infectionsFurther research involving interaction of the isolated purecompounds and antibiotics as well as in vitro determinationof mechanisms of action would be further investigated in ourlaboratory

Acknowledgments

The authors wish to acknowledge the financial support ofthe National Research Foundation and the University ofFort Hare South Africa Thanks are due to Professor DonGrierson for helping in the identification of the plant

References

[1] R Zhang K Eggleston V Rotimi and R J ZeckhauserldquoAntibiotic resistance as a global threat evidence from ChinaKuwait and the United Statesrdquo Globalization and Health vol 2article 6 2006

[2] A Kumar and H P Schweizer ldquoBacterial resistance to antibi-otics active efflux and reduced uptakerdquoAdvancedDrugDeliveryReviews vol 57 no 10 pp 1486ndash1513 2005

[3] R Edgar N Friedman S Molshanski-Mor and U QimronldquoReversing bacterial resistance to antibiotics by phage-mediateddelivery of dominant sensitive genesrdquo Applied EnvironmentalMicrobiology vol 78 no 3 pp 744ndash751 2012

[4] J A Karlowsky D C Draghi M E Jones C Thornsberry IR Friedland and D F Sahm ldquoSurveillance for antimicrobialsusceptibility among clinical isolates of Pseudomonas aerugi-nosa and Acinetobacter baumannii from hospitalized patientsin the United States 1998 to 2001rdquo Antimicrobial Agents andChemotherapy vol 47 no 5 pp 1681ndash1688 2003

[5] A Report from the NNIS System ldquoNational nosocomial infec-tions surveillance (NNIS) system report data summary fromJanuary 1992 through June 2004 issued October 2004rdquo TheAmerican Journal of Infection Control vol 32 no 8 pp 470ndash485

[6] C I Kang S H Kim B P Wan et al ldquoBloodstream infectionscaused by antibiotic-resistant gram-negative bacilli risk factorsfor mortality and impact of inappropriate initial antimicrobialtherapy on outcomerdquo Antimicrobial Agents and Chemotherapyvol 49 no 2 pp 760ndash766 2005

[7] S Gibbons ldquoAnti-staphylococcal plant natural productsrdquoNatu-ral Product Reports vol 21 no 2 pp 263ndash277 2004

[8] H D M Coutinho J G M Costa E O Lima V S Falcao-Silva and J P Siqueira Jr ldquoHerbal therapy associated withantibiotic therapy potentiation of the antibiotic activity againstmethicillinmdashresistant Staphylococcus aureus by Turnera ulmi-folia Lrdquo BMC Complementary and Alternative Medicine vol 9article 13 2009

[9] M F Bellini L N Cabrioti A P Terezan B Q Jordao L RRibeiro andM S Mantovani ldquoCytotoxicity and genotoxicity ofAgaricus blazeimethanolic extract fractions assessed using geneand chromosomal mutation assaysrdquo Genetics and MolecularBiology vol 31 no 1 pp 122ndash127 2008

[10] G P P Kamatou A M Viljoen S F van Vuuren and R L vanZyl ldquoIn vitro evidence of antimicrobial synergy between Salviachamelaeagnea and Leonotis leonurusrdquo South African Journal ofBotany vol 72 no 4 pp 634ndash636 2006

[11] E M Williamson ldquoSynergy and other interactions in phy-tomedicinesrdquo Phytomedicine vol 8 no 5 pp 401ndash409 2001

[12] J E C Betoni R P Mantovani L N Barbosa L C Di Stasiand A Fernandes Jr ldquoSynergism between plant extract andantimicrobial drugs used on Staphylococcus aureus diseasesrdquoMemorias do InstitutoOswaldo Cruz vol 101 no 4 pp 387ndash3902006

[13] G M Adwan B A Abu-Shanab and K M Adwan ldquoIn vitroactivity of certain drugs in combination with plant extractsagainst Staphylococcus aureus infectionsrdquo Pakistan Journal ofMedical Sciences vol 24 no 4 pp 541ndash544 2008

