University of Nigeria Virtual Library
Serial No ISSN: 1118-1028
Author 1 MBAH, Chika J.
Author 2
Author 3
Title The Effect of Ethylenediamine Tetraacetic Acid on the Antimicrobial Properties of Benzoic Acid and Cetrimide
Keywords
Description Pharmaceutical Chemistry
Category Pharmaceutical Sciences
Publisher
Publication Date 1999
Signature
* - ' 8 - . .
1 I r/ Journal of I PHARMACEUTICAL 1 RESEARCH AND i .DEVELOPMENT Journal of Pharmaceutical Research and Development 4: 1 (1 999) 1 -8 - 1 : The Effect offEthylenediamine Tetraacetic Acid 4 the Antimicrobial Properties of Benzoic ) Acid and Cetrimide
C. 0. Esirnonel* M. U. ~dikwu';D.B. Uzuegbu' and 0. P. Udeo 4 'Division of Pharmaceutical Microbiology Department of Pharmz Faculty of Phyackutical Sciences University of Nigeria, Ws I 'Department of ~harmacolo~~and Toxicology, Faculty of Pharmaceut
' . University of Nigeria,fisukka. i I
I : I. The effect of ethylenediamine tetraacetic acid (EDTA) on the in-vp antimicrobial activities of cetrimide and benzoic acid was evaluated by the checkerboardland killing curve method. The effect sf EDTA aid benzoic acid was evaluated against an isolate of Pseirdonionas aeruginosa (Ps. 021) which is highly resistant to either of the drugs alone. The effect of EDTA and cetrimide was evaluated against isolates of Aspergillus niger and Candidn albicarls resistant to either of the agents. The results show that in the'presence of EDTA, the bacteriostatic and bactericidal effects of benzoic acid and cetrimide against the test microorgan,isms were greatly enhanced. Checkerboard analy& revealed striking synergy (FIC indices > 1 and negative values of activity indices) between almost all the ratios of EDTA and the antimicrobial agents against the various test microorganisms. / / Keywords: Checkerboard, EDTA, Killing curve, Cetrimide, Benzoic acid, Pseu/hntn~r/lc aerrtginasa, Canditia ittbicms, Aspergillus niger. ;I I1
I Introduction The . developn~ent of resistant by the microo'rganisn,~ on prolong microorganisms on prolonged exposure to* there is the cheaper and often easier a existing antimicrobial agents has been known of improving the efficacy of for a long time (I). This has resulted in the antimicrobial agents via combination continual sexch for ways of eradicating An microbial c~nlbinationshave pro resistant strains of microorganisn~s. Apart 44 from the fastidious and often expensive option an efficient method in the comt of synthesizing new antimicrobial agents microbial resistance (2-4). The essenc against existing resistant microorganisms combinations is usually/ to pn i (which usually, ultimately results to resistance synergistic effect.
*C~rrespondingauthor 01 999 JPRD, ISSN: I 1 1 8- I.028 . C. 0.Esinlo~le et al I Journal of Phannaceulical Research and Development, Vol. 4.:No.I 1 (19991; 1-8 I j +. Ethylenediamine tetraacetic acid benzoic acid was evaluated against the Gram (EDTA) has been known to prc$uce a reversal negative bacteria, Pseudomonas aeruginosa 'I of antibiotic resistance in resistant strains of (strain Ps. 021, a resistant isplate obtained from Gram negative microorganism's including our laboratories). Preliminary investigations Pseudomoms aeruginosa (1,517). . It has been showed that the above stiiins of C, albicpnr postulated that the antimicrobia1,propertiesof and Asp. niger were very sensitive to benzoic EDTA is based on its chelating actions (7-9). acid, but, highly resistant to cetrimide; on the I I The integrity. of the lipopolysaccharide other hand, PS. 021 was very highly resistant to component of the outer membrane of Gram benzoic acld and cetrimide. These various I negative bacteria is strongly msintained by the observations occasioned our choice of test divalent cation, Mg2+(magnesium ion). The microorgan~smfor the vhious antimicrobial
chelation of Mg2' by EDTA therefore leads to combination evaluation. 4 the breakdown of the outer membrane structure (which is the major component of Gram I i negative bacteria cell wall). This usually result Materials and Methods i I in the lysis of the bacteria, (1, 10). The breakdown of the outertmembrane structure of Test Microorganisms the Gram negative bacterial cell wall also Clinical isolates of Candida albicans, i usually results in enhanced*;permeation of Aspergillus niger agd ~seudomonas antimicrobial agents which ordinarily could not aerugimsa (strain Ps. 02 1) were obtained from permeate the bacterial cell wall, This accounts the Department of Microbiology, University of for the observed potentiation of antimicrobial Nigeria, Nsukka. agents against resistant Gram negative bacteria :i in the presence of EDTA (l,3 - 12.). Studies Culture Mefia 1 on the antimicrobial properties'of EDTA and Nutrient agar: nuuimt broth, sabouraud its combinations with various antimicrobial dextrose agar, sibodd dextrose broth agents have nlostly been confined to bacteria (oxoid); Cetrimide agar (merck). All media (both Gram negative and Gram positive) (1,5 - were prepared according to manufacturer's 12). Attempts at evaluating these antimicrobial specifications. 8. effects against fungi (mould and yeast) have so Other materials uied as obtained from far, not been documented. The dependence of the suppliers without further processing fungal growth and/or at least a critical stage in include: EDTA (sodium salt), DMSO, Benzoic fungal growth cycle on divalent cations such as acid, Cetrimide (BDH chemicals).
