Mardones 2000

Journalof

MethodsMicrobiological

Journal of Microbiological Methods 40 (2000) 199–206www.elsevier.com/ locate / jmicmeth

Chromogenic plate assay distinguishing bacteriolytic frombacteriostatic activity of an antibiotic agent

*Gonzalo Mardones, Alejandro Venegas´ ´ ´ ´Laboratorio de Bioquımica, Departamento de Genetica Molecular y Microbiologıa, Pontificia Universidad Catolica de Chile,

Casilla 114-D, Santiago, Chile

Received 26 April 1999; received in revised form 20 December 1999; accepted 5 January 2000

Abstract

A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterialagent. The assay uses a bacterial culture harboring b-galactosidase enzyme as reporter of cellular lysis. When a drop ofbacteriolytic compound is placed on the agar, b-galactosidase is released from the bacteria to the external solid mediumwhere it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assaywas successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostaticcompounds consistently did not display blue halo at the inhibition zone. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Antibiotics; Apidaecin; Bacteriolytic assay; X-Gal plates

1. Introduction similar way to hormones by binding to specificcellular receptors which require specific peptide

The knowledge about the mechanism of action of conformation. In contrast, the mechanisms of actiona new antimicrobial agent is basic to understanding of other antibacterial agents is less dependent onthe events occurring during bacterial growth inhibi- such stringent structural requirement. Among thesetion. This issue is very important for the develop- compounds, cytolytic cationic peptides with a widement of any antibacterial compound for therapeutic spectrum of action have been isolated from mam-use. malian macrophages — the so called defensins

Recently, several efforts have focused on studying (Ganz et al., 1990), from insects, — melittin (Haber-the mechanisms of a number of new antibacterial mann, 1972), cecropins (Steiner et al., 1981) andpeptides. Surface active peptides which bind and sarcotoxins (Okada and Natori, 1985) and fromalter amphipatic surfaces, including membranes and amphibians, — magainin (Zasloff, 1987). The targetreceptors, have been extensively studied (DeGrado et for these surface-active peptides seems to be the lipidal., 1981; Kaiser and Kezdy, 1983, 1984; Kaiser, bilayer of the cellular membrane. It has been re-1988). Some of these antibacterial agents act in a ported that their activity is exclusively due to their

unique structural features, which allow them to bindto the corresponding cells, modulating the membrane*Corresponding author. Tel.: 1 56-2-686-2661; fax: 1 56-2-222-voltage and affecting membrane permeability (Wes-2810.

E-mail address: [email protected] (A. Venegas) terhoff et al., 1989; Ganz et al., 1990). Participation

0167-7012/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0167-7012( 00 )00125-1

200 G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 –206

of voltage-dependent ionic channels have also been tion. Only compounds causing cellular lysis produceproposed to explain lytic activity (Christensen et al., a blue-colored edge at the inhibition zone.1988; Cruciani et al., 1988; Duclohier et al., 1989; Some of the advantages of this method are lowKagan et al., 1990). cost, simplicity and the possibility to deal with

The entire structure of the bactericidal compound several samples at once in a single Petri dish,under study seems to be the most important feature, providing a comparative direct observation of theas found for some peptides, since their all-D-enantio- results on a particular bacterial strain.mers also have biological properties similar to thoseof the corresponding native L-enantiomers (Bessalleet al., 1990). This assumption, however, is not valid 3. Procedurefor the receptor-oriented-type of compound (Flouretand du Vigneaud, 1965; Morley et al., 1965; Stewart 3.1. Strainsand Woolley, 1965; Casteels and Tempst, 1994). At

2 2present, a variety of natural and synthetic products is Escherichia coli strain BL21 (DE3) (F ompT rB2under study, searching for new antibiotic com- m ) obtained from Novagen Inc. was used toB

pounds. standardize the assay. Also, E. coli strains BL212We propose here a simple, inexpensive assay to (Novagen Inc.), C600 lacY (Clowes and Hayes,

screen compounds with bacteriolytic activity. 1968), and UH302 (Cole et al., 1982), as well asErwinia carotovora spp. carotovora Ecc193 (kindlyprovided by Dr Chatterjee) and Citrobacter freundii

2. A new chromogenic plate test assay for and Shigella flexneri (provided by Dr Guido Mora)evaluation of bacteriolytic compounds were utilized.

