J Med Microbiol 53 (2004), 61-65; DOI: 10.1099/jmm.0.05250-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Antibacterial activity of the marine sponge constituent cribrostatin 6
Robin K. Pettit1,2,
Bridget R. Fakoury1,
John C. Knight1,
Christine A. Weber1,
George R. Pettit1,3,
Gary D. Cage4 and
Sandy Pon1
1,2,3Cancer Research Institute1 and Departments of Microbiology2 and Chemistry and Biochemistry3, Arizona State University, Tempe, AZ 85287-2404, USA 4Microbiology Lab, Phoenix Children's Hospital, Phoenix, AZ 85016, USA
Correspondence Robin K. Pettit pettitr{at}asu.edu
Received March 11, 2003
Accepted September 19, 2003
The antibacterial activity of the nitrogen heterocyclic sponge constituent cribrostatin 6 was examined. Cribrostatin 6 was bacteriostatic for a variety of Gram-positive species and was bactericidal for the majority of clinical isolates of Streptococcus pneumoniae, including penicillin-resistant strains. Minimum bactericidal concentration/MIC ratios were 
2 for 75 % of S. pneumoniae clinical isolates. Kill-curve analysis confirmed the bactericidal action of cribrostatin 6. Bactericidal activity was rather slow, beginning at 2, 4 or 8 h, depending on the strain. The frequency of occurrence of bacterial spontaneous mutations to resistance was 10-7. The maximum tolerated dose of cribrostatin 6 in mice was 750–1000 µg kg-1 day-1. Cribrostatin 6 is a promising lead antibiotic for Gram-positive bacteria, particularly S. pneumoniae, a leading cause of infection and mortality worldwide.
Abbreviations: BMHII, Mueller–Hinton II (cation-adjusted) broth containing 3 % lysed horse blood; MBC, minimum bactericidal concentration; MTD, maximum tolerated dose.
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Introduction
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Infections caused by antibiotic-resistant Gram-positive bacteria are a major cause of morbidity and mortality. Several of the more important resistance problems include vancomycin resistance in enterococci, penicillin resistance in streptococci and methicillin resistance in staphylococci. Streptococcus pneumoniae is the most common bacterial cause of acute respiratory infection and otitis media, and results in millions of deaths each year worldwide from pneumonia, bacteraemia and meningitis (Greenwood, 1999; Musher, 1992). Before 1990, most clinical isolates of S. pneumoniae in the US were susceptible to penicillin and various other antibiotics. As of 2000, 36 % of S. pneumoniae clinical isolates in the US were trimethoprim-sulfamethoxazole-resistant, 34 % were penicillin-resistant, 26 % were macrolide-resistant, 17 % were tetracycline-resistant, 9 % were clindamycin-resistant and 8 % were chloramphenicol-resistant (Doern et al., 2001). Also as of 2000, two-thirds of penicillin-resistant isolates were high-level-resistant and multiresistant (resistant to two or more non-ß-lactam antimicrobials) (Doern et al., 2001). There is an urgent need for new structural classes of antibiotics active against S. pneumoniae and other Gram-positive genera.
The blue marine sponge Cribrochalina sp. contains a variety of biologically active constituents, including the antimicrobial cribrostatins 2 and 4 (Pettit et al., 2000). We recently summarized the isolation and X-ray crystal structure determination of the dark-blue Cribrochalina constituent cribrostatin 6 (Pettit et al., 2003). Against a small panel of microbes, cribrostatin 6 demonstrated activity against Gram-positive bacteria (Pettit et al., 2003). Cribrostatin 6 was also weakly cytotoxic for human cancer cell lines (Pettit et al., 2003). Recently, interest in developing antineoplastics and their derivatives as antimicrobials, and vice versa, has increased. The antineoplastic agents methotrexate, cyclophosphamide, vincristine, bleomycin, daunorubicin, 5-fluorouracil, mitomycin C and taxol, for example, have antifungal activity (Cardenas et al., 1999). Tetracycline derivatives with human cancer and/or antifungal action are being pursued (Liu et al., 2002; Lokeshwar et al., 1998), and the lavage antibiotic taurolidine is currently being evaluated as a cancer chemotherapeutic in patients with glioblastoma and ovarian cancer (Calabresi et al., 2001). Our interest here is the in vitro development of cribrostatin 6 as a lead antibiotic for Gram-positive bacteria.
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Methods
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Cribrostatin 6.
Cribrostatin 6 (Fig. 1) was isolated in our laboratory as described previously (Pettit et al., 2003) and stored desiccated in the dark. Prior to each experiment, cribrostatin 6 was reconstituted in a small volume of sterile DMSO and then diluted in the appropriate growth medium.
