A.O. Vet. Fak. Derg.
34 (3): 513-518 • 1987
EFFECT OF EDTA.TROMETHAMINE ON THE SUSCEPTmILITY OF CAMPY. LOBACTER JEJUNİ AND CAMPYLOBACTER coL! TO ANTIMlCROBIAL
AGENTS
K. Serdar Diker'" Mustafa Ardaıl< '"
Caınpylobacter jejuni ve Caınpylobacter coli'nin antibiyotiklere duyarlılıtına EDTA-troınethaınin'in etkisi
Özet: Altı aııtimikrobiyal ajaıı (ampisilin, tetrasiklin, eritromısın, kloramfenikol, ııalidiksik asit ve kolistin sülfat) ve ED T A-tromethamin ile kombinasyonlarının Campylobacter jejuni ve C. coli üzerindeki sinerjistik etki-leri ince/endi. ED T A-ampisilin, ED T A-tetmsiklin, ED T A-eritromisin ve ED TA-kolistin süljat bileşimlerinin campylobacteder üzerinde belilgin siner-jistik etkileri görüldü. Kloramfenikol, ED T A-tromethamin ile birlikte
kullanıl-dığında zayıf bir ortak etki belirlendi. ED T A-tromethamin ve nalidiksik asit kombinasyonunda belirgin bir etki artışı bulunmadı. C. jejuni ve C. co/i'nin, ED T A-tromethamin ve antibiyotik/ere duyarlılıkları arasında bir fark belirlen-medi.
Sunınıary: Combinations of ED TA-lromethamine and 6 antimicrobial agents (ampicillin, tetmeyc!ine, eıythrom)'cin, chlommphenico/, nalidixic acid and colistin suljate) wae testedfor synergistic activities against CamjJylobacter jejuni and C. coli. A marked syneıgistic action was san when camp)'lobacters
were exposed to combinations of ampicillin-EDTA, tetraeycline-EDTA, erythromycin-ED TA and colistin suljate-ED TA. When chloramphenicol was mixed with ED TA-tromethamine, a slight synergistic effect was ılOticed. There was no synergistic effect rccorded with combination of ED T A-tromethamine and nalidixic acid. There was no difference between the sıısceptibility of C. jejuni and C. coli to combinations of ED TA-tromethamine and antimicrobial agents.
* Rcsearch Assistant, Ankara University, Vcterinary Faculty, Department of Mic-robiology, Ankara - 06110, Turkey .
•• Prof. Dr., Ankara University, Veterinary Faculty, Department of Microbiology, Ankara - 061LO,Turkey.
5H K. SERDAR DiKER -- MUSTAFA ARDA
Introduction
Cell surfaces of Gram-negative bacteria are demaged by expo-sure of ceııs to EDTA (ethylcnediaminetctraaccticacid) (8). Tromet-hamine (tris-hydroxymethyl-aminomethane) enhances the effectof EDTA (5). Gram-negative bacteria exposed to EDTA-tromethamine have increased permeability to extraceııular solutes and ıcakage of intraceııular solutes (7). Such bacteria become more sensitiye to the ("ffect of Iysosyme and antimicrobial agents (3). Combinations of cer-tain aııtibiotics and EDT A-tromethamine cause a decrease in the MIC (minimal inhibitory concentration) for Escherichia coli (I i), Pseudomonas aeTIIginosa (I 2) and Proteus vulgaris (I 4). Cilinicaııy, com-binations of EDTA-tromethamine and antibiotics have been used cffectively in the treatment of pseudomonas rhinitis (I O) and multiple fistulas (2) in dogs and metritis in mare (IS). Cell surfaces of Gram-positive bacteria are more resistant to the effect of EDTA-trometha-mine (13).
The purpose of the present study was to determine the inhibitory effect of combinations of 6 antimicrobial agents and EDTA-tromet-hamine on C. jejımi and C. coli.
