Electrochemical classification of antibacterial effects of betalactam antibiotics on Escherichia coli cultures

331

Bzoelecrrochernrsfry and Btoenergetrcs, 22 (1989) 331-340 A section of J Electroanal Chem , and constitutmg Vol 276 (1989) Elsevler Sequoia S A, Lausanne - Prmted m The Netherlands

Electrochemical classification of antibacterial effects of betalactam antibiotics on Escherichia coli cultures * Thierry Jouenne and Guy-Alain Junter Laboratorre de Chwnre MacrornolPcularre, U R A 500, Facultt! des Scrences de Rouen, B P 118, 76134 Mont Samt Algnan Cidex (France) (Received

27 September

1989)

ABSTRACT The potential-time responses of Escherrchra colr cultures exposed to various betalactam antibiotics were examined Thus systematic study led to an ongmal classification of the drugs mto three mam groups Antibiotics of the first group &d not affect bactenal reducing activity before causmg cell lysls The second group, the largest, involved ant¬ics whuzh stimulated both dissolved oxygen uptake and hpoic acid reduction before they revealed their bactenolytic potency The thud group, mcludmg only one antlblotic, cefatnzme, induced potentiometnc responses smular to those recorded m the presence of antllnotlcs actmg on protein synthesis These results are discussed by takmg mto account known data on the antibactenal properties and structural features of the drugs

INTRODUCTION

The potentlometnc measurement of hpolc acid (LA) reduction by bactenal cultures exposed to antlblotlcs 1s a simple procedure whtch has proven able to provide ongmal mformatlon on the antmncroblal effects of the mlubltors (see ref 1 for a review) In particular, the potential-time responses of cultures subnutted to antlblotlcs of the betalactam fanuly [2-41 are quite different from those induced by antlblotlcs acting on protein synthesis, e g ammoglycosldes [5] or macrohdes [6] In previous papers, we investigated the effects of cefotaxlme, a senusynthetlc cephalosporm, on the reducmg actlvlty of Escherzchuz colz cultures [2], and we analyzed these effects wlthm the framework of our modelhng of potential-time phenomena [4] The present study 1s devoted to a systematic survey of the potentlometnc responses induced by betalactam antlblotlcs, including both pemalhns and cephalosporms of different generatlons [7]

* Presented at the 10th September 1989

Bmelectrochenucal

0302-4598/89/$03

0 1989 Elsevler Sequma

50

Conference

SA

(BEC

X), Pont-8-Mousson

(France),

24-30

332 EXPERIMENTAL

Test orgamsm, culture medium and mcubatron condltlons The test orgamsm was a laboratory stram of E colr, provided by the Chmcal Bactenology Laboratory of the H&el-Dleu m Rouen Bactena were cultured at 37 o C m the nummal salt medmm already used m our previous studies, supplemented mth glucose (10 mM) and hpolc acid (10 PM) Standardized mocula were obtamed from these cultures (final cell concentration m the test flasks c lo6 orgamsms cme3 medmm) All measurements (time courses of potential, bactenal growth, dissolved oxygen and glucose consumption) were camed out m the rmmmal culture broth provided with glucose, LA and increasing amounts of antlblotlcs, under controlled aeroblosls [2], 1 e with a contmuous oxygen supply to the culture broth by transfer from residual an m the test flasks Antlblotlcs and coenzyme The antlblotlcs tested were kmdly supphed by the followmg laboratones Allard, Pans (cefazohn), Bayer Pharma, Sens (mezlocllhn), Bnstol-Myers S A, Pans (cefadroxd, cefatnzme), I C I -Pharma, Cergy (cefotetan), Lilly France, Samt-Cloud (cephalexm, cefaclor, cefamandole, moxalactam), Merck Sharp and Dohme-Chbret, Pans (cefoxltm), Roussel, Pans (cefotaxune) DL-a-hpolc (thloctlc) acid was a Merck product (Darmstadt) Determmatron of MICs (mmlmal mhlbltory concentrations) The conventional MICs of all antlblotlcs tested were determmed broth dilution method, as already described [6] Potential-time measurements The expenmental setup for the potent&-time previously [2,3] Momtormg of bacterial growth Optical density (OD) measurements Umcam SP6-550 spectrophotometer

measurements

were performed

by a standard

has been detaled

at 546 nm

ulth

a Pye

Measurements of dissolved oxygen consumption The time evolution of dissolved oxygen (DO) partial pressure dunng bactenal growth was momtored wth the expenmental setup described elsewhere [2,4] Measurements of glucose consumption Colonmetnc measurements of the glucose concentrations were performed at 500 nm usmg glucose oxldase/peroxldase enzymatic Color, Blotrol, Pans)

