7-O-Malonyl Macrolactin A, a New Macrolactin Antibiotic from Bacillus subtilis Active against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococci, and a Small-Colony Variant of Burkholderia cepacia

Strains and media.Microorganisms used to assess the antimicrobial activity of the macrolactins were from the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany), numbered as DSM, and the American Type Culture Collection (ATCC; Manassas, VA): methicillin-sensitive S. aureus (MSSA) strains (DSM 1104 and DSM 2569); Escherichia coli (DSM 498); P. aeruginosa (DSM 1117); Enterococcus faecalis (ATCC 29212); Candida parapsilosis (DSM 5784); Candida krusei (DSM 6128); Candida albicans (DSM 11225); and clinical isolates MSSA strain 32, MRSA strain 2, MRSA strain 3, E. faecalis strain E305 (vancomycin-resistant/ampicillin-sensitive [VRAS]), and Enterococcus faecium strain E315 (vancomycin-resistant/ampicillin-resistant [VRAR]). The parental wild-type (WT) and SCV pairs of P. aeruginosa and B. cepacia, as well as Stenotrophomonas maltophilia strain 1124, were isolated from cystic fibrosis patients in the Department of Medical Microbiology at the Medical School of Hannover (Germany). B. cepacia WT strain 139 was isolated from a bronchoalveolar lavage specimen, and its SCV form strain 141 from a blood sample from the same patient. P. aeruginosa WT strain 134 and its SCV form strain 137 were isolated from the same respiratory tract specimen. The clonal identities of the SCV and WTs were determined by pulsed-field gel electrophoresis, as previously described (12, 13).

The antibiotic-producing bacterium, ICBB 1582, was isolated from a soil sample obtained from a farmyard at Takalar, South Sulawesi Province, Indonesia in April 2000. The strain was deposited in the DSMZ under the accession number DSM 16696. Biochemical and phenotypic characterizations were made using Api 20E and 50CH kits (bioMérieux, Marcy l'Etoile, France). The 16S rRNA sequence determination and analysis, performed as previously described (40), identified the organism as a Bacillus subtilis strain. All organisms were maintained in glycerol broth at −80°C.

Bacteria were cultivated overnight at 37°C on Luria-Bertani (LB) agar, except staphylococci and enterococci, which were grown on Columbia blood agar (BD, New Jersey). Yeast strains were grown in DSM M186 medium (http://www.dsmz.de/microorganisms/html/media/medium000186.html ). The medium used for growth of B. subtilis strain DSM 16696 and production of secondary metabolites had the following composition: yeast extract (Difco Laboratories, Detroit, Mich.), 5 g/liter; tryptone (Difco), 20 g/liter; NaCl (Merck, Darmstadt, Germany), 5 g/liter; glucose (Merck), 5 g/liter; and XAD-16 adsorbent resin (Rohm & Haas, Frankfurt, Germany), 20 g/liter.

Antibiotics.Erythromycin (ERY), vancomycin (VAN), ampicillin (AMP), gentamicin, and miconazole were obtained from Sigma-Aldrich (Schnelldorf, Germany). Stock solutions, 10 mg/ml, were freshly prepared in sterile distilled water, except for erythromycin, which was prepared in ethanol. Erythromycin disks were obtained from A/S Rosco (Taastrup, Denmark) and contained 78 μg of diffusible antibiotic. Vancomycin and ampicillin disks were obtained from BBL (New Jersey) and contained, respectively, 30 and 10 μg/disk.

Cultivation of the Bacillus subtilis DSM 16696 strain and extraction of secondary metabolites.The preparation and recovery of secondary metabolites was carried out essentially as described by Sasse et al. (31). Briefly, this involved the inoculation of 500-ml Erlenmeyer flasks, each containing 200 ml of medium described above, with 0.5 ml each of an overnight culture of B. subtilis DSM strain 16696 and incubation with shaking at 30°C for 7 days. The adsorbent resin was recovered from the culture broths by decantation, transferred into a column, and washed with 50% aqueous methanol (MeOH). Adsorbed products were subsequently eluted with 100% MeOH and, after evaporation, the remaining aqueous mixture was extracted four times with ethyl acetate. Evaporation of the solvent under reduced pressure yielded approximately 300 mg of oily residue from a total culture volume of 4 liters. This residue was resuspended in 10 ml of MeOH, and the solution was extracted four times with the same volume of n-heptane to remove lipophilic products and contamination. The MeOH phase was retained for the isolation of natural products.