[14] Y Lu Y P Zhao Z C Wang S Y Chen and C X FuldquoComposition and antimicrobial activity of the essential oil ofActinidia macrosperma from Chinardquo Natural Product Researchvol 21 no 3 pp 227ndash233 2007

[15] Z H Mbwambo M J Moshi P J Masimba M C Kapinguand R S O Nondo ldquoAntimicrobial activity and brine shrimptoxicity of extracts of Terminalia brownii roots and stemrdquo BMCComplementary and Alternative Medicine vol 7 article 9 2007

[16] A Marchese and G C Schito ldquoResistance patterns of lowerrespiratory tract pathogens in Europerdquo International Journal ofAntimicrobial Agents vol 16 no 1 pp S25ndashS29 2000

[17] K Poole ldquoOvercoming antimicrobial resistance by targetingresistance mechanismsrdquo Journal of Pharmacy and Pharmacol-ogy vol 53 no 3 pp 283ndash294 2001

[18] P M Shah ldquoThe need for new therapeutic agents what isin the pipelinerdquo Clinical Microbiology and Infection vol 11supplement 3 pp 36ndash42 2005

[19] R S Paroda and B Mal ldquoNew plant sources for food andindustry in Indiardquo in New Crops for Food and Industry G EWickens N Haq and P Day Eds pp 135ndash149 Chapman ampHall London UK 1989

[20] E Palmer and N Pitman Trees of Southern Africa CoveringAll Known Indigenous Species in the Republic of South AfricaSouth-West Africa Botswana Lesotho and Swaziland vol 1ndash3AA Balkema Cape Town South Africa 1972

[21] P Tas H Stopper K Koschel and D Schiffmann ldquoInfluence ofthe carcinogenic oestrogen diethylstilboestrol on the intracellu-lar calcium level in C6 rat glioma cellsrdquo Toxicology In Vitro vol5 no 5-6 pp 463ndash465 1991


[22] A Hutchings A H Scott G Lewis and A B CunninghamZulu Medicinal Plants An Inventory University of Natal PressPeitermaritzburg South Africa 1996

[23] F Venter and J A VenterMaking the Most of Indigenous TreesBriza Pretoria South Africa 2002

[24] O O Olajuyigbe and A J Afolayan ldquoEthnobotanical surveyof medicinal plants used in the treatment of gastrointestinaldisorders in the Eastern Cape Province South Africardquo Journalof Medicinal Plants Research vol 6 no 18 pp 3415ndash3424 2012

[25] D F Basri and S H Fan ldquoThe potential of aqueous and acetoneextracts of galls of Quercus infectoria as antibacterial agentsrdquoIndian Journal of Pharmacology vol 37 no 1 pp 26ndash29 2005

[26] European Committee for Antimicrobial Susceptibility Testing(EUCAST) ldquoDetermination ofminimum inhibitory concentra-tions (MICs) of antibacterial agents by agar dilutionrdquo ClinicalMicrobiology and Infection vol 6 no 9 pp 509ndash515 2000

[27] National Committee for Clinical Laboratory Standards Meth-ods for Antimicrobial Susceptibility Testing for Bacteria ThatGrow Aerobically Approved Standard M7-A4 NCCLS Vil-lanova Pa USA 3rd edition 1997

[28] S Richard S M Lynn and C G Avery Antimicrobial Suscepti-bility Testing Protocols CRC Press New York NY USA 2007

[29] M Cheesbrough Medical Laboratory Manual for Tropi-cal Countries vol 2 of ELBS Tropical Health TechnologyButterworth-Heinemann Cambridge UK 2002

[30] National Commiettee for Clinical Labroratory Standards(NCCLS)Performance Standards for Antimicrobial Disc Suscep-tibility Tests Approved Standard NCCLS Villanova Pa USA1993

[31] British Society of Antimicrobial Chemotherapy (BSAC) ldquoDiscdiffusion method for antimicrobial susceptibility testingrdquo TheBritish Society for Antimicrobial Chemotherapy vol 2 pp 1ndash462002

