Journal of Microbiological Methods 40 (2000) 199–206

Journal of Microbiological Methods www.elsevier.com / locate / jmicmeth

Chromogenic plate assay distinguishing bacteriolytic from bacteriostatic activity of an antibiotic agent Gonzalo Mardones, Alejandro Venegas* ´ ´ ´ , Pontificia Universidad Catolica ´ , Departamento de Genetica Molecular y Microbiologıa de Chile, Laboratorio de Bioquımica 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 antibacterial agent. The assay uses a bacterial culture harboring b-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, b-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds 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 The knowledge about the mechanism of action of a new antimicrobial agent is basic to understanding the events occurring during bacterial growth inhibition. This issue is very important for the development of any antibacterial compound for therapeutic use. Recently, several efforts have focused on studying the mechanisms of a number of new antibacterial peptides. Surface active peptides which bind and alter amphipatic surfaces, including membranes and receptors, have been extensively studied (DeGrado et al., 1981; Kaiser and Kezdy, 1983, 1984; Kaiser, 1988). Some of these antibacterial agents act in a *Corresponding author. Tel.: 1 56-2-686-2661; fax: 1 56-2-2222810. E-mail address: [email protected] (A. Venegas)

similar way to hormones by binding to specific cellular receptors which require specific peptide conformation. In contrast, the mechanisms of action of other antibacterial agents is less dependent on such stringent structural requirement. Among these compounds, cytolytic cationic peptides with a wide spectrum of action have been isolated from mammalian macrophages — the so called defensins (Ganz et al., 1990), from insects, — melittin (Habermann, 1972), cecropins (Steiner et al., 1981) and sarcotoxins (Okada and Natori, 1985) and from amphibians, — magainin (Zasloff, 1987). The target for these surface-active peptides seems to be the lipid bilayer of the cellular membrane. It has been reported that their activity is exclusively due to their unique structural features, which allow them to bind to the corresponding cells, modulating the membrane voltage and affecting membrane permeability (Westerhoff et al., 1989; Ganz et al., 1990). Participation

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of voltage-dependent ionic channels have also been proposed to explain lytic activity (Christensen et al., 1988; Cruciani et al., 1988; Duclohier et al., 1989; Kagan et al., 1990). The entire structure of the bactericidal compound under study seems to be the most important feature, as found for some peptides, since their all-D-enantiomers also have biological properties similar to those of the corresponding native L-enantiomers (Bessalle et al., 1990). This assumption, however, is not valid for the receptor-oriented-type of compound (Flouret and du Vigneaud, 1965; Morley et al., 1965; Stewart and Woolley, 1965; Casteels and Tempst, 1994). At present, a variety of natural and synthetic products is under study, searching for new antibiotic compounds. We propose here a simple, inexpensive assay to screen compounds with bacteriolytic activity.

2. A new chromogenic plate test assay for evaluation of bacteriolytic compounds There are several approaches to establish the bacteriolytic or bacteriostatic nature of an antibacterial compound. For instance, bacterial lysis may be followed by permeability assays for the inner and outer membranes in liquid media (Lehrer et al., 1988), using electrophysiological techniques (Saberwal and Nagaraj, 1994), or by studying the enantiomer biological activities (Bessalle et al., 1990; Casteels and Tempst, 1994). However, some of these techniques are expensive and time-consuming. In this report we present a simple strategy based on the use of b-galactosidase as an appropriate marker of cellular lysis. The antibiotic to be tested is laid as a small drop (1–6 ml) on a plate with soft agar containing an Escherichia coli growing lawn which expresses b-galactosidase activity. If lysis occurs, then the enzyme activity is released outside the bacterium and detected on the plate. When the enzyme reaches the agar medium, it hydrolyzes the 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal), a chromogenic compound included in the agar and extensively used in alpha-complementation assays (Sambrook et al., 1989). After overnight incubation, X-Gal forms a blue circle staining the edge of the inhibition zone produced by the antibiotic applica-

tion. Only compounds causing cellular lysis produce a blue-colored edge at the inhibition zone. Some of the advantages of this method are low cost, simplicity and the possibility to deal with several samples at once in a single Petri dish, providing a comparative direct observation of the results on a particular bacterial strain.