m the culture broth enzyme luts (Gluco-

333 RESULTS

Potentzal-time measurements m E cob cultures exposed to mcreasmg amounts of dzfferent betalactam antlbrotxs The betalactam antlblotlcs tested could be classified into three main groups according to the potential-tune responses they induced m E cob cultures (1) Antlblotlcs belongmg to the first group (1 e mezlocllhn, cefoxltm, cefadroxll) exerted practically no mfluence on the potentlometnc lag time t(lOO mv), 1 e the time necessary for a 100 mV potential shift to appear after moculatlon of the culture broth Sudden potential U-turns (1 e the potential stopped falling and increased again) occurred after or dunng the wave (Fig 1) (u) “Paradoxical” effects on the t(lOO mV) values were recorded m the presence of a number of betalactams, e g cefotetan (Fig 2) Moreover, w&m a certam range of concentrations, increasing amounts of these antlblotlcs surpnsmgly decreased the potentlometnc lag time before the appearance of the potential U-turn (Fig 2) In addition to cefotetan, this second group included cefaclor, cefamandole, cefotaxlme, cephalexm, cefazohn and moxalactam (m) In contrast to the antlblotlcs of the second group, cefatmne did not induce potential U-turns and increased t(lOO mV) values (Fig 3) The maximum decrease m t(lOO mV) values was observed m the presence of cefaclor (Table 1) Thts antlblotlc, together with cefamandole, cefotaxlme and moxalactam, exerted its maxlmum stlmulatlve effect on bactenal reducing actlvlty at low concentrations, below the MIC, the other betalactams of the second group were more efficient at lugher concentrations, m the range of their MICs (Table 1) Effects of betalactams on bacterial growth Figure 4 shows the time courses of the OD of E colz cultures subnutted to betalactam antlblotlcs representative of the three groups dlstmgulshable from then

POTENTIAL

/fllv

Ag 1 Potentml-time signals recorded m E cok cultures prowded wth mcreasmg amounts of mezlocdhn Antlblotlc concentration pg cmp3 (1) 9, (2) 18, (3) 45, (4) 55 (- - - - - -) Control wthout antlblotx

334

POTENTIAL

/mV

t

Rg 2 Potential-time signals recorded m E co11 cultures supplemented wth mcreasmg amounts of cefotetan Antlblotlc concentration pg cme3 (1) 0 15, (2) 0 3, (3) 0 6, (4) 0 9, (5) 1 1 (- - - - - -) Control without antibiotic

potent&-time measurements, 1 e mezlocllhn, cefotetan and cefatnzme A single concentration was used for each antlblotlc, corresponding to a typical potential-time response (see Figs l-3) At the concentrations used, the three antlblotlcs caused the OD to decrease rapidly after a penod of increase snmlar to that of the control OD-time course Except cefatnzme, which did not induce potential U-turns (Fig 3), the time necessary for the sudden reduction of the OD to appear corresponded to the tune needed for the U-turn of the potential to appear BacterIaI consumption of dzssolved oxygen m the presence of betalactams Betalactams of the three groups exerted different effects on the kmetlcs of DO uptake by E colz cultures (Fig 5)

POTENTIAL

/mV

t

-4001

Rg 3 Potentlometnc responses of E colz cultures exposed to mcreasmg amounts of cefatnzme Antiblotlc concentration pg cmm3 (1) 1 7, (2) 2 5, (3) 2 7, (4) 2 9, (5) 3 1, (6) 3 4, (7) 4 8 (- - - - - -) Control without antlblotlc

335 TABLE

1

Classlflcatlon of the betalactams tested mto three groups according to the potential-time Induced m E co11cultures Antlblotlc

Max decrease of r(100 mV)/%

MIC /pg cmm3

responses they

Correspondmg antlblotlc cone /pg cmm3

Cefadroxd Cefoxltm Mezlocdhn Cefaclor Moxalactam Cefamandole Cefotaxlme Cephalexm Cefotetan Cefazolin Cefatnzme

6 -12 153 >lOO

86 _

0

50 -100 02505 10 - 20 3 -6 3 -6 01503 3 - 6 25-

48 0

5

41 34 26 25 25 24 22

22 9 007 35 0 14 40 0 14 30

0

In the presence of mezloalhn, the bactenal consumption of DO was slower than m the drug-free medium A sudden mhlbltlon of the bactenal respiration occurred, which caused the DO concentration to stop falling and increase agam The uptake of DO by bacterial cultures exposed to cefotetan was faster than m the absence of