Purification of macrolactins.Macrolactins were purified by preparative reversed-phase-high-pressure liquid chromatography (RP-HPLC) using a Nucleosil 100-7 C₁₈ column (250 by 21 mm; Macherey-Nagel, Düren, Germany) and a gradient of solvents A (0.5% acetic acid-MeOH 51%) and B (0.5% acetic acid-MeOH 56%) (gradient 0% B to 100% B in 60 min), with a flow rate of 20 ml/min, and UV detection at 280 nm. Between 40 and 60 mg of extract in 0.2 ml MeOH was used for the injection. Each macrolactin (5 to 7 mg) was further purified by LH-20 chromatography (column, 760 by 25 mm; solvent, MeOH-dichloromethane, [1:1]; flow rate, 5 ml/min).

Spectrometric analyses and structure determination.HPLC-UV-mass spectrometry (MS) analysis was performed on an HP model 1100 HPLC system (Hewlett-Packard, United States) with a UV diode array detector and a PE Sciex API 2000 LC/MS/MS system with an ACI electrospray ionization device (Perkin-Elmer, United States), using a Nucleosil 120-5 RP C₁₈ column (125 by 2 mm; Macherey-Nagel), at 40°C. MS data were obtained on a MAT 95 mass spectrometer in electron ionization and desorption chemical ionization modes (Finnigan, United States). Active compounds/peaks were identified with their molecular masses and UV data by searches in the Dictionary of Natural Products database (Chapman and Hall/CRC), Antibase 2000 (VCH Wiley), and the CrossFire Beilstein databases (MDL). For nuclear magnetic resonance (NMR) spectroscopy, the samples were dissolved in 99.95% MeOH-d₄, chloroform-d₃, or dichloromethane-d₂, and the data were obtained with an AVANCE DMX-600 spectrometer (Bruker, Karlsruhe, Germany). Optical rotation and UV spectra were measured in UV MeOH (Merck, Darmstadt, Germany) on a Polarimeter MC 241 (Perkin Elmer) (d = 10 cm) and a UV-2102 PC UV-VIS scanning spectrophotometer (Shimadzu, United States).

Determination of antibacterial activities by the agar diffusion method.Sterile disks (Schleicher & Schuell, Dassel, Germany) containing 10 μl of crude extract or 10 μl of purified macrolactin in MeOH (final compound concentration on the disk, 50 μg) were placed on fresh plates of Mueller-Hinton agar (Difco) seeded with suspensions (10⁵ CFU/ml) of overnight cultures of the test microorganisms. The diameters of the zones of inhibition of growth around the disks were measured after incubation periods of 18 h at 37°C.

MICs.The MICs were determined by the broth microdilution method as recommended by the National Committee for Clinical Laboratory Standards (24) using Mueller-Hinton broth (MHB). Microtiter plates containing 50 μl of serial twofold dilutions of each antimicrobial agent per well were inoculated with 50 μl of a bacterial suspension to yield a cell density of 5 × 10⁵ CFU/ml. MeOH and MHB alone (7 to 12 μl) had no effect and were used as controls. The microtiter plates were incubated for 48 h, and visible growth and optical density (OD) at 650 nm were recorded after 18, 24, 30, and 48 h of incubation and read with a model 3550 UV microtiter reader (Bio-Rad, Munich, Germany). The MIC was the lowest antibiotic concentration that completely prevented visible growth after incubation at 37°C for 18 h; the minimum restrictive concentration (MRC) was defined as the lowest antibiotic concentration that caused at least 50% retraction of growth at 37°C for 18 h (by visual observation and OD at 650 nm).

Kinetics of growth at subinhibitory concentrations.Bacterial growth at sub-MICs of 7-O-malonyl macrolactin A was also investigated. Overnight cultures were used to inoculate fresh MHB containing MMA at different sub-MICs to a cell density of ∼10⁶ CFU/ml, and the cultures were incubated at 37°C with gentle shaking. Aliquots were removed at 0, 1, 2, 4, 6, 8, and 24 h, and dilutions were plated in LB agar medium using a spiral plater (Spiral Biotech, United States). The plates were incubated for 24 h at 37°C, and the number of colonies developing was counted with a laser colony counter (CASBA 4; Spiral Biotech).