[32] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoTheAmerican Journal of Clinical Pathology vol 45 no 4 pp 493ndash496 1966

[33] CLSI Clinical and Laboratory Standard Institute ldquoPerformancestandards for antimicrobial susceptibility testing eighteenthinformational supplementrdquo CLSI Clinical and Laboratory Stan-dard Institute (M100-S18) vol 28 no 1 pp 46ndash52 2008

[34] M J Lee D H Bae D H Lee K H Jang D H Oh andS D Ha ldquoReduction of Bacillus cereus in cooked rice treatedwith sanitizers and disinfectantsrdquo Journal of Microbiology andBiotechnology vol 16 no 4 pp 639ndash642 2006

[35] W S Sung H J Jung I S Lee H S Kim and D G LeeldquoAntimicrobial effect of furaneol against human pathogenicbacteria and fungirdquo Journal of Microbiology and Biotechnologyvol 16 no 3 pp 349ndash354 2006

[36] H J Jung K S Choi and D G Lee ldquoSynergistic killingeffect of synthetic peptide P20 and cefotaxime on methicillin-resistant nosocomial isolates of Staphylococcus aureusrdquo Journalof Microbiology and Biotechnology vol 15 no 5 pp 1039ndash10462005

[37] P J Petersen P Labthavikul C H Jones and P A Bradford ldquoInvitro antibacterial activities of tigecycline in combination withother antimicrobial agents determined by chequerboard andtime-kill kinetic analysisrdquo Journal of Antimicrobial Chemother-apy vol 57 no 3 pp 573ndash576 2006

[38] S Mandal M D Mandal and N K Pal ldquoEvaluation of combi-nation effect of ciprofloxacin and cefazolin against Salmonella

enteric serovar typhi isolates by in vitro methodsrdquo CalicutMedical Journal vol 2 no 2 article e2

[39] G M Eliopoulos and C T Eliopoulos ldquoAbtibiotic combina-tions should they be testedrdquoClinicalMicrobiology Reviews vol1 no 2 pp 139ndash156 1988

[40] H D Isenberg ldquoSynergism testing broth microdilutioncheckerboard and broth macrodilution methodsrdquo in ClinicalMicrobiology Procedures Handbook J Hindler Ed Microbiol-ogy ASM 1992

[41] A Prinsloo A M S van Straten and G F Weldhagen ldquoAntibi-otic profiles ofmultidrug-resistantPseudomonas aeruginosa in anosocomial environmentrdquo South African Journal Epidemiologyand Infections vol 23 no 3 pp 7ndash9 2008

[42] D D Soejarto ldquoBiodiversity prospecting and benefit-sharingperspectives from the fieldrdquo Journal of Ethnopharmacology vol51 no 1ndash3 pp 1ndash15 1996

[43] O Said K Khalil S Fulder and H Azaizeh ldquoEthnopharmaco-logical survey of medicinal herbs in Israel the Golan Heightsand the West Bank regionrdquo Journal of Ethnopharmacology vol83 no 3 pp 251ndash265 2002

[44] M Elvin-Lewis ldquoShould we be concerned about herbal reme-diesrdquo Journal of Ethnopharmacology vol 75 no 2-3 pp 141ndash1642001

[45] K Chan ldquoSome aspects of toxic contaminants in herbal reme-dies A reviewrdquo Chemosphere vol 52 no 9 pp 1361ndash1371 2003

[46] E Pak K T Esrason and V H Wu ldquoHepatotoxicity of herbalremedies an emerging dilemmardquo Progress in Transplantationvol 14 no 2 pp 91ndash96 2004

[47] B Saad H Azaizeh and O Said ldquoTradition and perspectives ofArab herbal medicine a reviewrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2 no 4 pp 475ndash479 2005

[48] SMetz-Gercek AMaieron R Strauszlig PWieninger P Apfalterand H Mittermayer ldquoTen years of antibiotic consumption inambulatory care trends in prescribing practice and antibioticresistance in Austriardquo BMC Infectious Diseases vol 9 article 612009