Strains.
Non-duplicate clinical isolates and antibiotic resistance information were obtained from the Arizona Department of Health Services. Penicillin and methicillin resistance were verified in our lab by the broth microdilution assay outlined by the NCCLS (2000). Penicillin-resistant strains required MICs 
2 µg ml-1 (NCCLS, 2000). Methicillin resistance was evaluated with oxacillin and defined as an MIC 16 µg ml-1 (NCCLS, 2000). Invasive S. pneumoniae were cultured from sterile sites; their antibiotic resistance profiles are unknown. Reference strains were obtained from the American Type Culture Collection (Manassas, VA).
Susceptibility testing with cribrostatin 6.
Susceptibility testing with cribrostatin 6 was performed by the reference broth microdilution assay outlined by the NCCLS (2000). Isolated colonies from overnight cultures were suspended and diluted as recommended to yield final inocula of approximately 5 x 105 c.f.u. ml-1. Tests were performed in sterile microtitre plates containing twofold dilutions of cribrostatin 6 in Mueller–Hinton II (cation-adjusted) broth containing 3 % lysed horse blood (BMHII) (Arcanobacterium, Erysipelothrix, Lactobacillus, Streptococcus) or Mueller–Hinton II broth (all other bacteria). One well was left drug-free (but contained an equivalent volume of DMSO) for a turbidity control. Each well contained a total volume of 100 µl. Plates were incubated without agitation at 37 °C with 5 % CO2 (Arcanobacterium, Erysipelothrix, Lactobacillus) or at 35 °C (all other organisms). MICs were determined at 16–20 h for Bacillus, Corynebacterium, Enterococcus, Listeria, Rhodococcus equi and Staphylococcus, at 24 h for Arcanobacterium and Streptococcus and at 48 h for Erysipelothrix, Lactobacillus, Nocardia and Rhodococcus bronchialis. Broth microdilution assays were also performed in BMHII prepared at pH 6, 7 and 8, in BMHII with and without 25 % normal human serum (Lampire Biological Labs) and in BMHII with and without 20 and 40 µg BSA ml-1 (Sigma). The MIC was defined as the lowest concentration of drug that inhibited all visible growth of the test organism (optically clear). No trailing was observed. Minimum bactericidal concentrations (MBCs) were determined by subculture of 50 µl from each negative well and from the positive growth control well of the broth microdilution series onto drug-free plates. Plates were incubated at the appropriate temperature for 24–48 h. The MBC was defined as the lowest drug concentration that resulted in a 99.9 % reduction in the initial inoculum.
Frequency of spontaneous mutants.
The frequency of occurrence of single-step resistant mutants was determined. Overnight cultures of three strains of S. pneumoniae were diluted to an OD625 of 0.08–3. One hundred microlitres of each preparation was spread onto agar plates containing four times the broth microdilution MIC of cribrostatin 6. The starting inoculum for each organism was also diluted and plated onto drug-free plates for determination of c.f.u. ml-1. After 48 h incubation at the appropriate temperature, the number of bacterial colonies on drug-supplemented agar was counted. The frequency of occurrence of spontaneous resistant mutants was calculated by dividing the number of colonies on drug-containing plates by the number of c.f.u. in the inoculum. When no colonies were visualized on drug-containing plates, the calculation was (<)1 colony divided by the number of c.f.u. in the inoculum.
Time-kill studies.
Early log cultures in BMHII were inoculated into the same medium containing multiples of the broth microdilution MIC of cribrostatin 6 or an equivalent volume of DMSO. Cultures were shaken at 35 °C and aliquots were removed aseptically at various times for dilution plating. In addition, aliquots were plated directly from drug-treated flasks at the later time-points. Thus, the detection limit in these experiments was 10 c.f.u. ml-1. Standard errors of the means were calculated from at least two experiments.
Mouse toxicity evaluation.
Female CD-1 mice (5 weeks old) were obtained from Charles River Laboratories and assigned randomly into groups of five mice each. Cribrostatin 6 was dissolved in methanol and diluted in sterile PBS. Vehicle controls consisted of the same diluent mixture. Mice were given i.p. injections of 100, 200, 400, 500, 750 or 1000 µg cribrostatin 6 kg-1 day-1, 12 h apart, for 5 days. Mice were weighed daily and observed for gross signs of toxicity such as weight loss, dehydration and activity loss.
