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MICs of Selected Antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a Range of Clinical and Environmental Sources as Determined by the Etest

Peter C. B. Turnbull,^* Nicky M. Sirianni, Carlos I. LeBron, Marian N. Samaan, Felicia N. Sutton, Anatalio E. Reyes, and Leonard F. Peruski Jr.

Biological Defense Research Directorate, Naval Medical Research Center, Silver Spring, Maryland 20910-7500

Received 27 June 2003/ Returned for modification 7 August 2003/ Accepted 20 April 2004

	ABSTRACT

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This paper presents Etest determinations of MICs of selected antimicrobial agents for 76 isolates of Bacillus anthracis chosen for their diverse histories and 67, 12, and 4 cultures, respectively, of its close relatives B. cereus, B. thuringiensis, and B. mycoides derived from a range of clinical and environmental sources. NCCLS breakpoints are now available for B. anthracis and ciprofloxacin, penicillin, and tetracycline; based on these breakpoints, the B. anthracis isolates were all fully susceptible to ciprofloxacin and tetracycline, and all except four cultures, three of which had a known history of penicillin resistance and were thought to originate from the same original parent, were susceptible to penicillin. Based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria, all cultures were susceptible to amoxicillin-clavulanic acid and gentamicin and 99% (one with intermediate sensitivity) of cultures were susceptible to vancomycin. No group trends were apparent among the different categories of B. cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness). Differences between B. anthracis and the other species were as expected for amoxicillin and penicillin, with all B. anthracis cultures, apart from the four referred to above, being susceptible versus high proportions of resistant isolates for the other three species. Four of the B. cereus and one of the B. thuringiensis cultures were resistant to tetracycline and a further six B. cereus and one B. thuringiensis cultures fell into the intermediate category. There was a slightly higher resistance to azithromycin among the B. anthracis strains than for the other species. The proportion of B. anthracis strains fully susceptible to erythromycin was also substantially lower than for the other species, although just a single B. cereus strain was fully resistant. The Etest compared favorably with agar dilution in a subsidiary test set up to test the readings, and it compared with other published studies utilizing a variety of test methods.

	INTRODUCTION

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Little interest was shown in antimicrobial susceptibility profiles of Bacillus species until very recently. This was due to a combination of reasons: the low recognition of the ability of Bacillus species other than Bacillus anthracis to cause infections; the increasing rarity of human anthrax in industrialized, developed countries as a result of effective control programs over the past half century; and the high susceptibility of B. anthracis to penicillin coupled with the extreme rarity of reports of penicillin resistance. In developing countries where anthrax is endemic, penicillin has always been the drug of choice because of its reliability, low cost, and ready availability.

Concerns about bioaggression around the time of the 1991 Gulf War resulted in some examination of the effectiveness of more modern antimicrobials both in vitro (10, 22) and in vivo in animal models (13, 17, 20). The "anthrax letter" events of October and November 2001 in the United States further stimulated interest in antimicrobial therapy for anthrax and some debate on the appropriate therapies for different categories of patient infection (5, 6, 15, 16), with further in vitro susceptibility tests being carried out (4, 9, 12, 26). Unrelated to bioaggression, but also relevant, is the recent paper of Kadanali et al. (18) on the treatment of pregnant patients.

This study reported here, which had commenced before the anthrax letter events, was initiated to apply the Etest to as diverse a range of B. anthracis isolates as possible together with a set of its close relatives B. cereus, B. thuringiensis, and B. mycoides isolated from a range of clinical and environmental sources. The primary purpose of the study was to determine the susceptibilities of these species to a set of antibiotics selected to have the greatest guidance value to clinicians encountering anthrax, B. cereus, and possibly B thuringiensis infections in humans (B. mycoides has not been associated with infections). The generation of comparative susceptibility and resistance data on the members of the informally defined "B. cereus group" for academic purposes was the secondary aim of the work.

