Research Article A Biosurfactant-Sophorolipid Acts in...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 512495 8 pageshttpdxdoiorg1011552013512495

Research ArticleA Biosurfactant-Sophorolipid Acts in Synergy with Antibiotics toEnhance Their Efficiency

Kasturi Joshi-Navare and Asmita Prabhune

Biochemical Sciences Division National Chemical Laboratory Homi Bhabha Road Pune Maharashtra 411008 India

Correspondence should be addressed to Asmita Prabhune aaprabhunenclresin

Received 11 April 2013 Revised 22 July 2013 Accepted 2 August 2013

Academic Editor Ramkrishna Sen

Copyright copy 2013 K Joshi-Navare and A Prabhune 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

Sophorolipids (SLs) biosurfactants with antimicrobial properties have been tried to address the problem of antibiotic resistanceThe synergistic action of SL and antibiotics was checked using standard microdilution and spread plate methods WithStaphylococcus aureus SL-tetracycline combination achieved total inhibition before 4 h of exposure while tetracycline alonecouldnot achieve total inhibition till the end of 6 h The inhibition caused by exposure of bacterium to SL-tetracycline mixturewas sim25more as compared to SL alone In spite of known robustness of gram-negative bacteria SL-cefaclor mixture proved to beefficient againstEscherichia coliwhich showedsim48more inhibitionwithin 2 h of exposure as compared to cefaclor alone Scanningelectron microscopy of the cells treated with mixture revealed bacterial cell membrane damage and pore formation Moreover SLsbeing a type of asymmetric bola they are expected to form self-assemblies with unique functionalityThis led to the speculation thatSLs being amphiphilic in nature can span through the structurally alike cell membrane and facilitate the entry of drug molecules

1 Introduction

The advent of antibiotics for treating bacterial infections isconsidered as one of the major advances in modernmedicine[1] Owing to the use andmisuse of antimicrobials during pastdecades the majority of clinically important bacteria havedeveloped resistance to multiple antibiotics thus reducingthe utility of antibiotics Such infections are severe difficultto manage and require longer and more complex treatments[1ndash4]

In this scenario it is imperative to discover fresh antimi-crobials or new practices that are effective for the treatment ofinfectious diseases caused by drug-resistant microorganisms[3]

For the problem of antibiotic resistance differentapproaches such as nanotechnology genomics are beingtried out [1 5]These require detailed study for each drug andresponse by the target organism and are specific in natureCombined antibiotic therapy is an alternative approachIn order to control multi-drug resistant pathogens suchas tuberculosis and MRSA that is Methicillin resistantStaphylococcus aureus clinicians have increasingly turned

to multi antibiotic therapies [6] The approach is beingpractised against Tuberculosis for over 50 years involving thedrugs with different modes of action Based on this approachthe drug synergism between antibiotics and bioactive plantextracts has been demonstrated [7]

Different biosurfactants possess antimicrobial propertyVarious biosurfactants for example from Bacillus circulansB subtilis B licheniformis Candida antarctica and Pseu-domonas aeruginosa have been reported to have potentantimicrobial activity Some of them are effective againstMultidrug resistant strains By its structure biosurfactantis supposed to exert its toxicity on the cell membranepermeability as a detergent-like effect [8] Biosurfactants arecoming up as emerging class of biomedical compoundsThey are a suitable alternative to synthetic medicines andantimicrobial agents and could be used as safe and effectivetherapeutic agents or probiotics especially at a time whendrug resistance among causal organisms for many life-threatening diseases is on the rise [9] Sophorolipid (SL) isa promising candidate for such applications being producedby nonpathogenic yeasts such as Candida bombicola Capicola and C bogoriensis They are generally present in the

2 BioMed Research International

form of disaccharide sophoroses (2-O-120573-D-glucopyranosyl-D-glucopyranose) linked 120573 glycosidically to the hydroxylgroup at the penultimate carbon of fatty acids [10] Like otherbiosurfactants SLs do possess antimicrobial property Theantimicrobial action is notmerely restricted towards bacteriathey also act as antifungal antialgal antimycoplasma andantiviral agents [11] The proposed primary mechanism ofaction of these surfactants is membrane lipid order pertur-bation which compromises the viability of microorganisms[12] Moreover SLs offer the advantages of biodegradabilitylow ecotoxicity and the production based on renewable-resource substratesTheUSFDAhas also approved biosurfac-tantssugar esters for the use in food andpharmaceuticals SLsare not irritating to the skin do not trigger allergic reactionsand have an oral safety level which is greater than or equalto 5mLkg weight Cytotoxicity was evaluated with humanepidermal keratinocytes and was proven to be low [13]

Sun et al 2004 have demonstrated the synergistic effectsof combination of SL and loess for harmful algal bloommitigation to bring down the effective dose of both whenused individually [14] Mannosylerythritol lipid-A a typeof glycolipid biosurfactant containing cationic liposomespromoted the gene transfection efficiency five to seven timeswith mammalian cultured cells [15] Liposomes stand aspromising candidates with wide applicability based on thedrug delivery approach [16] SLs bear 2 different polar headson the 2 ends of the lipophilic core thus referred to asldquoasymmetric bolasrdquo Being amphiphilic in nature they tend toform self-assemblies with unique structural and physiochem-ical properties as well as functionality [17] Further uponmodification they can possibly be tuned into liposomesThusthey have promising potential to be used as a drug deliverysystem Moreover SLs possess a range of beneficial propertieswhich make them suitable for pharmaceutical applicationsNo studies have evaluated the use of SLs in combinationwith antibiotics [18] Therefore here we have attempted todetermine whether SLs can improve antibiotic efficiency