Sub-MIC effects.Sub-MIC effects (SME) were determined by the postantibiotic effect method of Craig and Gudmundsson (5). Briefly, 7-O-malonyl macrolactin was added at sub-MICs to tubes containing MHB, to which either MRSA 3 or VRAR E. faecium E315 at ∼10⁶ CFU/ml was subsequently added. Medium without antibiotic was used as a control. After incubation for 1 h at 37°C, samples were diluted 1:1,000 in warmed drug-free MHB to dilute out the antibiotic and incubated further at 37°C. Viability counts were made before exposure, immediately after dilution (0 h), and then hourly, by plating on LB agar plates as described above. The SME was defined by the relationship SME = Ts − C, where Ts is the time it takes for cultures exposed to sub-MICs to increase 1 log₁₀ unit above the counts observed immediately after antibiotic removal by dilution and C is the corresponding time for the unexposed control.

Cytotoxicity assays.HeLa human epithelial cells and L929 mouse fibroblasts, obtained originally from the ATCC, were cultured in Dulbecco's modified Eagle medium (Gibco BRL, Life Technologies, Karlsruhe, Germany) with low (1 mg/liter) and high (4.5 mg/liter) glucose, respectively, supplemented with 10% (vol/vol) fetal bovine serum (Gibco), at 37°C in a 5% CO₂ atmosphere. Cell suspensions were obtained by treatment of monolayer stock cultures with 0.25% trypsin, with and without 0.2 g/liter of EDTA (Gibco) for HeLa and L929, respectively, diluted to obtain suspensions of 2 × 10⁵ cells/ml; 100 μl of the suspension was added to each Nunc 96-well microtiter plate containing (or not containing) serial dilutions of the macrolactins in MeOH (100 μl). Dilutions of MeOH and cell culture medium were used as controls; no effect of these solutions on cell growth was observed. Morphological changes in cells after exposure to the compounds for 1, 2, and 5 days were assessed by phase-contrast microscopy.

Cell counts after 5 days of exposure were also made using the CyQUANT cell proliferation assay (Molecular Probes, United States), a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells (17) that employs CyQUANT dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using fluorescein excitation. The fluorescence emissions of the dye-nucleic acid complexes correlated linearly with the cell numbers. The linear range of the assay under our experimental conditions was 50 to 250,000 cells per 200-μl sample. For this test, the supernatant fluid was carefully removed, the cells were washed with phosphate-buffered saline, the buffer was removed, and cells were frozen at −80°C. For the assay, cells were thawed at room temperature and lysed in buffer containing the CyQUANT dye prepared according the manufacturer's instructions. Fluorescence was measured with a fluorometric plate reader (Titertex Fluoroskan II; excitation, 480 nm; emission, 520 nm). The values obtained were used to calculate the percentage of inhibition of cell proliferation in the presence of macrolactins, according to the formula: 100 − [(cell growth in the presence of drug/cell growth in drug-free medium) × 100].

Transmission electronic microscopy.For transmission electron microscopy, the macrolactin compounds were added at sub-MICs to tubes containing MHB, inoculated with either MRSA 3 or VRAR E. faecium E315 at densities of ∼10⁶ CFU/ml, and incubated at 37°C for 4 h. Tubes without antibiotic were used as controls. The cells were harvested by centrifugation at 12,000 rpm and fixed with 1.0% (vol/vol) glutardialdehyde in phosphate-buffered saline, pH 7.0, and processed as previously described (40). The samples were examined using an energy filter transmission electron microscope (Zeiss CEM 902, conventional mode; objective aperture, 30 μm; acceleration voltage, 80 kV; Zeiss Oberkochen, Germany). Electron micrographs were recorded digitally with a high-resolution 1024 by 1024 charge-coupled-device camera (Proscan; Electronic Systems, Scheuring, Germany).

Producer strain DSM 16696.The DSM strain 16696 was characterized as a gram-positive rod (0.5 to 1 μm by 1 to 3 μm), motile with flagella, forming endospores (diameter, ∼0.2 μm) and producing opaque, milky-white colonies with undulating and rough edges. In the API system, the positive reactions were for oxidase, ornithine, mannitol, Voges-Proskauer, citrate, tryptophan deaminase, and hydrolysis of starch, whereas negative reactions were for nitrate, lysine, H₂S production, glucose, xylose, beta-galactosidase, indole, and urease. According to the biochemistry tests and 16S rRNA sequence homology searches in the FASTA program for sequence database searches, DSM 16696 is a strain of B. subtilis (99% 16S rRNA sequence identity to B. subtilis strain accession no. AY775778; Gene Bank/EMBL/DDBJ).