Materials' and Methods
ThreeC. jejuııi and 6C.coli strains isolated from anirnal origirls by selective method were usecl in this study. Aıı of C. j~jll7li and 4 of 6 C. coli were isolatecl from chickens, one C. coli came from a cow with mastitis and one C. coli came from an aborted sheep ktus. Campylo-bacler spp. wc re identifiecl accorcling to the established criteria (9), and stOlTd at - 70 oC until usecl. Cephalathin resistant7 nalidixie acicl sensi-tive organisms that hydrolysed hippurate were identified as C. jejuni
and those did not as C. coli. .
The antimicrobial agents tested were ampicillin, colistin sl.,llfate, nalidixic acid, tetracyeline, erythromycin and chloramphenicol.
Minimal inhibitory concentrations (MIC) were determined by stanclard methods for agar clilution tests on Mueller-Hinton agar containing 5% defibrinated sheep bload. Aıı strains stored frozen were subeu1tured on this medium under microaerophilic atmasphere. These bacteria were suspended to a concentration of 108 CFU
ımı
(colony forming unit per ml) in bnıceııa broth. A 0.01 ml calibrated loop was used to spot the suspensian onto plates of Mucııer-Hinton
EFFECT OF EDTA - TROMETHAMINE ... 515
agar containing- two-fold" concentrations of antibiotics. This inoculum correspondcd to 106 CFU per spot. The plates were incubated at 37 oC in mieroaerophilic atmosphere for 36 hours. The MIC was defincd as the lowest concentration of drug aUowing no visible growth. The MICs for the 6 antimicrobial agents wcre also tcsted on MueUer-Hinton agar supplcmented with
ı
mM EDTA and 50 mM tromet-hamine in the same procedure. Appropriate eontrols for bacteria, antibiotics and EDT A-tromethamine were included 111 each test. Each experiment was perfarmed twicely.Results
Thc MIC for the 6 antimicrobial agcnts and antimicrabial agents plus EDT A-tromethaınıne are shawn 111 Table I. The addition of
Tablc i" In "itro ,usccptibiliıy of 3 C.jrciımi and 6. C. cbli ,trains to antimicrobial agcnts with or wİıhout EDTA-tromcthaminc.
ANTIBIOTIC Number of sıraim
MIC (tLg/ml)
",,"iıhoııl EDTA With EDTA-lroma.
AmpiciIlin C. jcjııni
i
3 8 2 Colistin Erythromycin Chloraınphenicol TctracycIinc Nalidixic acid C coli C. jcjuni C coli C. jcjuni C coli C. jcjuni C coli C. jejuni C coli C. jejuni 2 2 i i i 2 6 3 6 3 i 2 6 3 4 2 8 32 512 512 512 4 4 64 2 2 2 4 i 0.5 2 8 512 128 128 0.25 0.25 2 2 32 2 0.5 0.5 4 C coli 6---nı
mM EDTA plus 50 mM ırümeıhamine516 K. SERDAR DİKER - MUSTAFA ARDA
EDTA-tromethamine to medium with nalidixic acid show ed no dec-rease in MIC. Marked decreases (4-fold) in the MIC wcre seen with combinations of erythromycin plus EDTA-tromethamine and ampi-cillin plus EDTA-trometha~ine. A significant decrease (4-fold) in the MIC also occured with combinations of tetracycline plus EDT A-tromethamine and colistin sulfate plus EDT A-tromethamine except one C. jejuni strain. Chloramphenicol, when eomhined with EDT A-tro-methamine had a two-fold dccrease (not signifieant) in MIC for C. jeju71i and C. coli. There was no difference hctween the susceptibility of
e.
Jejuni and C. coli to the additive effect of EDTA-tromethamine. Theorigin of the organisms had also no cffcct on the results. In control group,
ı
mM EDTA plus 50 mM tromcthamine did not inhibit the growth ofe.
jejuni and C. coli.Discussion and Conclusion
Sublethal injury to bacteria has significant practical consequen-ces. Injury can include distruption of mcbrans, DNA or RNA mo-lecules and the loss of a number of metabolic functions (6). The prin-cipal action of EDT A-tromethamine on Gram-negative bacteria appe-ars to be the extraction oHunetional divalent cations (Ca+2, Mg+2,Zn2+) from the eeIl wall, whieh in turn eauses the release of phospholipid and a protein lipopolysaccharide eomplcx (8). The loss of thcsc su bstanees affects the structural integrity of the ccIl wall and causes the formatiOll of an osmotieally fragilc cell.