There are several approaches to establish the 3.2. Antibioticsbacteriolytic or bacteriostatic nature of an antibacter-ial compound. For instance, bacterial lysis may be Ampicillin, carbenicillin, cephaloridine, cephalo-followed by permeability assays for the inner and sporin C, cephradine, chloramphenicol, erythro-outer membranes in liquid media (Lehrer et al., mycin, gentamicin, kanamycin, kasugamycin, mox-1988), using electrophysiological techniques (Saber- alactam, nalidixic acid, spectinomycin, streptomycin,wal and Nagaraj, 1994), or by studying the enantio- sulfadiazine, tetracycline and trimethoprim weremer biological activities (Bessalle et al., 1990; from Sigma (St. Louis, MO). Apidaecin Ib (CasteelsCasteels and Tempst, 1994). However, some of these et al., 1989), cecropin B (Gazit et al., 1994) andtechniques are expensive and time-consuming. cecropin P1 (Christensen et al., 1988) were chemi-

In this report we present a simple strategy based cally synthesized by Bios Chile I.G.S.A. (Santiago,on the use of b-galactosidase as an appropriate Chile). Cephamezine and ceftizoxime were frommarker of cellular lysis. The antibiotic to be tested is Instituto Beta (Santiago, Chile). The stock solutionslaid as a small drop (1–6 ml) on a plate with soft of the antimicrobial compounds were prepared inagar containing an Escherichia coli growing lawn ethanol or distilled water depending on their solu-which expresses b-galactosidase activity. If lysis bility properties.occurs, then the enzyme activity is released outsidethe bacterium and detected on the plate. When the 3.3. Reagentsenzyme reaches the agar medium, it hydrolyzes the5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal), X-Gal was from Promega (Madison, WI).a chromogenic compound included in the agar and Isopropyl-b-D-thiogalactopyranoside (IPTG) wasextensively used in alpha-complementation assays from Sigma. Bacto-agar, bacto-tryptone were pur-(Sambrook et al., 1989). After overnight incubation, chased from Difco (Detroit, MI). NaCl was from

¨X-Gal forms a blue circle staining the edge of the Merck (Darmstadt, Germany). Yeast extract powderinhibition zone produced by the antibiotic applica- was from HiMedia (Bombay, India).

G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 –206 201

3.4. Minimal inhibitory concentration (MIC) stored at 48C for several weeks without loss of thedeterminations blue color.

This was done in liquid cultures following theprocedure described by Braude (1981) with few 4. Results and discussionmodifications. Ten microliters of an overnight cul-ture of E. coli BL21(DE3) cells were diluted into 1 The method presented here allowed us to dis-ml Luria broth and then, aliquots of 100 ml were tinguish a bacteriolytic from a bacteriostatic mode-transferred to eight sterile tubes. The first tube of-action of antimicrobial compounds. To test thecontains the highest antimicrobial concentration to be new assay, a selected group of antibiotics wastested. To this tube additional 100 ml of bacterial analyzed (Table 1). All the antibiotics assayed gavecells were added and after mixing, 100 ml were the expected pattern, a blue edge at the inhibitionwithdrawn and transferred to the next tube. The zone for bacteriolytic agents, and no color fortwo-fold serial dilution was repeated for the other bacteriostatic compounds. The only exception to thetubes. The tubes were incubated 12 h at 378C and the pattern was apidaecin which behaved as a bac-bacterial growth was measured at 600 nm. The MIC teriolytic agent, in contrast to the proposed non lyticvalue (a-b) expresses the highest antimicrobial con- mode-of-action (Casteels and Tempst, 1994).centration at which cells were able to grow (a) and Fig. 1 presents the assay plate for some of thethe lowest concentration at which no growth was antibiotics listed in Table 1, including two bacterio-detected (b). static agents (chloramphenicol and tetracycline) and

five bacteriolytic compounds (nalidixic acid, ampicil-lin, cecropin B, cecropin P1 and apidaecin Ib).