3. Procedure

3.1. Strains Escherichia coli strain BL21 (DE3) (F 2 ompT r B2 m ) obtained from Novagen Inc. was used to standardize the assay. Also, E. coli strains BL21 (Novagen Inc.), C600 lacY 2 (Clowes and Hayes, 1968), and UH302 (Cole et al., 1982), as well as Erwinia carotovora spp. carotovora Ecc193 (kindly provided by Dr Chatterjee) and Citrobacter freundii and Shigella flexneri (provided by Dr Guido Mora) were utilized. 2 B

3.2. Antibiotics Ampicillin, carbenicillin, cephaloridine, cephalosporin C, cephradine, chloramphenicol, erythromycin, gentamicin, kanamycin, kasugamycin, moxalactam, nalidixic acid, spectinomycin, streptomycin, sulfadiazine, tetracycline and trimethoprim were from Sigma (St. Louis, MO). Apidaecin Ib (Casteels et al., 1989), cecropin B (Gazit et al., 1994) and cecropin P1 (Christensen et al., 1988) were chemically synthesized by Bios Chile I.G.S.A. (Santiago, Chile). Cephamezine and ceftizoxime were from Instituto Beta (Santiago, Chile). The stock solutions of the antimicrobial compounds were prepared in ethanol or distilled water depending on their solubility properties.

3.3. Reagents X-Gal was from Promega (Madison, WI). Isopropyl-b-D-thiogalactopyranoside (IPTG) was from Sigma. Bacto-agar, bacto-tryptone were purchased from Difco (Detroit, MI). NaCl was from ¨ Merck (Darmstadt, Germany). Yeast extract powder was from HiMedia (Bombay, India).

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3.4. Minimal inhibitory concentration ( MIC) determinations This was done in liquid cultures following the procedure described by Braude (1981) with few modifications. Ten microliters of an overnight culture of E. coli BL21(DE3) cells were diluted into 1 ml Luria broth and then, aliquots of 100 ml were transferred to eight sterile tubes. The first tube contains the highest antimicrobial concentration to be tested. To this tube additional 100 ml of bacterial cells were added and after mixing, 100 ml were withdrawn and transferred to the next tube. The two-fold serial dilution was repeated for the other tubes. The tubes were incubated 12 h at 378C and the bacterial growth was measured at 600 nm. The MIC value (a-b) expresses the highest antimicrobial concentration at which cells were able to grow (a) and the lowest concentration at which no growth was detected (b).

3.5. Bacteriolytic plate assay Escherichia coli strain BL21(DE3) which contains a chromosomal IPTG-inducible b-galactosidase gene, was used for most of the assays. Other lacZ 1 strains tested were E. coli UH302, E. coli C600 (a lacY 2 derivative), Citrobacter freundii, Erwinia carotovora sp. carotovora Ecc93 and Shigella flexneri. First, an inoculum with this strain was grown overnight in 2 ml LB media (10 g / l tryptone, 5 g / l NaCl, 5 g / l yeast extract powder), at 378C with shaking. Then, a soft agar-incubation mix containing 10 ml of 0.8% agar previously melted at 458C with 50 ml of the bacterial cell inoculum, 10 ml of 1 mM IPTG, and 50 ml of 50 mg / ml X-Gal was vortexmixed and carefully overlaid on LB plates containing 20 ml of 1.5% agar prepared the day before. Once the soft agar was solidified and dried (2–3 h), single 1-, 1.5-, 3-, or 6-ml drops (depending upon the antibiotic tested), containing the appropriate concentration of the antibiotic, were deposited on the soft agar layer using fine disposable tips. Then the plates were incubated at 378C for 9–16 h. After incubation, the inhibition zones were visually inspected by color formation along the edge of spots and the plates were photographed. Plates can be

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stored at 48C for several weeks without loss of the blue color.

4. Results and discussion The method presented here allowed us to distinguish a bacteriolytic from a bacteriostatic modeof-action of antimicrobial compounds. To test the new assay, a selected group of antibiotics was analyzed (Table 1). All the antibiotics assayed gave the expected pattern, a blue edge at the inhibition zone for bacteriolytic agents, and no color for bacteriostatic compounds. The only exception to the pattern was apidaecin which behaved as a bacteriolytic agent, in contrast to the proposed non lytic mode-of-action (Casteels and Tempst, 1994). Fig. 1 presents the assay plate for some of the antibiotics listed in Table 1, including two bacteriostatic agents (chloramphenicol and tetracycline) and five bacteriolytic compounds (nalidixic acid, ampicillin, cecropin B, cecropin P1 and apidaecin Ib). Notice the sharp blue halos around the bacteriolytic compounds. We used tetracycline and ampicillin as bacteriostatic and bacteriolytic agents, respectively, to determine appropriate conditions for the assay. Different strains in the bacterial lawn, incubation time and suitable amount of X-Gal for color detection and sensitivity were also used to determine conditions. Results shown in Table 2 validated the assay for different Gram negative lac 1 strains. E. coli HB101 strain was included as a lac 2 control. In order to find the most appropriate X-Gal concentration, plates containing soft agar with 62.5, 125, 250 and 375 mg / ml were assayed (not shown). At the highest X-Gal concentration only the plate background was increased with rather modest improvement of color intensity at the inhibition zone. Under the standard X-Gal concentration described for the assay (250 mg / ml), no blue color appeared at the inhibition zone when the bacteriostatic compound was tested. In addition, plate incubation was also tested at 28 and 428C, keeping other assay conditions as described in Section 3.5, with no significant improvement of the assay. To evaluate the sensitivity of the method (the smallest inhibition zone at which the blue color