Antimicrobial agents are divided into groups based on their mode of action on hacterial cell (I). Thcse sites of antimicrobial acti-vity include; the bacte'rial ecll wall (ampicillin); the eytoplasmie membran e (eolistin sulfate); inhibition of protein synthesis at the ri-bosome levcl (chloramphenieol, tctraeycline, crythromyein) and DNA inhibitors (nalidixie acid). In the prcsent study, at lcast one antibiotic was chosen from eaeh group, to observe whether the mode of action would affeet the aetivity or not.
A significant decrease in the MIC occured with combinations of ampieillin and EDT A-tromethamine, in all strains of C. jejuni and C. coli studied. In other studies perl'ormed by using penicillin which acts on cell waIl like ampicillin, it has been found that Renicillin plus EDT A-tromethamine had decreased MI C for E. coli (Iı), Ps.
aeru-ginosa (12) and Pr. vulgaris (14), but no effect on MIC for Gram-po-sitiye bacteria (13). The activity of EDTA-tromethamine plus
ampi-EFFECT OF EDTA.- TROMETHAMINE ... 517
ciııin on C.jejuni and C. coli may be explained by the site ofaction these agents on bacterial ccll waıı synthesis. Ampicillin inhibits the bacterial ceıı waıı formation preventing cross linking of peptidogluycan strands (I). The action of EDTA may occur in the first step of the peptidogly-can biosynthesis where divalcnt cations (Mg+2 or Mn+2) are. required. The chelation of these cations by EDTA plus the action of ampicillin may explain their synergistic actian on C jejuni and C coli.
No synergistic effect on campylobacters was recorded with com-binations of nalidixie acid and EDT A-tromethamine. This has been supported by the findings of other studies in which it has beendeter-mined that combination of nalidixie acid with EDT A-tromethamine did not alter the MIC for Ps. aeruginOStl (12) and Pr. vu!gclris (14). But, a slight decrease in
Mıc
for E. coli has been observed when the con-centration of EDTA was inercaseel (ll).The synergistic action, observed in the present study, with com-binations of EDT A-tromethamine and colistin sulfate was not unex-pected since both agents act on the ceıı walI. Variable results have been obtained in other studies (1 1,12, 14), when EDTA-tromethamine was combineel with polymyxin-B, another agent of which primary site of action is cytoplasmic membrane (4).
In the present study, the synergistic actions seen with tetracycline plus EDTA or erythromycin plus EDTA were alsa not unexpected. Tetracyclinc, erythromycin and chloramphenicol act at the ribosome lcvel as inhibiting protein synthesis (1). This action, combined with the effect of EDTA which enhances the cell waıı permeability and degrades ribosomes, may explain their synergistic actian. It has been reported that synergistic effect occurs bctween combinations of EDT A-tromethamine and oxytetracycline or chloramphenicol on E. coli (I 1). When the findings were compared, no significant difference was observed between two species, C. jejuni and C. coli, tested. This may show that there is no major structural elifference between the cell waH composition of C. jejuni and C. coli.
In summary, the enhancement of antimicrobial activity in the pres en ce of EDT A-tromethamine appears to be a function of inc-reased permeability of bacterial cell, aııowing greater penetration of antimicrobial agent. From a clinical view, this combined activity may have limited use became local infections due to C. jejvni and C. coli are rather scare. But, it must be kept in mind that the addition of
518 K. SERDAR DiKER - MUSTAFA ARDA
EDTA and tromcthamİne to selcctİve mcdİa whİch contaİn sevel'a! antibiotics, may inhibit the growth of campylobacte~s.
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