magnesium (Mg 2+)and calcium (Ca2+)is well I' known (1 3, 14). It is possible therefore, that EDTA, with its chelating actions on cations Maintenance and Standardisation of could. affect fungal growth indirectly via Microorganism j 3I chelation of essential cations. i I In this work therefore. we evaluated the Stock of all test microorganism were combined antimicrobial effect of EDTA and maintained, and prior to each experiment, cetrimide against fungi (Asp. niger and C. standardized according to reported methods (8, albicans). The combined effect of EDTA and 15, 16). Ii C. 0. / and Esimone et a1 Journal of Pharmaceutical Research Development, Vol. 4i';. 0. 1 (1999);1-8 Deterhinotion of the ~inimurnInhibitoty FIC index + FIC, Concentration' (MIC) of Benzoic Acid, where A and B are two antimicrobial agents :, . , :,' Cetrimide and EDTA agaihst the Test being combined. !I,.)...... Microorganisrtts I FICA- Frnctional Inhibitory (:oqccntrationi I of Dmg A. Stock solutions of ED~A(4mgIml). i I benzoic acid (2mg/ml) and cetrimide (4mglml) RC, ,MIC of Drug A in combination wilh Dmg B were prepared in double; strength nutrient MIC of Drug ~ialone ., broth. Benzoic acid was soiubulized with I ; 1Omls of DMSO prior to incoporation into the FIC, - Fractional Inhibitory Concentration with Drug B. . . nutrient broth. The stock solutions were t? i MIC of Drug B in cornbjnation with Drug A sterilized in' an autoclave and their MICs FIG, , against the test microorganisms determined by MIC of Drug B alone the broth dilution method (17). The innoculum size of each test microo&anism was approximately 1 x ld cclUml. I The Activity Index j [A:I) - Log FIC index. ( 1 I:wdrrntiorr of rlrc Corrrhincd Acti\*ity of I I.,'lITAwith eirlr er Hett;oic Acid or Cetrirrridc I:'valration of the ~nctehcidnlActivity OJ by rite Checkerboard Method , . Nenzoic acid, EDTA and !heir corrrhirtntiorrs Stock solutioni of I berizoic acid by the Death Curve Metlrod Reaction mixtul-es tiitli final volumes . ' (6nq'1iil), cetriniidr: (6nig/nl,l) ;~ricl EDTA ~f IOmls were prepared tiiviwioils (81iip/nil) were firepared in dbuble- strength concentrations of EDTA alone. belizoic acid nutrient broth and sterilized in ill1 u~~toclii\~eat alonz and their conibinatio~iin sterile ~iirtl-ienr I 2 1 "c for 1 5 minutes. The~~ilfte~..\'iuying broth. Thereafter. I1111 yl il s:.~nctiudizciI proportions ranging from 0: 10 to 10:O of suspension (I x 10' crl!s'nil) of' rlie test riiicro EDTA and either benzoic acih or cetriniide organisni (Ps.acr-i~yittostc strain P.s. 02 I ) was solution were ni.ixed accoimdjng to the introduced into the . reaction niixture i111cl continuous variation Checkerboard niethod introduced into 5niI of ndrrient broth in test (18). Each proportion of the antiniicrobial tubes, serially diluted ten-folds and the bacteria count estimated by the niethod of Miles and. agent combination was serially diluted (2-fold) Misra (1 9). with sterile nutrient broth, after which 0. I rnl of
" *.. the standardized culture (10' ~ellsln~l)of the Results and Discussions I test microorganisms (either C. olbicms, Asp. The combined effects of EDTA with niger or Ps. ~krrr~inosa)was incorporated into cetrimide and benzoic acid against each of the dilutions. After 24 hours Pseudomonas aenrginosci ;I (Ps. 02 1) wero incubation at the appropriate temperature, the shown in Tables 1 and 2 respectively. tubes were observed for presence or absence of In the evaluationi of interactions microbial growth and the MIC of the various between two antimicrobial agents by the combination proportions ; determined. checkerboard method. values of FIC index Interaction between the antimicrobial less than one indicate synergism, the degree of synergy increasing as the value turns agents was assessed by determining their D I toward zero (1 8.20). A ! ,negative value for ~ractional Inhibitory C~ncentmtion (FIC) activity index also indicates synergism. It ' the relationship: follows therefore from Table -1. :that3 I '1 I i 'II : Colnbinctl effect ol',Cctrinlitlc i1n0 IJIYl'A against P,seudomonu.s ueru~fnosu(Ps. 02 1 ). I 1
