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Journal of Clinical Microbiology, October 2006, p. 3814-3818, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00881-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vitro Activities of Daptomycin, Ciprofloxacin, and Other Antimicrobial Agents against the Cells and Spores of Clinical Isolates of Bacillus Species

Diane M. Citron^1* and Maria D. Appleman²

Los Angeles County/University of Southern California Medical Center,¹ USC Keck School of Medicine, Los Angeles, California²

Received 26 April 2006/ Returned for modification 22 June 2006/ Accepted 3 August 2006

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Daptomycin inhibited 67 of 70 clinical isolates of Bacillus species at 1 µg/ml and 100% of them at 2 µg/ml. It showed bactericidal activity similar to that of ciprofloxacin against vegetative cells but not against spores. For 2 strains, the ciprofloxacin MICs were >4 g/ml, and 10 strains were resistant to erythromycin.

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Daptomycin is a cyclic lipopeptide with rapid bactericidal activity against most gram-positive bacteria (8, 22, 23, 24, 25). The drug acts through a calcium-dependent disruption of the cell membrane potential resulting in cell death (3, 23). While extensive research has been done with daptomycin against the well-established gram-positive pathogens (22), limited data are available on other, less frequently encountered, opportunistic gram-positive pathogens such as Bacillus species. Heine and colleagues demonstrated that the drug has activity against Bacillus anthracis by using a murine model of spore inhalation (H. S. Heine, J. Bassett, L. Miller, G. P. Andrews, and W. R. Byrne, Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. A-051, 2004). We investigated the activities of daptomycin against Bacillus cereus and other Bacillus species since these organisms are increasingly being implicated in gastrointestinal disease and various other serious infections, such as bacteremia (2, 4, 13, 14, 16), meningitis (5, 13), pneumonia (12), endophthalmitis (11, 20), periodontitis (15), and other infections (1, 9, 10, 17, 18, 21), especially in immunocompromised patients.

Study isolates. Seventy clinical isolates of Bacillus species recovered from blood, respiratory, eye, intraabdominal, and soft-tissue infections of patients at the Los Angeles County-University of Southern California Medical Center during the past 10 years were included in this study. The in vitro activities of daptomycin and comparator antimicrobial agents was determined by the CLSI broth microdilution method (6, 7). Testing was done with overnight cultures of the strains and also with spore preparations to see if daptomycin at any concentration showed activity against spores. A second method for testing sporicidal activity in water was investigated as well.

Spore-forming, gram-positive bacilli were identified to the species level by sequencing the16S rRNA genes and comparing the sequences to those in the GenBank database. The B. cereus group, which could not be well differentiated by 16S rRNA gene sequencing, was further identified by using tests for motility, hemolysis, and the presence of parasporal crystals demonstrated by brilliant green staining and confirmed by phase microscopy (19). The species and numbers of strains tested are listed in Table 1.

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TABLE 1. In vitro activities of daptomycin, ciprofloxacin, and eight comparator antimicrobial agents against Bacillus species

Measuring antibiotic effects on vegetative cells (MIC determinations). The comparator antimicrobial agents selected if previous studies and clinical trials suggested their efficacy included ciprofloxacin, doxycycline, vancomycin, penicillin, erythromycin, chloramphenicol, gentamicin, tobramycin, and amikacin. Laboratory reference standard powders were used to prepare MIC trays with the Quick-Spense apparatus (Sandy Springs Instrument Co., Germantown, MD). Conventional MICs were determined in cation-adjusted Mueller-Hinton broth (CAMHB) that was further supplemented to a final concentration of 50 µg/ml Ca²⁺ in the daptomycin wells. Twofold antibiotic dilutions of 0.03 to 16 µg/ml were tested. Overnight cultures were suspended in CAMHB and added to the wells for a final inoculum concentration of 5 x 10⁵ CFU/ml. Control wells were subcultured quantitatively to verify the colony counts. After overnight incubation at 35°C, the MICs were determined visually. Staphylococcus aureus ATCC 29213 was included as a control on each day of testing.

Measuring the activity of daptomycin against spores. Daptomycin and ciprofloxacin were tested for sporicidal activity. A heavy suspension of cells adjusted to a McFarland no. 2 standard was prepared in sterile distilled water from 5-day-old plates. This suspension was incubated in a water bath for 0.5 h at 80°C to kill vegetative cells and then centrifuged for 10 min to sediment the cell debris. The supernatant was quantitatively subcultured on blood agar to determine the number (CFU per milliliter) of viable spores and stored at 4°C overnight. The next day, the supernatant was diluted in CAMHB and added to the wells containing 200 µl of each twofold dilution of the antibiotic for a final inoculum concentration of approximately 10⁴ CFU/ml. A drug-free growth control well was included. After 0, 1, 2, 4, 8, and 24 h, the wells were mixed and a 10-µl aliquot was transferred from each well to a 0.2-µm-pore-size grid filter (Pall Corp., Ann Arbor, MI) and placed onto the pad of the Corning Filter System (Corning Inc., Corning, NY) with vacuum pressure. Each was carefully washed with 1 ml sterile distilled water to remove residual drug and distribute the 10 µl over the filter. The filters were placed onto Mueller-Hinton agar and incubated overnight at 35°C for colony count determinations.