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Table 1 Pattern of halo at inhibition zone for various antibacterial agents in the chromogenic plate assay a Antibacterial agent

Amount added (mg)

Ampicillin Apidaecin Ib Carbenicillin Cecropin B Cecropin P1 Cephamezine Ceftizoxime Cephaloridine Cephalosporin C Cephradine Chloramphenicol Erythromycin Gentamicin Kanamycin Kasugamycin Moxalactam Nalidixic acid Spectinomycin Streptomycin Sulfadiazine Tetracycline Trimethoprim

6 15 20 6 6 30 30 20 30 20 37.5 100 10 20 20 10 37.5 20 20 100 9.5 2

Type of halo b

Mechanism (Reference)

Observed

Expected

1 1 1 1 1 1 1 1 1 1 2 1/2 1 1 2 1 1 2 1 2 2 2

1 2 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 2 1 2 2 2

Tomasz, 1979 Casteels and Tempst, 1994 Maki et al., 1978 Gazit et al., 1994 Christensen et al., 1988 Mandell and Sande, 1991 Ogawa et al., 1981 Rolinson, 1980 Flynn, 1972 Neiss, 1973 Pratt and Fekety, 1986 ¨ and Trieu-Cuot, 1988 Brisson-Noel Rosselot et al., 1964 Bryan, 1989 Bakker, 1992 Labia, 1982 Hooper et al., 1987 Schoutens et al., 1972 Bryan, 1989 Woods, 1962 Chopra and Howe, 1978 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 was assayed 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 concentrations were tested. Results showed that the inhibition zone should be at least 3 mm or larger in diameter for the blue halo to be noticed (spot of 0.6 mg ampicillin in Fig. 2A). In the case of a bacteriostatic agent, no color was detected over a wide range of tetracycline (0.15–4.5 mg in Fig. 2B). In addition, it should be mentioned that the appropriate amount of ampicillin to be used in the testing plate is close to that of the MIC value determined in liquid media (referable to 1 ml of cultured cells) in such a way that the halo can be easily distinguished. For instance, 1 ml containing 1.3 mg ampicillin (MIC value determined as 1.3 mg / ml for BL21(DE3) strain) showed an inhibition blue halo of 5 mm in diameter (Fig. 2A). Since IPTG is a strong inducer of b-galactosidase activity, the effect of this compound was evaluated. It was found that 1 mM IPTG in the soft agar containing BL21(DE3) cells was just enough to

optimize the blue color at the inhibition zone compared to a plate without IPTG (not shown). This effect may vary due to the particular E. coli strain used. Moreover, when BL21(DE3) cells were used, concentrations higher than 1 mM IPTG increased the blue background of the plate rather than the blue color of the halo. Concentrations higher than 50 mM IPTG stained blue the entire plate, precluding any discrimination between bacteriostatic and bacteriolytic agents (not shown). The reproducibility of this method was tested at least four times for several antibiotics with well characterized mode-of-action giving the results summarized in Table 1. An interesting point to be mentioned is that among 22 tested antibiotics (Table 1) only apidaecin Ib behaved differently with respect to its assigned mode-of-action (Casteels and Tempst, 1994). Apidaecin Ib is a unique antibacterial peptide found in immune honeybee lymph. It consists of 18 amino

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Fig. 1. Plate assay showing bacteriolytic or bacteriostatic activity for some antibiotics. The assay was done as described in Section 3.5. 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 of 25 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, nalidixic acid, 1.5 ml of 25 mg / ml. Ten milliliters of 0.7% soft agar containing 50 ml of 50 mg / ml X-Gal and 50 ml of saturated culture of E. coli BL21(DE3) were added on the top of a Luria-agar plate and incubated at 378C for 16 h.