f' l)l I0:O l2.W I :O 1.00 1 0 . 9. I I 12.5: I 12.5 o.o.o.os 0.95 T -0.022 ,,, , x:2 ~rnm o.x:o. I - 0.90 -0.046 , 0.4 75:Sfl 0.0 0.2 0.80 -0.097 , . I " 5:5 t 02..5:02.5 0.5:0.25 0.75 ''! 0.1249 4: 0 50:SO 0#2:0.4 0.6 Ct -0.22 2:X 25:2Oi) 0.2:0.8 1 .oO 'I 0 I :o . . 12.5:225 0. I :0,9 1 .OO 'I 0 I): 10 . omi o:I !.oo 1 4)' '. i . - Kcy: C'I:'l' - ('c~rit~~itlc. 'i I. . 11 1 :I 1 five oul ol. [llc scvm di-ug ct,~llhiniltit,n respectively. For the ~ther~combinationratios. ratios (cetritnitlc: A exhibited it is equally~observeclthat the MIC of EDTA synergism against Pscadotnr~n~sueruaino.3~ is always reduced to a &eater extent. The strain Ps. 021. Ra~io4:6 (thilt is 4 pi1rt.S of implication of this findine: is that both agents cetrimide and 6 parts of EDTA) exhibited the modify thi ictivity of eaGh other against pr.. greatest degree of synergy while 9: I exhibited 021 and that cetrimide modifies the aclipity of the leasi. The other drug combination ratios EDTA to a much larger;jextent than EDTA exl!ibited additivity. A closer look at ratio 4:6 (the combination ratio that gave the highest does to it. In other words! cetrimide could be degree of synergy) reveal:; that the said to'sensitize this Gram negative organism * ,n combination ' reduces the MI^ of (Ps. aeruginosa strain Ps.. 021) to the actions cetrimide and EDT,A,. by 2.5 and 5 timi; of EDTA. This -hdwe;br is not the case. I I i I I C I l . I 1 I I Table 21 Combined activity of Benzoic acid and EDTA against Pseudomonas aeruginosa (Ps.02 I). , t Drug Ratio MIC (mdml) FIC FIC index 11 Activity index . (BA: EDTA) 1:9 ' 25:225 0.0 1250.9 1.9125 -0.040 , I I . 0:lO : 0: 250 0: 1 1 0 . - .. > I Key: BA - Benzoic acid. I I 1 I I . . I t- ' I with the combinations of by?zoic and EDTA (1.8- 12). This dis~uptio/' res%. Its fron! ... the fact Table 2 where six out of. the seven ratios that EDTA chelates 'plagnesium. ions' that exhibited synergisni and EqTA is observed to maintain the integrity of the lipopolysaccharide sensitize Ps. 02 1 to the actions of bcnzoic acid. (LPS) of GI.~IIIInegativb bacteria cell wall (8, For instance in the drug ratio that exhibited 1 1. 12). Our observation that cetrimide the greatest synergy (5:5) the MICs of bcnzoic enhances (sensitizes) the effect of EDTA acid and EDTA are reduced by 16 and 2 times against our test strain of,Ps. Aeruginosa could respectively. Several reports have shown that be because cetriniiide (a surfactant) EDTA sensitizes Gram negative organisms. enii~lsifies/solublize~the vast proportion .of especially Ps. nrrugitlosnl to the action of lipid coniponents of whiqh' the outer membrane several antimicrobial agents (5, 7, 8, 10). It of Gram negative bacteria cell :Wall is made, has been proposed that EDTA exerts this thereby possibly incdasingti . the ease with sensitizing effect by ,! disrupting the !. pem~eability barrier which the outer which EDTA !gains access to the membrane of Gram negative bacterial cell lipopolysaccharide component of the' outer wall imposes on most antinkmbial agents I ,I Table 3: Combined effect df Cetrirnide and EDTA against Codida albicam I I Drug Ratio MIC (mg'nil) FIC FIC index Activity index ! (CET: EDTA) I i I Key: CET - Cetriniide Table 4:'~onibined'effectof Cetrirn ide and EDTA against A.spergilkrs,t~i~e,. . . '/ Drug Ratio MIC (n~g'nil) FIC 1-IC intlcx ( Aclivily index I! (CET: EDTA) :II i I I l i, t C. 0.Esimone et a1 !Journal'of Pharnrclceuricczl Research arid Developnwrrr. Vql. 4. No. 1 (1999); 1-8, I % ! Tables 3 and 4 shdw the combined ions. It is possible theiefove that EDTA, by effect ' of. cetrimide and EDTA at seven chelating the metal ions essential for the different proportions against '~ardidaalbic& growth of fungi contributes in inducing fungi and Aspergillus niger respectively. The results stasis. However as shown from the very high are identical and reveal therefore that the effect MIC value of EDTA alone against the fungi of the combination against (ungi is the same (2000 n~dnil)one can conclude that it h(?