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The Effect Off Ethylenediamine Tetraacetic Acid 4 the Antimicrobial Properties of Benzoic

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The Effect Off Ethylenediamine Tetraacetic Acid 4 the Antimicrobial Properties of Benzoic 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<ll(; ,@ MI(' (11igh11) I;M' FIC indcx Activity indcx : l<A'l'l()(C'l-.'l':lil YI'A) ! i 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 .
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