acids including six proline residues, and is very stable at high temperature and at low pH (Casteels et al., 1989). Casteels and Tempst (1994) have proposed that apidaecin functions as a bacteriostatic agent, specifically toward Gram negative bacteria, through a ‘non-pore forming’ mechanism. In contrast, our results suggest a lytic mechanism. However, there are few differences in the assays that may explain the divergence in the results. First, our plate assay was evaluated after 10–12 h of incubation with the peptide, while Casteels and Tempst measured ONPG hydrolysis spectrophotometrically after 25 min. Second, our assay was done in solid medium, the other was done in solution. Third, we used E. coli BL21(DE3), a lacZ 1 derivative, and the other authors, E. coli ML-35p. These strains may differ in membrane permeability. Independently determined, apidaecin MIC values in liquid cultures were 0.6–3 mg / ml for BL21(DE3) (our data) and 0.05–0.1 mg /

Fig. 2. Sensitivity of the assay. The assay was done as described in Section 3.5, but inhibition zones were formed adding a 1-ml drop on the lawn. (A) Drops containing 0.3 (center), 0.6, 1.3, 2.5, 5, 10 and 20 mg of ampicillin. (B) Drops containing 0.08 (center), 0.15, 0.3, 0.6, 1.2, 2.3 and 4.5 mg of tetracycline.

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Table 2 Chromogenic plate assay done with different lacZ 1 bacterial strains to distinguish a bacteriolytic agent from a bacteriostatic compound a Strain

Citrobacter freundii Escherichia coli B E. coli BL21 E. coli BL21(DE3) E. coli C600 E. coli HB101 (lacZ 2 control strain) E. coli K-12 E. coli UH302 Erwinia carotovora spp. carotovora Ecc193 Shigella flexneri

Halo b Ampicillin (10 mg / drop)

Tetracycline (2.3 mg / drop)

1 1 1 1 1 2 1 1 1 1

2 2 2 2 2 2 2 2 2 2

a One microliter was laid on the bacterial lawn using ampicillin as a bacteriolytic agent and tetracycline as a bacteriostatic antibiotic and the 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 addition to the differences mentioned above, we have expressed a synthetic apidaecin Ib gene in BL21(DE3) cells (unpublished results) and 2 h after IPTG induction of the apidaecin gene we detected b-galactosidase activity in the supernatant fraction. This result indicates that bacterial lysis is induced by cytoplasmic expression of apidaecin and lysis can be detected as early as 2 h after IPTG induction. We also found that expression of apidaecin drastically affected bacterial growth in a similar way as reported by other authors (Taguchi et al., 1994), during expression of apidaecin fused to the inhibitor of Streptomyces subtilisin. A different case is erythromycin which, in addition to its bacteriolytic effect on Gram-positive bacteria, has shown a bacteriostatic effect at very low concentration such as 0.001 mg / ml (Brisson¨ and Trieu-Cuot, 1988). This result cannot be Noel evaluated in our assay conditions because it is beyond the sensitivity of the method, but the method allowed us to detect the bacteriolytic effect described for erythromycin, suggesting that, in some cases, the actual effect on bacterial cells depends on the concentration of the agent used. The formation of a blue halo when a bacteriolytic compound is being tested could be explained by X-Gal hydrolysis occurring at the edge of the inhibition zone. This could be due to two factors: (1) the radial diffusion of bacteriolytic agent that gener-

ates a concentration gradient at which an equilibrium between growing and lysed cells is reached, and (2) certain number of lysed cells release a sufficient amount of b-galactosidase enzyme able to hydrolyze a visible quantity of X-Gal substrate. Bacterial cells located close to the center of the inhibition zone did not have the chance to grow nor to accumulate the enzyme, because of the diffusion of the antimicrobial compound in a radial way, starting from the application point on the agar plate. The light blue background observed at concentrations of X-Gal higher than 125 mg / ml in the plate, may be due to a slight and slow X-Gal diffusion into the dividing cells, providing a soft blue background rather than the dark blue circle in the inhibition zone. Another explanation could be that the X-Gal may enter the bacterial cells using the lactose permease system. Regarding this point, we tried E. coli C-600 which is a lacY 2 mutant. However, no improvement to reduce the blue background was observed. We favour the explanation that the background may be related to the intrinsic X-Gal permeability for a defined strain. For instance, we did not observed a lawn with blue background when Shigella flexneri was used in our standard assay conditions, even at X-Gal concentrations higher than 125 mg / ml. We conclude that the method described here allows discrimination between a bacteriolytic or bacteriostatic mechanism-of-action of antimicrobial molecules. The assay is simple, economical, and

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reliable. It requires only a minimal amount of the compound to be tested and facilitates the analysis of an extensive number of different compounds at the same time. We believe that this method should facilitate investigations of the mechanism of action of new antibiotics.

Acknowledgements This research was supported by grants from Fondo ´ de Chile (FONNacional de Ciencia y Tecnologıa DECYT [1940713 and [1971010). We gratefully acknowledge Dr Jorge Delgado and Steve Nguyen for critical reading of the manuscript.

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