3.5. Bacteriolytic plate assay Notice the sharp blue halos around the bacteriolyticcompounds.

Escherichia coli strain BL21(DE3) which contains We used tetracycline and ampicillin as bacterio-a chromosomal IPTG-inducible b-galactosidase static and bacteriolytic agents, respectively, to de-

1gene, was used for most of the assays. Other lacZ termine appropriate conditions for the assay. Differ-strains tested were E. coli UH302, E. coli C600 (a ent strains in the bacterial lawn, incubation time and

2lacY derivative), Citrobacter freundii, Erwinia suitable amount of X-Gal for color detection andcarotovora sp. carotovora Ecc93 and Shigella flex- sensitivity were also used to determine conditions.neri. First, an inoculum with this strain was grown Results shown in Table 2 validated the assay for

1overnight in 2 ml LB media (10 g/ l tryptone, 5 g/ l different Gram negative lac strains. E. coli HB1012NaCl, 5 g/ l yeast extract powder), at 378C with strain was included as a lac control. In order to find

shaking. Then, a soft agar-incubation mix containing the most appropriate X-Gal concentration, plates10 ml of 0.8% agar previously melted at 458C with containing soft agar with 62.5, 125, 250 and 37550 ml of the bacterial cell inoculum, 10 ml of 1 mM mg/ml were assayed (not shown). At the highestIPTG, and 50 ml of 50 mg/ml X-Gal was vortex- X-Gal concentration only the plate background wasmixed and carefully overlaid on LB plates containing increased with rather modest improvement of color20 ml of 1.5% agar prepared the day before. Once intensity at the inhibition zone. Under the standardthe soft agar was solidified and dried (2–3 h), single X-Gal concentration described for the assay (2501-, 1.5-, 3-, or 6-ml drops (depending upon the mg/ml), no blue color appeared at the inhibitionantibiotic tested), containing the appropriate con- zone when the bacteriostatic compound was tested.centration of the antibiotic, were deposited on the In addition, plate incubation was also tested at 28soft agar layer using fine disposable tips. Then the and 428C, keeping other assay conditions as de-plates were incubated at 378C for 9–16 h. After scribed in Section 3.5, with no significant improve-incubation, the inhibition zones were visually in- ment of the assay.spected by color formation along the edge of spots To evaluate the sensitivity of the method (theand the plates were photographed. Plates can be smallest inhibition zone at which the blue color


Table 1aPattern of halo at inhibition zone for various antibacterial agents in the chromogenic plate assay

bAntibacterial Amount added Type of halo Mechanismagent (mg) (Reference)

Observed Expected

Ampicillin 6 1 1 Tomasz, 1979Apidaecin Ib 15 1 2 Casteels and Tempst, 1994Carbenicillin 20 1 1 Maki et al., 1978Cecropin B 6 1 1 Gazit et al., 1994Cecropin P1 6 1 1 Christensen et al., 1988Cephamezine 30 1 1 Mandell and Sande, 1991Ceftizoxime 30 1 1 Ogawa et al., 1981Cephaloridine 20 1 1 Rolinson, 1980Cephalosporin C 30 1 1 Flynn, 1972Cephradine 20 1 1 Neiss, 1973Chloramphenicol 37.5 2 2 Pratt and Fekety, 1986

Ërythromycin 100 1 / 2 1 Brisson-Noel and Trieu-Cuot, 1988Gentamicin 10 1 1 Rosselot et al., 1964Kanamycin 20 1 1 Bryan, 1989Kasugamycin 20 2 2 Bakker, 1992Moxalactam 10 1 1 Labia, 1982Nalidixic acid 37.5 1 1 Hooper et al., 1987Spectinomycin 20 2 2 Schoutens et al., 1972Streptomycin 20 1 1 Bryan, 1989Sulfadiazine 100 2 2 Woods, 1962Tetracycline 9.5 2 2 Chopra and Howe, 1978Trimethoprim 2 2 2 Ferone et al., 1969

a All antibacterial compounds were tested as described in Section 3.5, using E. coli BL21(DE3) strain, except for erythromycin which wasassayed in E. coli UH302.