2 Materials and Methods

21 Microorganisms andTheir Maintenance

(a) Nonpathogenic yeast Candida bombicola (ATCC22214) was used for the production of SLs Itwas maintained on MGYP (malt extractmdash03 gglucosemdash2 g yeast extractmdash03 g peptonemdash05 g and agarmdash20 g) slants The microorganismwas sub cultured in every 4 weeks and maintained at4∘C in a refrigerator

(b) The test microorganisms E coli (ATCC-8739) andS aureus (ATCC-29737) were procured from theNational Collection of Industrial MicroorganismsNCL The cultures were maintained on nutrient agarslantsThemicroorganismswere subcultured in every4 weeks and maintained at 4∘C in a refrigerator

22 Chemicals and Reagents All media chemicals andsolvents used in this study were of analytical grade and

supplied by either Hi-media pvt Ltd India or Merck Indialtd

The fatty acid precursor Oleic acid was purchased fromSigma AldrichThe antibiotics tetracycline HCl and cefaclorwere also purchased from Sigma and stored in refrigerator tillrequired Tetracycline is known to be soluble in water whilecefaclor is partially soluble in water

23 Synthesis and Extraction of SLs For SL production seedculture was prepared by inoculating 10mL of fresh MGYPnutrient medium with C bombicola ATCC 22214 followedby incubation at 30∘C 180 rpm for 24 h This preinoculumwas added to 90mL MGYP nutrient medium in a 500mLErlenmeyer flask and incubated further for 48 h Cells wereharvested and washed twice with sterile distilled water Thecell pellets (biomass sim15 g dry weight in 100mL medium)were redispersed in 100mL of 10 glucose solution supple-mented with 1mL of Oleic acid (dispersed in 1mL ethanol)and again incubation was continued for 96 h when a brownand viscous SL mass was seen settled at the bottom of theflask It was separated using a pipette tip cut at nozzle andsubjected to ethyl acetate extraction Culture medium wascentrifuged at 5000 rpm at 10∘C for 20minThe supernatantwas extracted twice with equal volumes of ethyl acetate theorganic layer was dried over anhydrous Na

2SO4 and the

solvent was removed by rotary vacuum evaporation Theyellowish brown semicrystalline product was washed twicewith n-hexane to remove unconverted fatty acid [19]

24 Characterization of SLs

241 HPLC Analysis to Determine the Relative Proportion ofLactone and Acid Components The SL sample was subjectedto HPLC analysis to get an idea about relative percentagesof lactonic and acidic components of SLs based on standardsample run The chromeline-Hitachi HPLC system was usedalong with C18 column (5 120583m 150 times 46mm) The solventsystem used was MilliQ water-Acetonitrile (ACN) Total runtime was 65 minutes For the first 15 minutes ACN wasmaintained at 20 and then it was gradually raised to 80up to 40minutes further brought to 100 till 50minutes andthereafter maintained for 15 minutesThe run was performedat flow rate 05mLminute and 25∘C The compounds weredetected by L-2490 UV detector at 207 nm

242 Matrix Assisted Laser DesorptionIonization Mass-Spectrometry (MALDI-MS) Study SL sample 1mg was dis-solved in 1mL of methanol Further 5 120583L of the sample wasmixed with 20120583L of dithranol matrix andMALDI-MS studywas done on AB SCIEX TOFTOF 5800

25 Assay of Conjugative Action of SL and Antibiotic For theassay of conjugative action of SL and antibiotic cefaclor andtetracycline were used as antibiotics differing in their modeof action Cefaclor inhibits the bacterial growth by preventingbacteria from forming the peptidoglycan cell walls [20] Likebeta lactam antibiotics cefaclor targets the penicillin bindingproteins or PBPs a group of enzymes found anchored in

BioMed Research International 3

Table 1 Dilution scheme used for the assay of conjugative action of SL and tetracycline against S aureus

Sr no Test reactiondescription

Volume of SLstock (120583L)

Volume ofantibiotic stock

(120583L)

Volume ofsterile distilledwater (120583L)

Volume ofbacterial

suspension (120583L)

Total volume(120583L)

1 Control mdash mdash 800 200 10002 SL alone 30 mdash 770 200 10003 SL + tetracycline 30 15 755 200 10004 Tetracycline mdash 15 785 200 1000

the cell membrane which mediate the cross-linking of thecell wall while tetracycline inhibits the protein synthesis bypreventing the attachment of charged aminoacyl-tRNA tothe ribosomal attachment (A) site [21] Thus tetracyclineprevents introduction of new amino acids to the nascentpeptide chain

As can be seen from the modes of action cefaclormolecules have to reach cell membrane while tetracyclinemolecules have to cross cell membrane and reach ribosomesto exert the inhibitory actionThese 2 antibiotics were chosenin order to assess the role of SLs in enhancing the action ofantibiotics differing in mode as well as site of action