Isolation of three macrolactin compounds.Secondary metabolites produced by B. subtilis strain DSM 16696 were obtained from small-scale cultures and screened for antimicrobial activities as described in Materials and Methods. HPLC fractionation of the XAD extracts and identification of the biologically active fractions by bioassays revealed 10 different antibacterial compounds, which were identified by their UV and MS spectra as bacillomycins, oxydifficidins, aromatic lipopeptides, and a new compound, together with two other known compounds belonging to the macrolactin group. Since the initial screening revealed the new compound to be inhibitory towards MRSA and VRE, subsequent efforts focused on its characterization. In addition, since it was subsequently identified as a macrolactin, the three macrolactins (compounds I, II, and III) were isolated from a 4-liter culture, purified by preparative RP-HPLC and LH-20 chromatography, and compared.

NMR data of compound II, the new macrolactin.NMR analysis was performed on the purified macrolactin obtained by RP-HPLC and LH-20 chromatography. The data obtained were as follows: ¹H NMR (600 MHz, methanol-d₄): δ (ppm), J (Hz) = 5.59 (d, J = 11.7, 2-H), 6.67 (t, J = 11.3, 3-H), 7.25 (dd, J = 14.7, 11.7, 4-H), 6.15 (dt, J = 15.4, 7.2, 5-H), 2.60 (m, 6-H₂), 5.50 (ddd, J = 6.0, 6.0, 6.0, 7-H), 5.75 (dd, J = 15.3, 5.5, 8-H), 6.71 (dd, J = 15.1, 11.3, 9-H), 6.13 (t, J = 10.2, 10-H), 5.63 (dt, J = 10.6, 8.4, 11-H), 2.63 (m, 12-Ha), 2.33 (ddd, J = 13.0, 7.2, 5.5, 12-Hb), 3.84 (ddd, J = 10.6, 6.0, 5.7, 13-H), 1.66 (m, 14-H₂), 4.39 (dt, J = 6.3, 6.3, 15-H), 5.60 (dd, J = 15.1, 6.4, 16-H), 6.21 (dd, J = 15.1, 10.6, 17-H), 6.10 (dd, J = 15.1, 10.6, 18-H), 5.69 (ddd, J = 14.9, 7.0, 6.8, 19-H), 2.23 (td, J = 14.0, 6.8, 20-Ha), 2.15 (td, J = 14.4, 7.2, 20-Hb), 1.54 (m, 21-H₂), 1.70 (m, 22-Ha), 1.62 (m, 22-Hb), 5.05 (ddq, J = 4.5, 7.1, 6.1, 23-H), 1.30 (d, J = 6.0, 24-H₃), 2.90 (m, 2′-H₂). ¹³C NMR (150 MHz, methanol-d₄): δ (ppm) = 167.94 (C-1), 118.52 (C-2), 144.50 (C-3), 130.79 (C-4), 140.51 (C-5), 40.13 (C-6), 74.72 (C-7), 132.06 (C-8), 128.09 (C-9), 130.91 (C-10), 129.78 (C-11), 36.39 (C-12), 69.51 (C-13), 43.84 (C-14), 69.77 (C-15), 135.32 (C-16), 131.27 (C-17), 131.78 (C-18), 135.10 (C-19), 33.03 (C-20), 25.81 (C-21), 36.08 (C-22), 72.37 (C-23), 20.14 (C-24), 169.64 (C-1′), 44.74 (C-2′, from heteronuclear multiple quantum coherence [HMQC] spectrum), not observed (C-3′).

Identification of the new compound as MMA.The chemical structures and physical properties of the three macrolactin compounds are compared in Fig. 1 and Table 1, respectively.

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FIG. 1.

Chemical structures of the macrolactin compounds produced by B. subtilis DSM 16696.

The molecular ion m/z of 402 and the UV absorptions at 227 and 261 nm enabled the identification of compound I as macrolactin A or its 10E isomer. The latter was ruled out by its optical rotation of [α]²²D = −138, compared to a result of about −10 found for macrolactin A. A direct comparison of the ¹H and ¹³C NMR data of the macrolactin A (data not shown) produced by the DSM 16696 strain with the previous reports was hampered, because only ¹H NMR data in benzene-d₆ and ¹³C NMR data in pyridine-d₅ are available in the literature (10).