b Halo indicated as ( 1 ) blue color, (2) colorless, and ( 1 / 2 ) faint blue.

could be detected in the halo), lower ampicillin optimize the blue color at the inhibition zone com-concentrations were tested. Results showed that the pared to a plate without IPTG (not shown). Thisinhibition zone should be at least 3 mm or larger in effect may vary due to the particular E. coli straindiameter for the blue halo to be noticed (spot of 0.6 used. Moreover, when BL21(DE3) cells were used,mg ampicillin in Fig. 2A). In the case of a bacterio- concentrations higher than 1 mM IPTG increased thestatic agent, no color was detected over a wide range blue background of the plate rather than the blueof tetracycline (0.15–4.5 mg in Fig. 2B). In addition, color of the halo. Concentrations higher than 50 mMit should be mentioned that the appropriate amount IPTG stained blue the entire plate, precluding anyof ampicillin to be used in the testing plate is close to discrimination between bacteriostatic and bac-that of the MIC value determined in liquid media teriolytic agents (not shown).(referable to 1 ml of cultured cells) in such a way The reproducibility of this method was tested atthat the halo can be easily distinguished. For in- least four times for several antibiotics with wellstance, 1 ml containing 1.3 mg ampicillin (MIC value characterized mode-of-action giving the results sum-determined as 1.3 mg/ml for BL21(DE3) strain) marized in Table 1.showed an inhibition blue halo of 5 mm in diameter An interesting point to be mentioned is that among(Fig. 2A). 22 tested antibiotics (Table 1) only apidaecin Ib

Since IPTG is a strong inducer of b-galactosidase behaved differently with respect to its assignedactivity, the effect of this compound was evaluated. mode-of-action (Casteels and Tempst, 1994).It was found that 1 mM IPTG in the soft agar Apidaecin Ib is a unique antibacterial peptide foundcontaining BL21(DE3) cells was just enough to in immune honeybee lymph. It consists of 18 amino


Fig. 1. Plate assay showing bacteriolytic or bacteriostatic activityfor some antibiotics. The assay was done as described in Section3.5. A, ampicillin, 1.5 ml of a stock solution 20 mg/ml; T,tetracycline, 1.5 ml of 6.3 mg/ml; C, chloramphenicol, 1.5 ml of25 mg/ml; API, apidaecin Ib, 1.5 ml of 4 mg/ml; CB, cecropin B,6 ml of 1 mg/ml; CP, cecropin P1, 6 ml of 1 mg/ml; N, nalidixicacid, 1.5 ml of 25 mg/ml. Ten milliliters of 0.7% soft agarcontaining 50 ml of 50 mg/ml X-Gal and 50 ml of saturatedculture of E. coli BL21(DE3) were added on the top of aLuria-agar plate and incubated at 378C for 16 h.

acids including six proline residues, and is verystable at high temperature and at low pH (Casteels etal., 1989). Casteels and Tempst (1994) have pro-posed that apidaecin functions as a bacteriostaticagent, specifically toward Gram negative bacteria,through a ‘non-pore forming’ mechanism. In con-trast, our results suggest a lytic mechanism. How-ever, there are few differences in the assays that mayexplain the divergence in the results. First, our plateassay was evaluated after 10–12 h of incubation withthe peptide, while Casteels and Tempst measuredONPG hydrolysis spectrophotometrically after 25min. Second, our assay was done in solid medium,the other was done in solution. Third, we used E.