251 Conjugative Effect of SL and Tetracyclineagainst S aureus

(a) Stock preparation tetracycline stock was prepared bydissolving tetracycline HCl in sterile distilled waterat the stock strength 1mgmL SL stock was preparedin sterile distilled water by dissolving the appropriateamount of SL in sterile distilled water supplementedwith 3 vv alcohol SL stock strength used was10mgmL

(b) Determination of minimum inhibitory concentra-tions of individual SL and tetracycline in the first stepA600

that is absorbance of bacterial suspension giv-ing isolated colonies was fixed and the same A

600was

maintained throughout the experiment Based on theprior experimentation the SL concentration rangewas fixed for MIC determination as 100ndash600 120583gmLwhile the concentration range used for tetracyclinewas 5ndash100120583gmL Minimum inhibitory concentra-tion (MIC) is defined as the lowest concentration ofcompound that inhibits visible growth of microor-ganisms on the culture plate The concentration ofSL or antibiotic at which no bacterial colony wasobserved on the plate was considered as minimuminhibitory concentration [22] Based on the resultsof MIC determination experiments the sublethalconcentrations of both bioactive compounds wereidentified and used during the assay of conjugativeeffect

(c) Time dependent assay of synergistic action alongwithcontrols 4 test reactions were set up The dilutionscheme has been mentioned in Table 1 Cells withoutexposure to any bioactive agent served as the control

During the sequential additions SL and tetracycline weremixed thoroughly followed by addition of sterile distilledwater and suspension Thus the cells were exposed to theaction of SL and tetracycline Reaction mixtures were incu-bated at 28∘C 180 rpm for 6 h The samples were removedat periodic intervals 2 4 and 6 h and number of colonyforming units (CFUs) were determined by spreading 50 120583L ofmixture on nutrient agar plates The plates were incubated at28∘C and colonies were visualized after 24 h All antibacterialactivity tests were performed in triplicates and average valueswere noted to certify the reproducibility Colonies werecounted and percentage cell survival was calculated using thefollowing formula [23]

cell survival = no of colonies on test plate lowast 100no ofcolonies on control plate

252 Conjugative Effect of Cefaclor and SL against E coliSimilar protocol as mentioned in the above experimentwas followed The SL stock was prepared at 10mgmLconcentration while cefaclor stock was prepared at 1mgmLconcentration The concentration range used for MIC deter-mination of cefaclor against E coli was 20ndash80120583gmL whilethe concentration range used for SL was 100ndash1000 120583gmL

Based on the results of MIC determination experimentthe sublethal concentrations of SL and cefaclor were decidedand used for further experiment 4 test reactions were setnamely control SL alone SL with cefaclor and cefacloralone Please refer to the Supplementary Information (avail-able online at httpdxdoiorg1011552013512495) for thedilution scheme The sampling intervals protocol and dataevaluation method were the same as mentioned above

26 Scanning Electron Microscopy of Treated Cells The cellswere subjected to the action of SL and antibiotic combina-tions in respective proportions as per the protocolmentionedin Sections 251 and 252 After 4 h incubation with SL-antibiotic combination the bacterial cell suspension wascentrifuged Cell pellet was resuspended in 200 120583L of steriledistilled water and 10ndash15 120583L of it was drop-casted onto asilicon wafer for easier locating of bacterial cells and allowedto air-dry Sampleswere sputter coated till a fine layer of 10 nmwas formed (sputter coater makemdashEMITECH source- Au-Pd gas-argon) The E-SEMs of the samples were thenrecorded at the resolution 3 nm at 30 kV under high vacuum(SEM makemdashFEI model-Quanta 200 D Dual beam ESEMwith EDAX sourcemdashtungsten thermionic emission) The


untreated healthy cells were also subjected to SEM as per thesame procedure They served as the control

3 Results

31 Characterization of SL

311 HPLC Analysis to Determine Relative Proportions ofLactone and Acid Components As per the HPLC analysisit was found that the SL sample contains around 75 oflactone form and remaining 25 of acidic form The acidicSL forms get eluted first while the lactonic SLs especiallythe acetylated ones show longer retention times because ofhigher hydrophobicity [24] Thus the peaks lying in the laterhalf region were considered to be of different lactonic forms(Refer to supplementary information for chromatogram)

312 MALDI-MS Study of SL Prominent peaks from themass spectrum were correlated to sodium adducts [M+ + H++ Na+] of the expected forms of SLs Four different forms ofOleic acid derived SLs were detected Diacetylated lactonicSL of Oleic acid that is 17-L-(-oxy)-octadecanoic acid 1410158401015840-lactone 61015840610158401015840-diacetate was detectedwithmaximumabun-dance Diacetylated acidic form monoacetylated lactonicSL and monoacetylated acidic SL were also detected inrelatively small proportions Apart from Oleic acid derivedSLs different SL structures having Linoleic (C182) stearic(C180) and palmitic (C160) acids as the hydrophobic partwere also detected (Refer to supplementary information formass spectrum)The findingwas in accordancewith previousreports [25]

Dengle-Pulate et al 2013 have synthesized SL usingLauryl alcohol as hydrophobic precursor and assessed itssurface tension reducing property along with the SL derivedfrom Oleic acid (SLOA) as a control They have reported theCritical Micelle Concentration value of SLOA as 012 gL andlowest surface tension as 34mNm [26]