A

,l --

Fig 4 Time courses of optical density of cultures exposed to different betalactams Antibiotics (0) mezlocilhn (45 pg cm-‘), (0) cefotetan (0 1 pg cme3), (W) cefatrvme (17 pg cmm3) (0) Control without antlblotlc

336

02

01

(1,

0

TIME/mm t 120

240

360

Fig 5 Bacterial consumption of dissolved oxygen m the presence of different betalactams (17 pg cmm3) (2) mezlocdhn (45 pg cmm3), (3) cefotetan (0 1 pg cm -3). (4) cefatnzme wthout antlblotlc

Antlblotlcs (1) Control

360 0 240 120 Fig 6 Bactenal consumption of glucose m the presence of different betalactams Antlblotlcs (0) mezlocdhn (45 pg cmm3) (0) cefotetan (0 1 pg cme3), (w) cefatnzme (1 7 pg cmw3) (0) Control wthout antlblotlc

337

the drug This stlmulatlve effect on DO consumption was followed by a drastic reduction of the nucroblal respiration, disclosed by a U-turn m the DO concentratlon On the contrary, cefatnzme reduced the DO uptake rate without mducmg U-turns m the DO concentration Injluence of betalactams on bacterial consumption of glucose Figure 6 shows the time evolution of the glucose concentration m E colz cultures exposed to mezlocdhn, cefotetan or cefatIlvne The three antlblotlcs mtiblted glucose consumption However, the consumption of glucose by the orgamsms was increased by cefotetan dunng the u-&al stages of mcubatlon, whde mezlocllhn and cefatnzme did not exlnblt ths stlmulatlve effect DISCUSSION

The present mvestlgatlon of the potentlometrlc behavlour of E cob cultures exposed to vmous betalactam antlblotlcs shows that ths farmly of antlblotlcs 1s not homogeneous as Judged from the LA-based potentlometnc procedure, since the betalactams tested could be classlfled into three groups according to the potential-time responses of the cultures We have already analyzed the typlcal potentlometnc responses of E cob cultures exposed to antlblotlcs classlfled m the second group, takmg cefotaxlme as an example [2,4] In bnef, the potential U-turn corresponds to the lysls of abnormal forms of bactenal cells induced by bmdmg of the antlblotlcs to their protein targets (pemclllm-bmdmg proteins PBPs) [8] Cell lyns, hlghhghted by OD measurements (Fig 4), 1s also responsible for the U-turn of the DO concentration (Fig 5) The decrease m the t(100 mV) lag time values can be ascribed to stlmulatlon by the drugs of the reducing actlvlty of organisms, 1 e DO consumption (Fig 5) and, more particularly, LA reduction (1 e , LA transmembrane transport, considered to be the hrmtmg step of the LA reduction process) [4] In agreement with our previous observations on cefotaxlme [2], antlblotlcs of the second group increased glucose consumption (Fig 6) before cell lysls occurred Antlblotlcs belongmg to the first group did not exert a stunulatlve effect on bactenal reducing actlvlty and glucose uptake, although they showed bactenolytlc potency The case of cefatmme 1s questlonable The potentlometnc responses of E cob cultures exposed to ths antlblotlc (Fig 3) were quite sumlar to those of cultures incubated m the presence of antlblotlcs actmg on protem synthesis [5,6] We have attnbuted these effects to the mhlbltlon of bacterial growth by the drugs, the speclflc growth rate of the orgamsms bemg a decreasmg function of the antlblotlc concentration The time evolution of the OD of cultures subrmtted to cefatmne (Fig 4) shows that this antlblotlc exerted a bactenolytlc action wluch did not appear on either the potential-time curves or the kinetics of DO uptake (Fig 5) probably, the bactenal started to grow agam very rapldly durmg the potentlometnc lag penod followmg thts lytlc effect, thus obhteratmg the potential U-turn

338

Two mm questions arise from the results presented here The first one 1s whether the potentlometnc classlflcatlon of the tested betalactams reflects known differences m the antlbactenal propertles of the drugs Cephalosporms have been classified mto several groups based on their pharmacologlcal and antlbactenal properties such as parenteral or oral admmlstratlon and betalactamase resistance [8] More usually, they are divided into three “generation” groups on the basis of then antlbactenal spectrum [9] The different groups and generations of antlblotlcs were represented m the present study, e g betalactamasesensitive, parenteral fn-St-generation agents (cefazohn), betalactamase-resistant, orally absorbed antlblotlcs of the first (cefaclor, cefadroxd) or the second (cephalexm, cefatnzme) generation, and thud generation antlblotlcs (cefotaxlme, cefotetan, moxalactam) No correspondence could be found between ths conventional classlfication of cephalosporms and the three groups determmed by the potential-time measurements The structure-activity relatlonshps m betalactams, 1 e the effects of structural modlflcatlons on the blolo@cal propertles of the drugs, have also been widely investigated [lo] The cephalosporms are essentially blcychc rmg systems composed of the four-membered lactam rmg to which a six-membered rmg 1s fused Table 2 gves the structures of the different cephalosporms used m the present work It seems difficult to correlate the structure of these antlblotlcs with the intensity of their stlmulatlve actlon on bacterial reducing actlvlty For example, the R, and R, substltuents m cefazolm are quite different from those m cephalexm (Table 2), whereas these two antlblotlcs display slmdar antibacterial properties according to both the potential-time measurements and conventional susceptlblhty tests (MICs) On the contrary, the structures of cephalexm and cefadroxll, which do not belong to the same “potentlometnc group”, differ only slightly m the R, substltuent (Table 2)