[49] C Costelloe C Metcalfe A Lovering D Mant and A DHay ldquoEffect of antibiotic prescribing in primary care on antimi-crobial resistance in individual patients systematic review andmeta-analysisrdquoThe British Medical Journal vol 340 Article IDc2096 2010

[50] D J Anderson and K S Kaye ldquoControlling antimicrobialresistance in the hospitalrdquo Infectious Disease Clinics of NorthAmerica vol 23 no 4 pp 847ndash864 2009

[51] YWChinM J BalunasH B Chai andADKinghorn ldquoDrugdiscovery from natural sourcesrdquo AAPS Journal vol 8 no 2 ppE239ndashE253 2006

[52] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007

[53] A L Harvey ldquoNatural products as a screening resourcerdquoCurrent Opinion in Chemical Biology vol 11 no 5 pp 480ndash4842007

[54] Y Park H J Kim and K S Hahm ldquoAntibacterial synergismof novel antibiotic peptides with chloramphenicolrdquo Biochemicaland Biophysical Research Communications vol 321 no 1 pp109ndash115 2004

[55] F Perez A M Hujer K M Hujer B K Decker P NRather and R A Bonomo ldquoGlobal challenge of multidrug-resistant Acinetobacter baumannirdquo Antimicrobial Agents andChemotherapy vol 51 no 10 pp 3471ndash3484 2007


[56] J W Baddley and P G Pappas ldquoAntifungal combinationtherapy clinical potentialrdquo Drugs vol 65 no 11 pp 1461ndash14802005

[57] W Setzer and B Vogler ldquoBioassays for activityrdquo in NaturalProducts from Plants L Cseke A Kirakosyan B Kaufman SWarber J Duke and H Brielmann Eds pp 390ndash413 CRCPress Boca Raton Fla USA 2006

[58] L Principe S DrsquoArezzo A Capone N Petrosillo and P ViscaldquoIn vitro activity of tigecycline in combination with variousantimicrobials against multidrug resistant Acinetobacter bau-manniirdquoAnnals of ClinicalMicrobiology andAntimicrobials vol8 article 18 2009

[59] O Aiyegoro A Adewusi S Oyedemi D Akinpelu andA Okoh ldquoInteractions of antibiotics and methanolic crudeextracts of Afzelia Africana (Smith) against drug resistancebacterial isolatesrdquo International Journal of Molecular Sciencesvol 12 no 7 pp 4477ndash4487 2011

[60] O O Olajuyigbe and A J Afolayan ldquoSynergistic interactions ofmethanolic extract of Acacia mearnsii with antibiotics againstbacteria of clinical relevancerdquo in International Journal of Molec-ular Sciences vol 13 pp 8915ndash8932 2012

[61] S Mandal M DebMandal N K Pal and K Saha ldquoSynergisticanti-Staphylococcus aureus activity of amoxicillin in combina-tion with Emblica officinalis and Nymphae odorata extractsrdquoAsian Pacific Journal of Tropical Medicine vol 3 no 9 pp 711ndash714 2010

[62] G M Eliopoulos and R C Moellering Jr ldquoAntimicrobialcombinationsrdquo in Antibiotics in Laboratory Medicine pp 330ndash338 Williams amp Wilkins Baltimore Md USA 4th edition1996

[63] S Hemaiswarya A K Kruthiventi and M Doble ldquoSynergismbetween natural products and antibiotics against infectiousdiseasesrdquo Phytomedicine vol 15 no 8 pp 639ndash652 2008

[64] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

[65] V Kak I You M J Zervos R Kariyama H Kumon and JW Chow ldquoIn-vitro synergistic activity of the combination ofampicillin and arbekacin against vancomycin-and high-levelgentamicin-resistant Enterococcus faecium with the aph(2

10158401015840

)-IdgenerdquoDiagnosticMicrobiology and InfectiousDisease vol 37 no4 pp 297ndash299 2000