Compound II was identified as a macrolactin A type from its identical UV spectrum. Mass spectrometry indicated a molecular mass of 488 Da, which is 86 absolute mass units higher than the mass observed for compound I. Corresponding to the elimination of one H₂O from compound I, compound II showed the loss of malonic acid by a fragment ion at a m/z of 383 in the (−)-ESI spectrum. The NMR data of compound II in MeOH-d₆ were nearly identical to those of compound I. However, compared to compound I, the 7-H signal was shifted about 1.2 ppm downfield as a consequence of the acylation of 7-O. Because only one carboxy group was directly visible in the NMR spectra of compound II in MeOH-d₄, the residue at 7-O was identified by comparison with compound I from the ¹H and ¹³C NMR spectra in dichloromethane-d₂. Here the malonyl residue was clearly indicated by additional carboxy ¹³C signals at 166.33 and 169.15 ppm and a methylene ¹³C signal at 42.17 ppm, which correlated to two ¹H doublet signals at 3.51 and 3.40 ppm (J = 15.5 Hz). The ¹H NMR data of compound III in CDCl₃ (data not shown) were found to be identical to those described by Jaruchoktaweechai et al. (16) for 7-O-succinyl macrolactin A (SMA).

MMA is a new bacteriostatic antibiotic active against MRSA, VRE, and a small-colony variant of B. cepacia.The agar diffusion method was used to compare the antimicrobial activities of the crude extract from strain DSM 16696, the three purified macrolactins, and, as controls, relevant antibiotics in clinical use against reference strains and clinical isolates (Table 2). All macrolactins showed good inhibition activity against both MSSA and MRSA but, while MMA and SMA also inhibited VRE, unsubstituted macrolactin A did not. However, the inhibition zones observed with the staphylococcal test strains were turbid, rather than clear, suggesting a growth inhibition rather than a bactericidal activity. To rule out the possibility of the turbidity resulting from the development of resistant variants, the small colonies developing in the inhibition zones were purified and retested for sensitivity. In all cases, they gave turbid inhibition zones, confirming that the macrolactins inhibit growth rather than kill (data not shown).

These qualitative experiments were subsequently confirmed by quantitative determination of MICs, the antibiotic concentrations that totally prevent microbial growth in liquid cultures (Table 3). In fact, the MICs of MMA for staphylococci and enterococci were higher than 128 μg/ml, but a strong inhibition of growth of both types of organisms was observed at much lower concentrations. We therefore determined the lowest concentrations of the new compound that resulted in 50% inhibition of bacterial growth, the MRC. The MRCs were between 1 and 64 μg/ml for the S. aureus reference strains and the MRSA strains and between 0.06 and 4 μg/ml for E. faecalis ATCC strain 29212 and the clinical isolates VRAS E305 and VRAR E315 (Table 3). No changes in the endpoints occurred when the incubation was extended to 48 h, which shows that the bacteria inhibited by the drugs do not reinitiate growth (data not shown). The liquid growth test also confirmed that MMA and SMA exhibited higher activities than macrolactin A (MA) against MRSA and VRE, suggesting the importance of substituents on the C-7 for the biological activity of this group of compounds (data not shown).

MMA was not active against the majority of the gram-negative isolates tested (data not shown). Interestingly, however, a small-colony variant of B. cepacia, but not the parental normal colony type, was inhibited, as was a Candida krusei strain, but not other Candida strains tested (Table 3).

Long-term inhibition of staphylococci and enterococci at MMA sub-MICs.To investigate the growth inhibition effect of 7-O-malonyl macrolactin A, time courses of the effects on bacterial viability of antibiotic at several sub-MICs were followed (Fig. 2). MMA at 1 μg/ml rapidly reduced the counted number of viable cells of the methicillin-susceptible S. aureus strain by almost 2 orders of magnitude over the first 2 hours of exposure. Over the next 6 h, bacterial multiplication occurred at the same rate as that of the nontreated control, although levels remained about 1 log unit lower than those of the controls over the 24-h period of the experiment. In the case of the MRSA 3 strain, no significant reduction in counts of viable cells occurred, but no significant growth was observed either, and by the end of the experiment there was a difference of >2 log units in the number of viable cells of the treated and control cultures. In the case of E. faecalis ATCC strain 12912, the reduction in the number of viable cells observed, ca. 50%, was less than that seen with the S. aureus reference strain, but otherwise the picture was similar. Also, MMA had an inhibitory effect on VRAR E. faecium strain E315 similar to that on the MRSA strain, namely, complete inhibition of growth. Essentially, the same patterns of viable cell numbers were seen with both subinhibitory concentrations of antibiotic, 4 and 16 μg/ml. Thus, interestingly, MMA was bacteriostatic for the antibiotic-resistant strains tested in liquid cultures but less inhibitory for the sensitive ones, although the sample is too small to generalize.