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Results and Discussion
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In broth microdilution assays, cribrostatin 6 inhibited the growth of all Gram-positive bacteria tested (Table 1). Cribrostatin 6 was most active against S. pneumoniae, including multidrug-resistant strains. MBC/MIC ratios were 2 for 75 % of S. pneumoniae clinical isolates, consistent with a bactericidal mechanism of action. We were puzzled by the appearance of increasing numbers of survivors at concentrations higher than the MBC for some S. pneumoniae strains (0.1 %) and for many Streptococcus pyogenes strains (68 %). This so-called Eagle effect has been described for other antimicrobials, including the ß-lactams, and is believed to be the result of high antimicrobial concentrations inhibiting protein synthesis to a degree that prevents the growth necessary for expression of the lethal effect of the antimicrobial (Amsterdam, 1996). There are apparently no therapeutic implications associated with this effect (Amsterdam, 1996). As no MBC could be defined for these strains, they were not included in MBC50 (MBC at which 50 % of the strains are killed), MBC90 (MBC at which 90 % of the strains are killed) and MBC/MIC ratio calculations.
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Table 1. Broth microdilution MICs and MBCs of cribrostatin 6 for reference strains and clinical isolates Where more than one strain was tested, the number of strains is given in parentheses; ranges are given in these cases.
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The effects of two host factors, pH and serum, on broth microdilution MICs were examined. For some strains, MICs increased slightly in acidic or alkaline medium (Table 2). Attempts were made to determine MICs at pH 6 for S. pyogenes, but these strains did not grow in acidic medium. MICs for S. pneumoniae increased three- to fourfold in 25 % human serum, while S. pyogenes MICs were unchanged (Table 2). Serum inactivation did not appear to be due to serum albumin binding, as MICs with or without 20 or 40 µg BSA ml-1 typically varied by no more than one, twofold dilution (data not shown).
The frequency of occurrence of single-step-resistant mutants at four times the MIC was < 10-7 for S. pneumoniae ATCC 6303, 9.3 x 10-7 for invasive clinical isolate 19486130 and 3 x 10-7 for clinical isolate 404090. Mutation frequencies were in the range expected for a compound in preclinical development.
Time-kill curves confirmed the bactericidal mechanism of action of cribrostatin 6. Fig. 2 summarizes the time-kill curves for three S. pneumoniae strains, ATCC 6303 (Fig. 2a), invasive clinical isolate 19486130 (Fig. 2b) and clinical isolate 404090 (Fig. 2c), and one S. pyogenes clinical isolate (30524975) (Fig. 2d). We attempted to perform kill-curves with the S. pneumoniae clinical isolates listed in Table 2, but the untreated controls did not yield typical growth curves. Killing was not concentration-dependent, with the exception of S. pneumoniae 404090 (Fig. 2c), where killing was concentration-dependent between one and four times the MIC. Killing was time-dependent for the three S. pneumoniae strains, but not for the single S. pyogenes strain during the first 8 h (Fig. 2d). Killing occurred slowly, after 2 h for the two clinical isolates of S. pneumoniae (Fig. 2b, c) after 4 h for S. pneumoniae ATCC 6303 (Fig. 2a) and after 8 h for the S. pyogenes clinical isolate (Fig. 2d). The number of survivors in cultures of S. pneumoniae ATCC 6303 and 404090 treated with an intermediate dose (four times the MIC) for 24 h varied greatly, with regrowth occurring in some cases (Fig. 2a, c). In vitro time-kill systems lack host parameters like immune defences. Thus, clinical studies will be necessary in order to define any in vivo significance of the regrowth phenomena.
The MTD of cribrostatin 6 in mice was 750–1000 µg kg-1 day-1. At all doses except 1000 µg kg-1 day-1, mice showed no signs of gross toxicity. Mice at the highest dose exhibited toxicity through lower overall activity and weight loss. Efficacy studies in topical and systemic models of Gram-positive infection await synthesis of cribrostatin 6. Synthesis of cribrostatin 6 and derivatives is in progress in our laboratories.
There has been an alarming increase in the number of drug-resistant bacteria. The novel nitrogen heterocyclic compound cribrostatin 6 is a promising lead compound for Gram-positive infections, particularly S. pneumoniae. In vivo studies are necessary in order to validate the in vitro properties of cribrostatin 6. The relative potency of the cribrostatins for cancer cells versus bacterial cells will be critical when selecting candidates for further preclinical development.
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ACKNOWLEDGEMENTS
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This research was supported by the Arizona Disease Control Research Commission, Outstanding Investigator Grant CA 44344-08-12 and grant R01 CA90441-02 from the Division of Cancer Treatment and Diagnosis, NCI, DHHS, and the Robert B. Dalton Endowment Fund. We thank Dr F. Hogan for preparing Fig. 1.
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References
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Introduction
Methods
) and at the MIC (
) and at four (
) and eight (
) times the MIC of cribrostatin 6. The results are means ± SE of at least two experiments.