[66] Z Q Hu W H Zhao Y Hara and T Shimamura ldquoEpigallo-catechin gallate synergy with ampicillinsulbactam against 28clinical isolates of methicillin-resistant Staphylococcus aureusrdquoJournal of Antimicrobial Chemotherapy vol 48 no 3 pp 361ndash364 2001

[67] M Sato H Tanaka R Yamaguchi K Kato and H EtohldquoSynergistic effects of mupirocin and an isoflavanone iso-lated from Erythrina variegata on growth and recovery ofmethicillin-resistant Staphylococcus aureusrdquo International Jour-nal of Antimicrobial Agents vol 24 no 3 pp 241ndash246 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


prevention of the development of resistant isolates is the com-bination of herbal remedies with the first-line antimicrobialagents to which most of them have become resistant WhileKamatou et al [10] showed that combination of antimicrobialagents had expressed significant interactionsWilliamson [11]reported that two or more compounds interact to producemutual enhancement amplification or potentiation of eachotherrsquos effects when combined Although these combina-tions could enhance the efficacy of the other antimicrobialagents and acted as alternative to treating infections causedby multidrug-resistant microorganisms having no effectivetherapy [12 13] the pharmacological effects of such mix-tures could have resulted from the diverse mechanisms ofaction resulting from the drug-herbal interactions Hencewhile natural products from plants are considered interestingalternatives for treatment of microbial infections [14 15]preventing the global increase of undesirable side effects ofcertain antibiotics and the emergence of previously uncom-mon infections [16 17] become imminent with the use of newcompounds which are not based on the existing syntheticantimicrobial agents [18]

The genus Ziziphus belongs to the Rhamnaceae familyThe members of the taxon are drought tolerant and veryresistant to heat [19] Ziziphus mucronata Willd subspmucronata Willd known as buffalo thorn is a small-to-medium-sized tree with a spreading canopy It is distributedthroughout summer rainfall areas of sub-Saharan Africaextending from South Africa northwards to Ethiopia andArabia In ethnomedicine the pastes of the roots and leavesare used to treat boils swollen glands wounds and soreswhilesteambaths from the bark are used to purify and improve skincomplexion [20] Its bark and roots are used for the treatmentof rheumatism gastrointestinal complaints and snake bites[21] In East Africa the roots are used for treating snakebites gonorrhea diarrhea and dysentery [22] Decoctionsof roots and leaves are used to ooze boils and treat soresand glandular swellings [23] In South Africa ethnobotanicalsurvey indicated that this plant is used for gastrointestinaldisorders including dysentery and diarrhoea [24] Unlikesome members of the Ziziphus genus there is a dearth ofscientific reports to indicate the pharmacological activitiesof this plant Hence this study was aimed at evaluatingthe combination effects of the ethanolic bark extract of Zmucronata and some first-line antibiotics to which microbeshave shown resistance against bacteria pathogens that areimplicated in clinical infections in order to determine theirpotential drug-herbal interactions

2 Materials and Methods

21 Collection of Plant Material The bark materials of Zizi-phus mucronata subsp mucronata were collected in August2010 from the plant growing within the University ofFort Hare campus in Alice South Africa The plant wasauthenticated in the Department of Botany and a voucherspecimen (OLAJ2010ZM01) was prepared and deposited inthe Griffin Herbarium of the University

22 Extract Preparation The bark sample was air-dried atroom temperature and pulverized using a milling machineThe extract of the bark material was prepared in accordancewith the description of Basri and Fan [25] About 100 g of thepulverized sample was extracted with 500mL of ethanol for48 h with shaking (Stuart Scientific Orbital Shaker UK) Theextract was filtered through Whatman no 1 filter paper andconcentrated under reduced pressure at 40∘C using a rotaryevaporator (Laborota 4000 efficient Heidolph Germany)The crude extract collected was allowed to dry at roomtemperature to a constant weight of 142 g The extract wasredissolved in absolute ethanol and made up to the requiredconcentrations for bioassay analysis using sterile deionizeddistilled water The reconstituted extract solution was steril-ized by filtering through 045120583mmembrane filter and testedfor sterility after membrane filtration by introducing 2mL ofthe extract into sterile nutrient broth before being incubatedat 37∘C for 24 h A sterile extract was indicated by the absenceof turbidity in the broth after the incubation period