It 1s also difficult to fmd a relation between the mtrmslc afflmtles of the tested betalactams for the PBPs of E colz and then antlbactenal activity according to the potential-time measurements Thus, most antlblotlcs of the second group, e g cefamandole, cefotaxlme and cephalexm, bmd pnmanly to PBP 3, causing the formation of fllamentous cells, the pnmary target of mezlocllhn, which belongs to the first group, 1s also PBP 3, and the morphological response of cultures subrmtted to this pemcillin is also cell filamentatlon [12] The second question addresses the mechamsms of action at the cellular level by whch betalactams of the second group stimulate bactenal reducing activity In our previous work, devoted to a hpolc acid-based potentlometnc study of the antumcroblal effects of cefotaxlme [2], we hypothesized that these betalactams rmght both affect energy transduction processes, acting as uncouplers, and permeabdlze the bacterial cell membrane, enhancing the transmembrane transport of LA By exposmg E cob and Staphylococcus aureus (a gram-posltlve organism) cultures to chenucals known to affect bactenal energy flow (e g uncouplers, arsenate) or to enhance the permeability of the outer membrane of gram-negative bactena (EDTA), we have shown recently that the stlmulatlon of the reducing activity of E colz by

339

TABLE

2

Structures of the cephalosponns tested (The oxacephalosponns cefoxltm, cefotetan R, =-OCH, For all other antlblotlcs bear a methoxy group m the 7a-posItIon Rohnson [111

Genenc

Rl

name

R2

-CH3

Cefadroxd

HO

N-N

Cefazohn

0/ \ -

CH-

-cH2s4S>CHJ

-CHg

Cephalexm

ilH2

-Cl

Cefaclor

N-N

Cefamandole hHj

Cefatnzme

-CH2S

c /

N \).,

El’

1 c)

’

CH2-

S

pNT=>\N

Cefoxltm

-CH2-O-

Cefotaxlme

AH20

!

C-NH2

COCH3

‘OCl$

NaoocxC+,, _ $lNOC'

N-N Cefotetan C”3 N-N

Moxalactam

-CH2Sl(N> &ij

and moxalactam R, =-H) From

340

betalactams of the second group can be attnbuted mamly to a permeablhzatlon of the outer membrane of the orgamsms These results ~11 be detaled m the near future [13] ACKNOWLEDGEMENT

This work was supported by a grant (Contrat de Recherche Externe No 872006) from the Instltut National de la Santt et de la Recherche MCdlcale (INSERM) REFERENCES 1 G A Junter m G A Junter (Ed ), Electrochenucal Detection Techniques m the Apphed Blosclences, Vol 1, Analysis and Chmcal Apphcatlons, Elhs Honvood, f&Chester, 1988, p 167 2 GA Junter and M C Bogaczyk-Hbbert, J Antlrmcrob Chemother , 15 (1985) 685 3 G A Junter, Path01 Blol , 34 (1986) 549 4 GA Junter, Bloelectrochem Bmenerg , 17 (1987) 43 5 M C Bogaczyk, E Selegny and G A Junter, Bloelectrochem Bloenerg , 12 (1984) 93 6 T Jouenne, D Lemome and GA Junter, Bloelectrochem Bloenerg , 21 (1989) 161 7 B G Spratt, J Gen Mlcroblol , 129 (1983) 1247 8 C H O’Callaghan, J Antmucrob Chemother , 5 (1979) 635 9 L Balant, P Dayer and R Auckenthaler, Chn Pharmacokmet , 10 (1985) 101 10 J R E Hoover, Handb Exp Pharmacol , 67 (1983) 119 11 G N Rohnson, J Antmucrob Chemother , 17 (1986) 5 12 N A C Curtis, D Orr, G W Ross and M G Boulton, Antnmcrob Agents Chemother , 16 (1979) 533 13 T Jouenne and G A Junter, m preparation