313 Conjugative Effect of SL and Tetracycline against Saureus MIC of tetracycline against S aureus (ATCC-29737)was found to be 150 120583gmL while that of SL was found to be400120583gmL Therefore it can be observed that both SL andtetracycline are capable of achieving inhibition In order torightly assess the time dependent trend of inhibitory effectof combination sublethal concentrations of SL (300 120583gmL)and tetracycline (15 120583gmL) were exercised during the assayThe time dependent bacterial inhibition on the action of SLand tetracycline has been represented in Figure 1 It can beobserved from Figure 1 that tetracycline alone cannot achievetotal inhibition even after 6 h of exposure SL alone wasefficient against S aureus at 300 120583gmL and showed totalinhibitionwithin 4 hHowever it wasworth noting that whenboth agents were used in combination at 2 h exposure sim22more inhibition was observed

314 Conjugative Effect of SL and Cefaclor against E coliMIC of cefaclor against E coli (ATCC 8739) was foundto be 200120583gmL while SL alone was not inhibitory to

0

20

40

60

80

100

120

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h

Figure 1 Synergistic action of tetracycline and SL against S aureusThe graphs represent reduction in colony forming units onexposure to different bioactive agents with respect to time 0 2 4and 6 h Bars represent Amdasheffect of tetracycline alone Bmdasheffect oftetracycline + SL and Cmdasheffect of SL alone

E coli till 1000120583gmL For the assay of conjugative effectsublethal concentrations of SL and cefaclor were decided tobe 500 120583gmL and 50120583gmL respectively Figure 2 representsthe comparative inhibitory action of cefaclor SL and theircombination against E coli Cefaclor achieved almost totalinhibition at the end of 6 h exposure SL alone was unable tototally inhibit the bacterial growth but when administeredalong with the antibiotic it resulted in faster killing of thebacterium It is worth to be noted that SL-cefaclor togethercould achieve sim98 killing within 4 h while with cefacloralone it required 6 h exposure to get equivalent effect

It has been observed through previous reports thatSLs have better antibacterial action against gram-positivebacteria as compared to that against the gram-negative onesSleiman et al 2009 have checked the action of SLs and SLderivatives against different clinically relevant bacteria andreported that some activity was found against gram-positivebacteria but against gram-negative bacteria considerablyhigh concentrations showed trace activity [18] Shah et al2007 also reported concurrent findings [27]

32 Scanning Electron Microscopy Images of the BacterialCells Treated with SL and Antibiotic Figure 3 shows thescanning electron micrographs of the S aureus cells treatedwith the tetracycline-SL mixture while Figure 4 shows thescanning electron micrographs of the E coli cells treatedwith the cefaclor-SL mixture Damage to cell membrane isevident from the images The consequences of disturbed cellmembrane integrity such as formation of membrane poresleading to leakage of cytoplasmic contents and accumulationof cell debris were also noted


0

20

40

60

80

100

120

140

160

180

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h

Figure 2 Synergistic action of cefaclor and SL against E coli Thegraphs represent reduction in colony forming units on exposureto different bioactive agents with respect to time 0 2 4 and 6 h Barsrepresent Amdasheffect of cefaclor alone Bmdasheffect of cefaclor + SL andCmdasheffect of SL alone

4 Discussion

Till date no studies have evaluated the use of SLs incombination with antibiotics Hence it is not known if thesecompounds might have antagonistic or synergistic effectswhen administered with antibiotics [18] We have used theSL mixture during the study knowing that natural synergismbetween SLs creates a better balance for many interfacialactivities [28]

In this study S aureus and E coli were used to evaluatethe synergistic action of SLs with different antibiotics Theseorganisms are commonly occurring pathogens S aureus is agram-positive coccus currently responsible for the majorityof skin and soft tissue infections (SSTIs) as per survey reportcarried out in the United StatesThis bacterium is commonlyfound asymptomatically in healthy individuals colonizingthe anterior nares and other sites of the body such as theskin and gastrointestinal tract However S aureus can beextraordinarily pathogenic causing a broad range of morbidstates from serious skin infections such as cellulitis andabscesses to endocarditis and sepsis S aureus is rapidlyevolving resistance to contemporary topical as well as sys-temic antibiotics [29] Another index bacterium used in thepresent study E coli is found in the lower intestine of warmblooded animalsE coli ismore than just a harmless intestinalinhabitant it can also be a highly versatile pathogen that canbe frequently deadly Several different E coli strains causediverse intestinal and extraintestinal diseases by means ofvirulence factors that affect a wide range of cellular processes[30] To include the bacterium here was important so as toobserve the antimicrobial effect of cefaclor and cefaclor incombination with SL on the gram-negative bacteria that have

a thin peptidoglycan layer adjacent to the inner cytoplasmicmembrane which makes them have little resistance againstcefaclor

The scanning electron micrographs exhibit a range ofmorphological changes suggestive of damage to cell mem-brane and the consequences of disturbed cell membraneintegrity such as formation of membrane pores leading toleakage of cytoplasmic contents and accumulation of celldebris were noted It should be noted that though theantibiotics differ in their action similar features of cellulardamage were observed in both cases So it can be saidthat the inhibitory action involved cell membrane lipidorder perturbation in addition to the action of antibioticThe inhibitory effect of antibiotics was observed at lowerconcentrations when coadministered with SLs Probably SLshave enhanced the drug action by facilitating the entryacross cell membrane thus achieving requisite intracellularantibiotic concentration at low dosage A hypothesis has beenproposed about the entry of antibiotic molecules in presenceof SL as schematically represented in Figure 5