23 Bacterial Strain The bacteria used in this study includedseven types of culture strainsmdashBacillus cereus ATCC 10702Enterococcus faecalis ATCC 29212 Escherichia coli ATCC8739 Klebsiella pneumoniae ATCC 10031 Proteus vulgarisATCC 6830 Pseudomonas aeruginosa ATCC 19582 and Ser-ratiamercescencesATCC9986 three environmental strainsmdashAcinetobacter calcoaceticus anitratus UP Bacillus subtilisKZN and S flexneriKZN and two clinical strainsmdashS aureusOK2a and S aureus OK

2b These organisms were obtainedfrom the Department of Biochemistry and MicrobiologyUniversity of Fort Hare Alice South AfricaThe antibacterialassayswere carried out usingMueller-Hinton IIAgar (Biolab)and broth The inocula of the test bacteria were preparedusing the colony suspension method [26] Colonies pickedfrom 24 h-old cultures grown on nutrient agar were used tomake suspensions of the test organisms in saline solution togive an optical density of approximately 01 at 600 nm Thesuspension was then diluted 1 100 by transferring 01mL ofthe bacterial suspension to 99mL of sterile nutrient brothbefore being used

24 Antibiotics Used in This Study Antibiotic powders ofamoxicillin chloramphenicol ciprofloxacin tetracyclinehydrochloride kanamycin and nalidixic acid were usedStock antibiotic solutions were prepared and dilutions madeaccording to the CLSI (Clinical Laboratory StandardizationInstitute) method or the manufacturerrsquos recommendations[27 28]

25 Antibiotic Susceptibility Testing (Agar Diffusion Method)Each of the isolateswas standardized using colony suspensionmethod Each strainrsquos suspension was matched with 05McFarland standards to give a resultant concentration of 15times 106 cfumLThe antibacterial activity was determined usingthe well diffusion method according to the modified Kirby-Bauer diffusion technique [29] and the National Committeefor Clinical Laboratory Standard [30] by swabbing theMueller-Hinton agar (MHA) (Oxoid UK) plates with the





3 Results






(a)



A 17 18 20 23 27 30 20 25 28 20 23 26 0 14 17B 17 18 20 20 25 30 22 26 29 14 16 21 0 13 16C 17 18 20 23 27 30 23 25 27 16 19 21 0 13 16D 15 16 18 21 27 30 23 25 29 15 18 20 13 15 18E 14 15 17 22 25 29 22 26 28 30 32 34 28 31 34F 16 17 19 22 26 30 20 26 29 0 15 18 13 16 18G 16 17 18 22 27 31 19 22 25 13 16 18 0 12 16H 15 17 19 23 25 27 20 23 26 28 30 32 28 30 33I 14 15 18 0 0 15 13 15 17 30 34 37 28 31 34J 15 17 18 23 25 27 0 15 17 30 33 35 27 31 33K 15 16 18 20 24 27 23 25 28 27 31 35 25 28 30L 23 25 27 33 37 40 38 40 42 31 36 42 29 33 35

(b)



A 17 18 20 0 15 18 0 15 17 27 30 35 20 22 25B 17 18 20 0 0 14 0 15 16 18 21 24 18 20 22C 17 18 20 0 0 15 13 15 17 13 15 19 21 24 26D 15 16 18 0 13 16 0 14 16 19 20 22 18 22 25E 14 15 17 25 28 32 20 22 25 23 26 32 15 17 20F 16 17 19 0 0 15 13 15 17 13 15 17 20 23 27G 16 17 18 0 15 17 13 15 19 18 21 24 20 23 25H 15 17 19 25 27 30 22 25 27 17 20 23 26 29 31I 14 15 18 0 13 16 0 15 17 17 21 24 15 18 22J 15 17 18 25 27 30 15 18 20 13 15 17 16 19 23K 15 16 18 25 28 31 20 23 25 25 27 30 15 16 20L 23 25 27 29 32 36 30 33 36 30 35 39 33 37 41