	MATERIALS AND METHODS

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Isolates. The B. anthracis isolates included 24 cultures (Table 1), kindly supplied by Martin Hugh-Jones and Pamala Coker, School of Veterinary Medicine, Louisiana State University, which represented all but one of the amplified fragment length polymorphism (AFLP) genotype clusters of B. anthracis (19). A further 52 isolates from the culture collection of the Centre for Applied Microbiology and Research, Health Protection Agency, Porton Down, Salisbury, United Kingdom, were chosen on the basis of being as diverse as possible in terms of (i) geographic source, (ii) year of isolation, (iii) type of source (human, animal, or environmental), (iv) known or likely laboratory manipulation (frequent passage or deliberate curing of one or both plasmids), and (v) known unusual characteristics, particularly penicillin or phage resistance. Of the 76 total cultures included, 59 were believed to be unrelated epidemiologically. All manipulations of B. anthracis were carried out in class 2 microbiological safety cabinets within the Biological Defense Research Directorate (BDRD) biosafety level 3 (BSL3) facility under strict safety protocols and meeting all the requirements of DHHS 42 CFR 73 (12a).

Etests. Cultures were grown on Mueller-Hinton agar (MHA) overnight at 36°C ± 1°C. For each test, growth from approximately five colonies was emulsified in 1 ml of sterile saline, and this was used to make a suspension, again in sterile saline, with turbidity equivalent to a 0.5 McFarland standard. This turbidity level was established with 18 of the cultures, representative of the different species included in the study, to fall within the range of 3 x 10⁶ to 20 x 10⁶ CFU/ml, compared against 1 x 10⁸ to 3 x 10⁸ CFU/ml for S. aureus ATCC 29213. Sterile swabs dipped into this suspension and squeezed against the side of the suspension tube to remove excess fluid were streaked across predried MHA plates (90 mm), three times for each plate, with the plate rotated approximately 90° between each streaking. After approximately 10 to 15 min, to allow absorption of excess moisture into the agar, two Etest strips (AB Biodisk North America Inc., N.J.) per plate in opposing directions were placed on either side of each plate.

The plates were incubated at 36°C ± 1°C for 18 to 20 h, and the MICs were read according to the manufacturer's instructions.

Inoculum size and incubation time and temperature. The Etest manufacturer's specifications for inoculum size for aerobes is based on bringing the culture to a turbidity equivalent to a 0.5 McFarland standard. Bacillus species, being comprised of large rods, had lower counts at this turbidity level than did smaller bacteria such as the gram-positive cocci or the Enterobacteriaceae. Being rapid growers, producing large colonies by 16 to 24 h, the lawns on the plates of Bacillus species also have different properties from lawns of more frequently encountered pathogenic aerobes. Additionally, the manufacturer specifies an incubation temperature of 35°C. This temperature is not necessarily a convenient specification for a laboratory incubator or the optimum incubation temperature for Bacillus species. Tests were therefore set up to assess the influence of inoculum size, temperature, and time of incubation. Fourteen of the strains, three clinical (F77/1589, F78/667, and F95/8201), two food poisoning (F72/4810 and F73/4433), and two environmental (F99/5739 and F00/3016) isolates of B. cereus and two B. thuringiensis (B1143 and F98/5750) and five B. anthracis (Ames, ASC 32, LSU102, LSU248, and LSU293) strains, chosen from the main set of tests as being representative of diverse susceptibility readings with the antibiotics, were retested by using inocula with turbidities of 0.5, 2.0, and 4.0 and with the test plates incubated at carefully controlled temperatures of 30, 35, and 37°C, followed by readings at 16 and 24 h. S. aureus ATCC 29213 was again included for comparison. The readings were analyzed statistically by the unpaired Student's t test.

Agar dilution MIC tests. For purposes of direct comparison of the Etest results with a conventional procedure, the MICs for 10 of the B. anthracis strains, 15 of the B. cereus strains (5 food and environmental, 5 food poisoning, and 5 nongastrointestinal infection isolates), 5 of the B. thuringiensis strains, and the 4 B. mycoides strains, together with S. aureus ATCC 29213, were tested by using an agar dilution method described previously (22). Each antibiotic was diluted and incorporated into 100 ml of MHA to create a series of plates (150 mm) ranging from 64 to 0.015 mg/liter. In practice, this involved adding 5 ml of 20x solutions in sterile deionized water, prewarmed to 44°C, to 95 ml of sterilized MHA also at 44°C prior to the solutions being poured onto the plates. Because preliminary trials established no difference in results between the use of 1:10 and 1:50 dilutions of these suspensions, the final inoculum chosen was 5 µl of a 1:25 dilution of the 0.5 McFarland standard suspensions, established by plate counts to be equivalent to approximately 1,000 CFU. Duplicate 5-µl drops of the diluted culture suspensions were placed onto each plate by using a location grid.