A second method for testing sporicidal activity was used with one strain each of B. cereus, B. thuringiensis, B. subtilis, and B. pumilus. Spores (10⁴/ml) were added to tubes containing concentrations of daptomycin (in water adjusted to a Ca²⁺ concentration of 50 µg/ml) equal to two, four, and eight times the respective MICs. Ciprofloxacin was tested in the same manner, and a drug-free water tube was included as a negative control. The tubes were incubated at 35°C and assayed by removing 0.1 ml and washing as described above, at 2, 4, 8, and 24 h. A positive control was designed as 1% Clorox (0.05% hypochlorite) and assayed after 30 s, 30 min, and 1 h.

Identification by 16S rRNA gene sequencing classified most of the Bacillus strains to the species level at 99% similarity. Six of the strains did not match any of the published species in the GenBank database, but their similarity to several different species within the genus Bacillus or Paenibacillus was at 97 to 99%.

The effects of the antibiotics on vegetative cells are presented as MICs in Table 1. The time-kill interactions showing the activities of daptomycin and ciprofloxacin against spores of representative strains of B. pumilus and B. subtilis and the vegetative cells and spores of a representative strain of B. cereus are shown in Fig. 1 to 4.

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FIG. 1. B. cereus MRL 18731 cells. Daptomycin (D) MIC, 0.5 µg/ml; ciprofloxacin (C) MIC, 0.125 µg/ml. Level of detection is 100 CFU/ml.

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FIG. 4. B. subtilis MRL 11803 spores. Daptomycin (D) MIC, 0.5 µg/ml; ciprofloxacin (C) MIC, 0.03 µg/ml. Level of detection is 100 CFU/ml.

MIC determinations demonstrated the effects of the antibiotics on vegetative cells. Daptomycin inhibited 96.7% of the strains tested at 1 µg/ml and 100% of them at 2 µg/ml. Ciprofloxacin likewise inhibited 97.1% of the strains tested at 1 µg/ml, although for one strain each of B. cereus and B. thuringiensis the MIC was >4 µg/ml. Doxycycline inhibited all except one strain of B. cereus at 1 µg/ml. Most strains in the B. cereus group were resistant to penicillin (MIC, 16 µg/ml), as was one strain each of B. subtilis and B. firmus. The erythromycin MICs for 10 strains were 8 µg/ml, including B. cereus, B. thuringiensis, B. halodurans, B. firmus, Paenibacillus lautus, and Paenibacillus species. All strains were inhibited by 8 µg/ml chloramphenicol, and all except the two strains of P. lautus were susceptible to the aminoglycosides. Our MIC results are similar to those obtained by Weber et al. (25) and Turnbull et al. (24), except that we found two ciprofloxacin-resistant strains.

Figures 1 and 2 show the contrast in the numbers of colonies (CFU) recovered from cell versus spore inocula of B. cereus. The initial studies of sporicidal activity demonstrated that daptomycin and ciprofloxacin were killing the spores. However, there is an apparent increased lag time from inoculation to CFU reduction with the spores compared to the very rapid killing observed with the vegetative cells. This suggests that there is no activity until the spores germinate. Thus, it appears that the spores germinated in CAMHB and the observed decrease in the number of CFU was more likely due to the death of vegetative cells from germinated spores. Moreover, the MICs measured after 24 h in the sporicidal experiments were within 1 dilution of those measured in the standard MIC tests. However, although spore germination was not inhibited, the tests showed that daptomycin and ciprofloxacin were both rapidly bactericidal against the B. cereus group, although killing was slower for some of the other species, for example, representative strains of B. pumilus and B. subtilis, as shown in Fig. 3 and 4, with very low numbers of cells persisting after 24 h.

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FIG. 2. B. cereus MRL 18731 spores. D, daptomycin; C, ciprofloxacin. Level of detection is 100 CFU/ml.

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FIG. 3. B. pumilus MRL 11052 spores. Daptomycin (D) MIC, 0.5 µg/ml; ciprofloxacin (C) MIC, 0.125 µg/ml. Level of detection is 100 CFU/ml.

The second method that tested antispore activity in water showed no difference in the number of CFU per milliliter between the drug-containing tubes and the drug-free control tube after 24 h of incubation. The positive control containing 1% Clorox (0.05% hypochlorite) killed three of the four strains within 30 s. However, only 94% of the B. cereus cells were killed after 30 s. All were killed at the end of the second assay period (30 min).

Daptomycin demonstrated very good in vitro efficacy against a variety of clinical isolates of Bacillus spp., comparable to that of ciprofloxacin. The in vitro activity was demonstrated against vegetative cells of this genus but not against spores. However, since the spores were apparently killed as they germinated and since this is how the disease process with spore-forming bacteria occurs, there is reason to investigate the drug's possible role in therapy for these infections. Daptomycin's clinical utility in infections with Bacillus spp. warrants further investigation.

 
This study was supported by a grant from Cubist Pharmaceuticals, Inc.

We thank Rosa Altomstone for excellent technical assistance.

 
* Corresponding author. Mailing address: Microbial Research Lab, 2G-24, LAC+USC Medical Center, 1801 Marengo Street, Los Angeles, CA 90033. Phone: (323) 226-3749. Fax: (323) 226-7021. E-mail: dcitron{at}usc.edu.

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Journal of Clinical Microbiology, October 2006, p. 3814-3818, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00881-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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