(c)



A 17 18 20 21 26 29 15 16 18 26 29 32 24 26 29B 17 18 20 21 25 29 13 15 17 24 25 28 23 24 28C 17 18 20 19 22 26 15 17 20 25 27 29 23 25 28D 15 16 18 20 23 25 13 15 17 24 27 30 24 26 28E 14 15 17 16 20 25 23 24 26 19 21 23 17 19 22


(c) Continued



F 16 17 19 18 21 25 14 16 17 25 26 28 22 24 27G 16 17 18 20 22 26 14 16 18 25 26 30 23 25 28H 15 17 19 17 20 23 20 21 25 20 23 25 18 20 23I 14 15 18 17 21 25 13 16 18 21 24 27 21 23 25J 15 17 18 15 17 20 25 28 30 19 20 23 13 15 19K 15 16 18 23 26 27 21 24 26 22 24 25 0 14 17L 23 25 27 22 24 28 25 28 34 30 32 36 32 25 38



4 Discussion











FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125










5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results






(a)



A 17 18 20 23 27 30 20 25 28 20 23 26 0 14 17B 17 18 20 20 25 30 22 26 29 14 16 21 0 13 16C 17 18 20 23 27 30 23 25 27 16 19 21 0 13 16D 15 16 18 21 27 30 23 25 29 15 18 20 13 15 18E 14 15 17 22 25 29 22 26 28 30 32 34 28 31 34F 16 17 19 22 26 30 20 26 29 0 15 18 13 16 18G 16 17 18 22 27 31 19 22 25 13 16 18 0 12 16H 15 17 19 23 25 27 20 23 26 28 30 32 28 30 33I 14 15 18 0 0 15 13 15 17 30 34 37 28 31 34J 15 17 18 23 25 27 0 15 17 30 33 35 27 31 33K 15 16 18 20 24 27 23 25 28 27 31 35 25 28 30L 23 25 27 33 37 40 38 40 42 31 36 42 29 33 35

(b)



A 17 18 20 0 15 18 0 15 17 27 30 35 20 22 25B 17 18 20 0 0 14 0 15 16 18 21 24 18 20 22C 17 18 20 0 0 15 13 15 17 13 15 19 21 24 26D 15 16 18 0 13 16 0 14 16 19 20 22 18 22 25E 14 15 17 25 28 32 20 22 25 23 26 32 15 17 20F 16 17 19 0 0 15 13 15 17 13 15 17 20 23 27G 16 17 18 0 15 17 13 15 19 18 21 24 20 23 25H 15 17 19 25 27 30 22 25 27 17 20 23 26 29 31I 14 15 18 0 13 16 0 15 17 17 21 24 15 18 22J 15 17 18 25 27 30 15 18 20 13 15 17 16 19 23K 15 16 18 25 28 31 20 23 25 25 27 30 15 16 20L 23 25 27 29 32 36 30 33 36 30 35 39 33 37 41

(c)



A 17 18 20 21 26 29 15 16 18 26 29 32 24 26 29B 17 18 20 21 25 29 13 15 17 24 25 28 23 24 28C 17 18 20 19 22 26 15 17 20 25 27 29 23 25 28D 15 16 18 20 23 25 13 15 17 24 27 30 24 26 28E 14 15 17 16 20 25 23 24 26 19 21 23 17 19 22


(c) Continued



F 16 17 19 18 21 25 14 16 17 25 26 28 22 24 27G 16 17 18 20 22 26 14 16 18 25 26 30 23 25 28H 15 17 19 17 20 23 20 21 25 20 23 25 18 20 23I 14 15 18 17 21 25 13 16 18 21 24 27 21 23 25J 15 17 18 15 17 20 25 28 30 19 20 23 13 15 19K 15 16 18 23 26 27 21 24 26 22 24 25 0 14 17L 23 25 27 22 24 28 25 28 34 30 32 36 32 25 38