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FIG. 2.

Growth curves for bacteria treated with MMA at sub-MIC levels. The curves are viable cell concentrations plotted against time. (A) MSSA 32; (B) MRSA 3; (C) E. faecalis ATCC 29212; and (D) E. faecium VRAR E315. The sub-MICs (MRC and 4× MRC) of MMA in μg/ml are indicated for each curve.

MMA induces significant cellular damage at sub-MICs.The postantibiotic effect and SME, the time taken to repair and recover from antibiotic damage, are important properties of antibiotics which reflect the degree of such damage inflicted by antibiotics at supra-MICs and sub-MICs, respectively (5, 25). The SME for VRAR E. faecium strain E315 and MRSA strain 3 after 1 h of exposure to 16 μg/ml MMA were 2.31 h and 0.42 h, respectively, indicating that the compound induced significant damage, particularly in enterococci, at sub-MICs.

MMA inhibits separation of daughter cells.MMA-treated cells of E. faecium VRAR E315 and MRSA 3 were examined by electron microscopy in order to obtain ultrastructural information about the damage caused. Sub-MICs of MMA had very marked effects on cell division, and separation of daughter cells was severely inhibited through incomplete septum formation (Fig. 3 and 4). Treated cells of MRSA 3 were larger than controls, and approximately 60% were observed in packets of nonseparated cells (data not shown), in which multiple asymmetric initiation points of septum formation were visible (Fig. 3d, e, and f). Similarly, treated E. faecium VRAR E315 showed chains of nonseparated cells in which several symmetric initiations of cell division are evident (Fig. 4d, e, and f). Moreover, treated cells of E. faecium had a smooth appearance, whereas untreated cells had rough surfaces (Fig. 4c and e). SMA, but not MA, induced similar morphological alterations in both MRSA and VRE, but to a lesser extent (data not shown).

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FIG. 3.

Transmission electron micrographs of MMA-treated methicillin-resistant Staphylococcus aureus. The MRSA strain 3 clinical isolate was treated for 4 h with 16 μg/ml of MMA. Micrographs a, b, and c are of untreated controls and d, e, and f are of treated bacteria. Different states and positions of septum formation are indicated with large arrowheads (b, c, e, and f). Asymmetrical initiations of septum formation are also observed in treated cells (small arrowheads) in e and f.

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FIG. 4.

Transmission electron micrographs of MMA-treated vancomycin- and ampicillin-resistant enterococci. The E. faecium E315 clinical isolate was treated for 4 h with 16 μg/ml of MMA. Micrographs a, b, and c are of untreated controls and d, e, and f are of MMA-treated bacteria. Septa are indicated with large arrowheads (c, e, and f). Asymmetric initiation of cell division (d, arrow) and pseudomulticellular chains (d, white asterisk) with many primordial septa are indicated (e and f, small arrowheads) in treated cells.

MMA exhibits weak cytotoxicity.Macrolactin A has previously been reported to exhibit cytotoxic and antiviral activities (10) and, as described above, MMA exhibited weak activity towards a Candida krusei strain. Possible activity of MMA towards mammalian cells was therefore assessed with L929 mouse fibroblasts and HeLa human epithelial cells. As can be seen in Fig. 5, all three macrolactins inhibit the growth of L929 mouse fibroblasts, with MA being the most active, which suggests that eukaryotic cytotoxicity may be modulated by varying the C-7 substituent. 7-O-Malonyl macrolactin A was less inhibitory for the human than the mouse cells tested: quantitation of the cytotoxic effect by means of the CyQUANT assay revealed that MMA inhibited the proliferation of HeLa cells partially at 31.25 μg/ml and totally at 62.5 μg/ml (Fig. 5). The antiproliferative effect of the macrolactins was reflected in a change in cell morphology to rounded cells.

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FIG. 5.

Cytotoxicity of macrolactins. Effect of macrolactin A, 7-O-malonyl macrolactin A, and 7-O-succinyl macrolactin A on L929 mouse fibroblasts and HeLa human epithelial cells.