Hypothesis aboutMechanism ofDrug Entry Facilitation by SLsNaturally evolution does not provide any active transporterfor antibiotics and a passive diffusion process facilitated bychannels must be invoked [31] Antibiotic agents are thoughtto diffuse freely through the cell wall of gram-positivebacteria However in gram-negative bacteria the diffusionof a given antibiotic agent depends on the permeability ofthe outer membrane This permeability is determined by theparticular structure of the membrane which is composedof proteins and an asymmetric lipid bilayer [32] The outermembrane of bacteria contains various protein channelscalled porins which are involved in the influx of variouscompounds including several classes of antibiotics Bacterialadaptation to reduce influx through porins is an increasingproblem worldwide that contributes together with effluxsystems to the emergence and dissemination of antibioticresistance Gram-negative bacteria are responsible for a largeproportion of antibiotic-resistant bacterial diseases Thesebacteria have a complex cell envelope that comprises anouter membrane and an inner membrane that delimit theperiplasm [33] Tetracycline resistance is often due to theacquisition of new genes which code for energy dependentefflux of tetracyclines [21] Therefore while addressing theissue of antibiotic resistance enhancing the permeability ofdrugs is of fundamental importance

The SLs as described earlier are capable of formingmicelles bilayer structures and self-assemblies which canenclose thewater soluble drugsWhen administered togetherSLs can span through the structurally alike cell membranelipid bilayer and deliver the drugmolecules to the cell interiorStructural studies of SL-antibiotic mixture with sophisticatedinstrumentation may throw some light on interaction ofthe two such as micellarization SLs are known to havebetter antibacterial action against gram-positive bacteriawhile large doses of the SLs are required to demonstrate anyantibacterial activity especially with Gram-negative bacteria[15 27] In agreement with this fact SL alone could not


(a) (b) (c)

Figure 3 SEM images of (a) controlmdashuntreated S aureus cells and (b) and (c) S aureus cells treated with the mixture of SL and tetracyclineCell membrane damage was evident from the images with accumulation of cell debris

(a) (b) (c)

Figure 4 SEM images of (a) controlmdashuntreated E coli cells and (b) and (c) E coli cells treated with the mixture of SL and cefaclor Disturbedcell integrity was evident from the images Formation of pores (indicated by arrows) was visible with leakage of cytoplasmic contents

Hydrophilic head Hydrophobic

tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400

Figure 5 Schematic diagram displaying the proposed mechanism of SL mediated drug entry facilitation across cell membrane


inhibit the growth of E coli But in case of cells treatedwith the combination of SL and cefaclor total inhibitionwas achieved much faster as compared to the sample treatedwith cefaclor alone Therefore it can be concluded that theenhanced inhibitory effect is not due to additive action oftwo antimicrobial agents Hence the enhanced efficiency ofcefaclor-SL combination against E coli can be considered asa proof for the argument that better performance is due tofacilitation of entry of drug molecules by SLs

Lactonic SLs have been reported to have better surfacetension lowering and antimicrobial activity as compared tothe acidic form [11] Therefore we can expect that increasingthe percentage of lactonic SL will improve the antibacterialproperty of SL

Lactonic SLs are more hydrophobic as compared to theacidic SL Packing constraints are associated with the lactonicSLs due to closed-ring structure of alkyl chain Self-assembledstructures of lactonic SLs can be unilamellar vesicles ortubules whereas acidic SLs generally form small globularmicelles Therefore pertaining to the hypothesis we havespeculated about the entry of drug molecules it can be saidthat varying the lactone acid percentage will also affect theaffinity ofmicellar structures towards water soluble andwaterinsoluble drugs [17]

Combined antibiotic therapy has been shown to delay theemergence of bacterial resistance and also produce desirablesynergistic effects in the treatment of bacterial infections[7] In case of nanoparticles when they are used togetherwith antibiotics advantage is conferred that if bacteria haveresistance against one of the components a further compo-nent could kill them in a different manner [22] SimilarlySLs being antimicrobial in nature when coadministered withantibiotics reduce the likelihood of bacterial survival as wellas development of resistance Also because of enhanced entryof antibiotic molecules the desired inhibitory effect may beachieved at low concentration of antibiotic

The structure of the surfactant dictates the HLB that ishydrophilic lipophilic balance value of the molecule Thisvalue indicates whether the molecule will form water inoil that is WO emulsions or oil in water that is OWemulsions Depending on the solubility of the antibioticchoice of surfactant can be made SLs are known as ldquotailor-made moleculesrdquo It is possible to achieve SL structuralvariation by varying the hydrophobic or hydrophilic carbonsubstrates fed during the synthesis procedure

Therefore due to enhanced entry and simultaneous actionof 2 inhibitory agents it can be expected that the coadminis-tration of suitable SL and antibiotic will handle the infectionefficiently SLs can be safely administered till considerablyhigh dosage that is 5mLkg of body weight which is anotherfact favouring its pharmaceutical usage [13]