4 Discussion











FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125










5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)



A 17 18 20 23 27 30 20 25 28 20 23 26 0 14 17B 17 18 20 20 25 30 22 26 29 14 16 21 0 13 16C 17 18 20 23 27 30 23 25 27 16 19 21 0 13 16D 15 16 18 21 27 30 23 25 29 15 18 20 13 15 18E 14 15 17 22 25 29 22 26 28 30 32 34 28 31 34F 16 17 19 22 26 30 20 26 29 0 15 18 13 16 18G 16 17 18 22 27 31 19 22 25 13 16 18 0 12 16H 15 17 19 23 25 27 20 23 26 28 30 32 28 30 33I 14 15 18 0 0 15 13 15 17 30 34 37 28 31 34J 15 17 18 23 25 27 0 15 17 30 33 35 27 31 33K 15 16 18 20 24 27 23 25 28 27 31 35 25 28 30L 23 25 27 33 37 40 38 40 42 31 36 42 29 33 35

(b)



A 17 18 20 0 15 18 0 15 17 27 30 35 20 22 25B 17 18 20 0 0 14 0 15 16 18 21 24 18 20 22C 17 18 20 0 0 15 13 15 17 13 15 19 21 24 26D 15 16 18 0 13 16 0 14 16 19 20 22 18 22 25E 14 15 17 25 28 32 20 22 25 23 26 32 15 17 20F 16 17 19 0 0 15 13 15 17 13 15 17 20 23 27G 16 17 18 0 15 17 13 15 19 18 21 24 20 23 25H 15 17 19 25 27 30 22 25 27 17 20 23 26 29 31I 14 15 18 0 13 16 0 15 17 17 21 24 15 18 22J 15 17 18 25 27 30 15 18 20 13 15 17 16 19 23K 15 16 18 25 28 31 20 23 25 25 27 30 15 16 20L 23 25 27 29 32 36 30 33 36 30 35 39 33 37 41

(c)



A 17 18 20 21 26 29 15 16 18 26 29 32 24 26 29B 17 18 20 21 25 29 13 15 17 24 25 28 23 24 28C 17 18 20 19 22 26 15 17 20 25 27 29 23 25 28D 15 16 18 20 23 25 13 15 17 24 27 30 24 26 28E 14 15 17 16 20 25 23 24 26 19 21 23 17 19 22


(c) Continued



F 16 17 19 18 21 25 14 16 17 25 26 28 22 24 27G 16 17 18 20 22 26 14 16 18 25 26 30 23 25 28H 15 17 19 17 20 23 20 21 25 20 23 25 18 20 23I 14 15 18 17 21 25 13 16 18 21 24 27 21 23 25J 15 17 18 15 17 20 25 28 30 19 20 23 13 15 19K 15 16 18 23 26 27 21 24 26 22 24 25 0 14 17L 23 25 27 22 24 28 25 28 34 30 32 36 32 25 38



4 Discussion











FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125










5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(c) Continued



F 16 17 19 18 21 25 14 16 17 25 26 28 22 24 27G 16 17 18 20 22 26 14 16 18 25 26 30 23 25 28H 15 17 19 17 20 23 20 21 25 20 23 25 18 20 23I 14 15 18 17 21 25 13 16 18 21 24 27 21 23 25J 15 17 18 15 17 20 25 28 30 19 20 23 13 15 19K 15 16 18 23 26 27 21 24 26 22 24 25 0 14 17L 23 25 27 22 24 28 25 28 34 30 32 36 32 25 38



4 Discussion











FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125










5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







FICE 025 025 025 025 0125 2 1 0125 025 0125 025 0125










5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions


Acknowledgments


References





































































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















































10158401015840











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

olufunmisoolusolaolajuyigbeandanthonyjideafolayan · 2019. 7. 31. ·...

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