1coli BL21(DE3), a lacZ derivative, and the otherFig. 2. Sensitivity of the assay. The assay was done as describedauthors, E. coli ML-35p. These strains may differ inin Section 3.5, but inhibition zones were formed adding a 1-ml

membrane permeability. Independently determined, drop on the lawn. (A) Drops containing 0.3 (center), 0.6, 1.3, 2.5,apidaecin MIC values in liquid cultures were 0.6–3 5, 10 and 20 mg of ampicillin. (B) Drops containing 0.08 (center),mg/ml for BL21(DE3) (our data) and 0.05–0.1 mg/ 0.15, 0.3, 0.6, 1.2, 2.3 and 4.5 mg of tetracycline.


Table 21 aChromogenic plate assay done with different lacZ bacterial strains to distinguish a bacteriolytic agent from a bacteriostatic compound

bStrain Halo

Ampicillin Tetracycline(10 mg/drop) (2.3 mg/drop)

Citrobacter freundii 1 2

Escherichia coli B 1 2

E. coli BL21 1 2

E. coli BL21(DE3) 1 2

E. coli C600 1 22E. coli HB101 (lacZ control strain) 2 2

E. coli K-12 1 2

E. coli UH302 1 2

Erwinia carotovora spp. carotovora Ecc193 1 2

Shigella flexneri 1 2

a One microliter was laid on the bacterial lawn using ampicillin as a bacteriolytic agent and tetracycline as a bacteriostatic antibiotic andthe assay conditions were done as described in Section 3.5, except for the Erwinia strain which was grown at 288C.

b Halo indicated as ( 1 ) blue color, and (2) colorless.

ml for ML-35p (Casteels and Tempst, 1994). In ates a concentration gradient at which an equilibriumaddition to the differences mentioned above, we have between growing and lysed cells is reached, and (2)expressed a synthetic apidaecin Ib gene in certain number of lysed cells release a sufficientBL21(DE3) cells (unpublished results) and 2 h after amount of b-galactosidase enzyme able to hydrolyzeIPTG induction of the apidaecin gene we detected a visible quantity of X-Gal substrate. Bacterial cellsb-galactosidase activity in the supernatant fraction. located close to the center of the inhibition zone didThis result indicates that bacterial lysis is induced by not have the chance to grow nor to accumulate thecytoplasmic expression of apidaecin and lysis can be enzyme, because of the diffusion of the antimicrobialdetected as early as 2 h after IPTG induction. We compound in a radial way, starting from the applica-also found that expression of apidaecin drastically tion point on the agar plate.affected bacterial growth in a similar way as reported The light blue background observed at concen-by other authors (Taguchi et al., 1994), during trations of X-Gal higher than 125 mg/ml in the plate,expression of apidaecin fused to the inhibitor of may be due to a slight and slow X-Gal diffusion intoStreptomyces subtilisin. the dividing cells, providing a soft blue background

A different case is erythromycin which, in addi- rather than the dark blue circle in the inhibition zone.tion to its bacteriolytic effect on Gram-positive Another explanation could be that the X-Gal maybacteria, has shown a bacteriostatic effect at very enter the bacterial cells using the lactose permeaselow concentration such as 0.001 mg/ml (Brisson- system. Regarding this point, we tried E. coli C-600

2¨Noel and Trieu-Cuot, 1988). This result cannot be which is a lacY mutant. However, no improvementevaluated in our assay conditions because it is to reduce the blue background was observed. Webeyond the sensitivity of the method, but the method favour the explanation that the background may beallowed us to detect the bacteriolytic effect described related to the intrinsic X-Gal permeability for afor erythromycin, suggesting that, in some cases, the defined strain. For instance, we did not observed aactual effect on bacterial cells depends on the lawn with blue background when Shigella flexnericoncentration of the agent used. was used in our standard assay conditions, even at

The formation of a blue halo when a bacteriolytic X-Gal concentrations higher than 125 mg/ml.compound is being tested could be explained by We conclude that the method described hereX-Gal hydrolysis occurring at the edge of the allows discrimination between a bacteriolytic orinhibition zone. This could be due to two factors: (1) bacteriostatic mechanism-of-action of antimicrobialthe radial diffusion of bacteriolytic agent that gener- molecules. The assay is simple, economical, and


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Mardones 2000