5 Conclusion

Coadministration of SL enhanced the action of antibioticsin representative gram-positive and gram-negative bacteriaThese bacteria differ in their cell membrane structuresand also the machinery to prevent the entry of antibiotic

molecules Considering the ability of SLs to form self-assemblies it has been speculated that self-assembled SLs canspan through the structurally alike bacterial cell membraneand thus facilitate the entry of drugmolecules Self-assembledsystem may enhance solubility of antibiotic or offer shieldingfrom the environment hence resulting in improved efficiencyAdditionally SLs are antimicrobial in nature Hence if co-administered with another inhibitory agent the mixturereduces the likelihood of bacterial survival and probably thedevelopment of resistance as the bacteria have to combatagainst 2 agents

Conflict of Interests

The authors have declared no conflict of interests

Acknowledgments

Kasturi Joshi-Navare would like to thank the UniversityGrants Commission for the research fellowship The authorsthank CMC division NCL for providing MALDIMS SEMfacility

References

[1] G Rossolini and MThaller ldquoCoping with antibiotic resistancecontributions from genomicsrdquo Genome Medicine vol 2 article15 2010

[2] S B Levy and B Marshall ldquoAntibacterial resistance worldwidecauses challenges and responsesrdquo Nature Medicine vol 10 no12 pp S122ndashS129 2004

[3] O Aiyegoro A Adewusi S Oyedemi et al ldquoInteractions ofantibiotics and methanolic crude extracts of Afzelia Africana(Smith) against drug resistance bacterial isolatesrdquo InternationalJournal of Molecular Sciences vol 12 no 7 pp 4477ndash4487 2011

[4] A J Alanis ldquoResistance to antibiotics are we in the post-antibiotic erardquo Archives of Medical Research vol 36 no 6 pp697ndash705 2005

[5] A M Allahverdiyev K V Kon E S Abamor M Bagirova andM Rafailovich ldquoCoping with antibiotic resistance combiningnanoparticles with antibiotics and other antimicrobial agentsrdquoExpert Review of Anti-Infective Therapy vol 9 no 11 pp 1035ndash1052 2011

[6] J Torella R Chait and R Kishony ldquoOptimal drug synergy inantimicrobial treatmentsrdquo PLOS Computational Biology vol 6no 6 Article ID e1000796 2010

[7] G Adwan and M Mhanna ldquoSynergistic effects of plant extractand antibiotics on Staphylococcus aureus strains isolated fromclinical specimensrdquo Middle-East Journal of Scientific Researchvol 3 pp 134ndash139 2008

[8] E Gharaei-Fathabad ldquoBiosurfactants in pharmaceutical indus-try (a mini-review)rdquo American Journal of Drug Discovery andDevelopment vol 1 no 1 pp 58ndash69 2011

[9] P Singh and S S Cameotra ldquoPotential applications ofmicrobialsurfactants in biomedical sciencesrdquoTrends in Biotechnology vol22 no 3 pp 142ndash146 2004

[10] K S Bisht R A Gross and D L Kaplan ldquoEnzyme-mediatedregioselective acylations of sophorolipidsrdquo Journal of OrganicChemistry vol 64 no 3 pp 780ndash789 1999


[11] I N A van Bogaert K Saerens C D Muynck D DevelterW Soetaert and E J Vandamme ldquoMicrobial productionand application of sophorolipidsrdquo Applied Microbiology andBiotechnology vol 76 no 1 pp 23ndash34 2007

[12] A Azim V Shah G F Doncel N Peterson W Gao and RGross ldquoAmino acid conjugated sophorolipids a new familyof biologically active functionalized glycolipidsrdquo BioconjugateChemistry vol 17 no 6 pp 1523ndash1529 2006

[13] I N A van Bogaert J Zhang and W Soetaert ldquoMicrobialsynthesis of sophorolipidsrdquo Process Biochemistry vol 46 no 4pp 821ndash833 2011

[14] X Sun Y Lee J Choi and E Kim ldquoSynergistic effect ofsophorolipid and loess combination in harmful algal bloomsmitigationrdquoMarine Pollution Bulletin vol 48 no 9-10 pp 863ndash872 2004

[15] Y Inoh D Kitamoto N Hirashima and M Nakanishi ldquoBio-surfactants of MEL-A increase gene transfection mediatedby cationic liposomesrdquo Biochemical and Biophysical ResearchCommunications vol 289 no 1 pp 57ndash61 2001

[16] S Loew A Fahr and S May ldquoModeling the release kinetics ofpoorly water-soluble drug molecules from liposomal nanocar-riersrdquo Journal of Drug Delivery vol 2011 Article ID 376548 10pages 2011

[17] P Dubey K Sevaraj and A Prabhune ldquoSophorolipids inself assembly and nanomaterial synthesisrdquo World Journal ofPharmacy and Pharmaceutical Sciences vol 2 no 3 pp 1107ndash1133 2013

[18] J N Sleiman S A Kohlhoff P M Roblin et al ldquoSophorolipidsas antibacterial agentsrdquoAnnals of Clinical amp Laboratory Sciencevol 39 no 1 pp 60ndash63 2009

[19] K Joshi-Navare A Shiras and A Prabhune ldquoDifferentiation-inducing ability of sophorolipids of oleic and linoleic acids usinga glioma cell linerdquo Biotechnology Journal vol 6 no 5 pp 509ndash512 2011