~very irrespective of whether the fungi involved is a n~inin~alactivity against fungi. It hqwever yeast (C. Albicans) or a mould (Asp. niger). does nlotiifi (enliances) the fungistatic activity These results show that EDTA modifies of cetrimide considerably. (enhances) the antimicrobial properties of The Death -!curves showing I ' cetrimide against the fungi. This activity may antibacterial activities; 1 of various not be unconnected to the fact that fungi, in concentrations of benzoic acid and their some stages of their growtb c cle depend conlbinat ion against Yscrr~lonrona.~ucrugino.su considerably on calcium ion' (CaY+ ) (13, 21). Tallentire and Chiori (14) have also (Ps. 021) is presented in; ~i~ureI. Benzoic demonstrated that sponllation:of heat resistant acid (0.21ng/liil) exhibited a slight spores be affected' by divalent metallic ion (bactericidal) action up 1 bo the fourth hour, content of the growth medium. In their after which the resistant cells exhibited a work, they showed that Mg2+is essential for significant increase in viable number. EDTA sporulation together with iron and manganese I (0.2mg/ml) on the other hand exhibited . I)I . +A ...A-.. 13 -o-C +D - *.. E bactericidal action up to, the sixth hour. The various combinations of BDTA and Benzoic I ', ', acid exhibited the best bactericidal activity. r , . , , xl! the combinations recorded the lowest number of bacterial SU~.~VO~S(compared to the antimicrobial agents alone) at the end of the fourth hour. The combination containing equal proportion of EDTA &d Benzoic acid (0.2mgIml each) seems to possess the greatest efficacy, recording th~lowes< numb& of survivors at the end of the third hour. Conclusively, these combinations could be said to possess synergistic bactericidal- .-..- properties against Pseudontonas aeruginosa (Ps. 02 1) I 5.00 I,, because wlien conipnrcd tq!the.n~osteffective 1 2 3 41 5 a single antimicrobial agent (EDTA) they caused a decrease of at least 41 og," in viable cell count at the end of the sixth hour;] Fig. 1. Death Curves Showing Antibacterial Activities I of Various Concenuarions of Benzoic Acid (BA). .' I EDTA and Various ~6mbi"ationsof EDTA ad ~~~~l~~i~~~. I: Bencoic Acid. , . i , . There is striking synergy-. between A - 0.2mg/ml BA; combinations of EDTA and either cetrimide or B - 0.2mg/ml EDTA; ' C - 0.1 6mglml BA + 0 24mglml EDTA benzoic acid against C. albicans, Asp. niger D - 0.36mglnil BA + 0.04n1d,ml EDTA; and Ps. nenginosn. The effeit is both' .. E - 0.2mglml BA + 0.2mdnd EDTA . biostatic and biodical. This finding will be of : ' I J I 'I yp./. ., I ! C. 0.Esimone el ul I Journd of Plforntuceuticul Ressearch and Developmenf,Vol. 4. No. 1 (1999); 1,8 great significance in educing kesistance of 5. Gerberick, G. F. an$I Cap, P. A. various microorganisms to the ac Ions of fairly, (1980). In-virro ju~ceptibilit~of . is t weak pmsewativ& like bebzoib kid, while the Pscudomonas a f ru inosa to spectrum of fairly strong agents like cetrimide. carbeniallin, Glycin and EDTA could be increased. I combinations. J. ~nfimicrohialAgent and Chemotherapy, 17: 732-73S. I Acknowlcdgmcnt 1 We acknowledge the asiistance of all 6. McBride, J. R. and Richards, R. M. 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