[20] K E Vivekanandan K Gokul Raj S Kumaresan andM PandildquoBiosynthesis of silver nanoparticle activity against bacterialstrain cephalexin antibiotic synergistic activityrdquo InternationalJournal of Current Science vol 4 pp 1ndash7 2012

[21] I Chopra and M Roberts ldquoTetracycline antibiotics modeof action applications molecular biology and epidemiologyof bacterial resistancerdquo Microbiology and Molecular BiologyReviews vol 65 no 2 pp 232ndash260 2001

[22] A Rai A Prabhune and C C Perry ldquoAntibiotic mediatedsynthesis of gold nanoparticles with potent antimicrobial activ-ity and their application in antimicrobial coatingsrdquo Journal ofMaterials Chemistry vol 20 no 32 pp 6789ndash6798 2010

[23] R Gupta S Uma and A Prabhune ldquoAntibacterial properties oflinolenic sophorolipid and its chemically esterified methyl esterformrdquo Research Journal of Biotechnology vol 7 no 3 pp 40ndash452012

[24] Y Hu and L Ju ldquoSophorolipid production from differentlipid precursors observed with LC-MSrdquo Enzyme and MicrobialTechnology vol 29 no 10 pp 593ndash601 2001

[25] R Merchant and I M Banat ldquoBiosurfactants a sustainablereplacement for chemical surfactantsrdquo Biotechnology Lettersvol 34 no 9 pp 1597ndash1605 2012

[26] V Dengle-Pulate S Bhagwat and A Prabhune ldquoMicrobialoxidation of medium chain fatty alcohol in the synthesis ofsophorolipids by candida bombicola and its physicochemicalcharacterizationrdquo Journal of Surfactants and Detergents vol 18pp 173ndash181 2013

[27] V Shah D Badia and P Ratsep ldquoSophorolipids havingenhanced antibacterial activityrdquo Antimicrobial Agents andChemotherapy vol 51 no 1 pp 397ndash400 2007

[28] Y Hirata M Ryu K Igarashi et al ldquoNatural synergism of acidand lactone type mixed sophorolipids in interfacial activitiesand cytotoxicitiesrdquo Journal of Oleo Science vol 58 no 11 pp565ndash572 2009

[29] L Englander and A Friedman ldquoNitric oxide nanoparticletechnologyrdquo Journal of Clinical and Aesthetic Dermatology vol3 no 6 pp 45ndash50 2010

[30] J B Kaper J P Nataro and H L T Mobley ldquoPathogenicEscherichia colirdquo Nature Reviews Microbiology vol 2 no 2 pp123ndash140 2004

[31] M Ceccarelli A V Vargiu and P Ruggerone ldquoA kinetic MonteCarlo approach to investigate antibiotic translocation throughbacterial porinsrdquo Journal of Physics vol 24 no 10 Article ID104012 2012

[32] L Gutmann R Williamson and E Collatz ldquoThe possible roleof porins in bacterial antibiotic resistancerdquo Annals of InternalMedicine vol 101 no 4 pp 554ndash557 1984

[33] J Pages C E James and M Winterhalter ldquoThe porin and thepermeating antibiotic a selective diffusion barrier in Gram-negative bacteriardquo Nature Reviews Microbiology vol 6 no 12pp 893ndash903 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

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Stem CellsInternational



Enzyme Research



Microbiology


form of disaccharide sophoroses (2-O-120573-D-glucopyranosyl-D-glucopyranose) linked 120573 glycosidically to the hydroxylgroup at the penultimate carbon of fatty acids [10] Like otherbiosurfactants SLs do possess antimicrobial property Theantimicrobial action is notmerely restricted towards bacteriathey also act as antifungal antialgal antimycoplasma andantiviral agents [11] The proposed primary mechanism ofaction of these surfactants is membrane lipid order pertur-bation which compromises the viability of microorganisms[12] Moreover SLs offer the advantages of biodegradabilitylow ecotoxicity and the production based on renewable-resource substratesTheUSFDAhas also approved biosurfac-tantssugar esters for the use in food andpharmaceuticals SLsare not irritating to the skin do not trigger allergic reactionsand have an oral safety level which is greater than or equalto 5mLkg weight Cytotoxicity was evaluated with humanepidermal keratinocytes and was proven to be low [13]

Sun et al 2004 have demonstrated the synergistic effectsof combination of SL and loess for harmful algal bloommitigation to bring down the effective dose of both whenused individually [14] Mannosylerythritol lipid-A a typeof glycolipid biosurfactant containing cationic liposomespromoted the gene transfection efficiency five to seven timeswith mammalian cultured cells [15] Liposomes stand aspromising candidates with wide applicability based on thedrug delivery approach [16] SLs bear 2 different polar headson the 2 ends of the lipophilic core thus referred to asldquoasymmetric bolasrdquo Being amphiphilic in nature they tend toform self-assemblies with unique structural and physiochem-ical properties as well as functionality [17] Further uponmodification they can possibly be tuned into liposomesThusthey have promising potential to be used as a drug deliverysystem Moreover SLs possess a range of beneficial propertieswhich make them suitable for pharmaceutical applicationsNo studies have evaluated the use of SLs in combinationwith antibiotics [18] Therefore here we have attempted todetermine whether SLs can improve antibiotic efficiency

2 Materials and Methods

21 Microorganisms andTheir Maintenance

(a) Nonpathogenic yeast Candida bombicola (ATCC22214) was used for the production of SLs Itwas maintained on MGYP (malt extractmdash03 gglucosemdash2 g yeast extractmdash03 g peptonemdash05 g and agarmdash20 g) slants The microorganismwas sub cultured in every 4 weeks and maintained at4∘C in a refrigerator

(b) The test microorganisms E coli (ATCC-8739) andS aureus (ATCC-29737) were procured from theNational Collection of Industrial MicroorganismsNCL The cultures were maintained on nutrient agarslantsThemicroorganismswere subcultured in every4 weeks and maintained at 4∘C in a refrigerator

22 Chemicals and Reagents All media chemicals andsolvents used in this study were of analytical grade and

supplied by either Hi-media pvt Ltd India or Merck Indialtd

The fatty acid precursor Oleic acid was purchased fromSigma AldrichThe antibiotics tetracycline HCl and cefaclorwere also purchased from Sigma and stored in refrigerator tillrequired Tetracycline is known to be soluble in water whilecefaclor is partially soluble in water

23 Synthesis and Extraction of SLs For SL production seedculture was prepared by inoculating 10mL of fresh MGYPnutrient medium with C bombicola ATCC 22214 followedby incubation at 30∘C 180 rpm for 24 h This preinoculumwas added to 90mL MGYP nutrient medium in a 500mLErlenmeyer flask and incubated further for 48 h Cells wereharvested and washed twice with sterile distilled water Thecell pellets (biomass sim15 g dry weight in 100mL medium)were redispersed in 100mL of 10 glucose solution supple-mented with 1mL of Oleic acid (dispersed in 1mL ethanol)and again incubation was continued for 96 h when a brownand viscous SL mass was seen settled at the bottom of theflask It was separated using a pipette tip cut at nozzle andsubjected to ethyl acetate extraction Culture medium wascentrifuged at 5000 rpm at 10∘C for 20minThe supernatantwas extracted twice with equal volumes of ethyl acetate theorganic layer was dried over anhydrous Na

2SO4 and the

solvent was removed by rotary vacuum evaporation Theyellowish brown semicrystalline product was washed twicewith n-hexane to remove unconverted fatty acid [19]

24 Characterization of SLs

241 HPLC Analysis to Determine the Relative Proportion ofLactone and Acid Components The SL sample was subjectedto HPLC analysis to get an idea about relative percentagesof lactonic and acidic components of SLs based on standardsample run The chromeline-Hitachi HPLC system was usedalong with C18 column (5 120583m 150 times 46mm) The solventsystem used was MilliQ water-Acetonitrile (ACN) Total runtime was 65 minutes For the first 15 minutes ACN wasmaintained at 20 and then it was gradually raised to 80up to 40minutes further brought to 100 till 50minutes andthereafter maintained for 15 minutesThe run was performedat flow rate 05mLminute and 25∘C The compounds weredetected by L-2490 UV detector at 207 nm

242 Matrix Assisted Laser DesorptionIonization Mass-Spectrometry (MALDI-MS) Study SL sample 1mg was dis-solved in 1mL of methanol Further 5 120583L of the sample wasmixed with 20120583L of dithranol matrix andMALDI-MS studywas done on AB SCIEX TOFTOF 5800

25 Assay of Conjugative Action of SL and Antibiotic For theassay of conjugative action of SL and antibiotic cefaclor andtetracycline were used as antibiotics differing in their modeof action Cefaclor inhibits the bacterial growth by preventingbacteria from forming the peptidoglycan cell walls [20] Likebeta lactam antibiotics cefaclor targets the penicillin bindingproteins or PBPs a group of enzymes found anchored in






(120583L)


Volume ofbacterial










600was










3 Results







0

20

40

60

80

100

120

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h






0

20

40

60

80

100

120

140

160

180

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h


4 Discussion








(a) (b) (c)


(a) (b) (c)



tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400









5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






(120583L)


Volume ofbacterial










600was










3 Results







0

20

40

60

80

100

120

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h






0

20

40

60

80

100

120

140

160

180

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h


4 Discussion








(a) (b) (c)


(a) (b) (c)



tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400









5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results







0

20

40

60

80

100

120

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h






0

20

40

60

80

100

120

140

160

180

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h


4 Discussion








(a) (b) (c)


(a) (b) (c)



tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400









5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100

120

140

160

180

A B C

Redu

ctio

n in

colo

ny fo

rmin

g un

its (

)

0 h2 h

4 h6 h


4 Discussion








(a) (b) (c)


(a) (b) (c)



tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400









5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b) (c)


(a) (b) (c)



tail

+ Drug molecules

Sophorolipid

Bacterial cell

R2O

HO

HO

O

O O

O

OH

OH

OH

R1OCH3

CH(CH2)6CHCH

(CH2)7COOH

1998400

2998400

3998400

4998400

5998400

6998400

1

9

10

17

18

1998400998400

2998400998400

3998400998400

4998400998400

5998400998400

6998400998400









5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








5 Conclusion





Acknowledgments


References










































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article A Biosurfactant-Sophorolipid Acts in...

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