	RESULTS

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The Etest results are summarized in Table 3. Where NCCLS breakpoints have been established for B. anthracis (ciprofloxacin, penicillin, and tetracycline) (27), the interpretation of susceptibility has been based on those breakpoints. For the other antibiotics in the case of B. anthracis and for all the antibiotics in the case of the nonanthrax Bacillus species, the susceptibility and resistance judgments are based on NCCLS interpretive standards for gram-positive and/or aerobic bacteria as given in the Etest manufacturer's product inserts.

An analysis of results for the different categories of B. cereus (isolates from food poisoning incidents and nongastrointestinal infections and food and environmental specimens not associated with illness) did not reveal any group trends (details not presented). Apparent species differences between B. cereus, B. thuringiensis, and B. mycoides with cefotaxime (29% of B. cereus isolates were susceptible versus none of the B. thuringiensis and B. mycoides isolates) may simply reflect the relatively small numbers of B. thuringiensis and B. mycoides strains included.

In relation to penicillin sensitivity, ASC 32, ASC 70, and ASC 183 were counted as a single strain so as not to distort the percentage of the total of strains that were penicillin resistant. The unusual resistance of ASC 32 and ASC 70 to penicillin has already been noted (22). A single isolate, LSU 62, was fully susceptible and a second isolate, ASC 65, had intermediate susceptibility to cefotaxime. This may be a good strain marker for these cultures, which in fact have other slightly unusual characteristics; LSU 62 is the only strain of B. anthracis that we have encountered which will not grow on the well-established polymyxin-lysozyme-EDTA-thallous acetate (PLET) agar used for selective isolation of B. anthracis from environmental samples, and ASC 65 produces colonies resembling those of Enterobacteriaceae. The inhibitory component of PLET for LSU 62 was shown not to be polymyxin.

ASC 32, ASC 70, and ASC 183 exhibited complete resistance with no zones of clearing. One of the strains (LSU 102) reported to be penicillin resistant by Coker et al. (9) did exhibit a resistant subpopulation with colonies present in the ellipse. This strain was therefore deemed resistant to penicillin. In this study, resistance was not noted with the other two strains Coker et al. recorded as being resistant (LSU 248 and LSU 293). All three LSU strains were included in the subsidiary study on the effect of inoculation size and incubation temperature and time. While the four resistant cultures had elevated MICs of amoxicillin and clavulanic acid compared with the others, they still fell well within the susceptible category as it is presently defined.

None of the Ames or Vollum strain reisolates ASC 394 to ASC 399, from guinea pigs which died after the cessation of ciprofloxacin or doxycycline prophylaxis following infection by the inhalational route (17), had developed observable resistance.

In the tests carried out to assess the effect of inoculum size and temperature and time of incubation, no significant differences in the readings were found for any of the starting inocula (turbidity equivalents of 0.5, 2.0, and 4.0), incubation temperature (30, 35, or 37°C), or reading times (16 or 24 h) (P = 0.16 to 1.00 for all, except for the comparison of vancomycin tests read at 30 and 35 or 37°C, for which P = 0.06).

In the subsidiary tests done to compare Etests with a conventional MIC approach, although only 78% of the Etest and agar dilution MIC readings were within 1 agar dilution unit of each other (96% in the case of the tests on B. anthracis alone), the only disagreement in terms of judgements as to susceptibility or resistance were that B. anthracis and B. cereus cultures, which were seen as having intermediate susceptibility to cefotaxime by agar dilution, were fully resistant by Etests (Tables 4 and 5).

	DISCUSSION

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Cavallo et al. (4) recorded a surprisingly high proportion (11.5%) of penicillin-resistant isolates of B. anthracis in their series, but they did not give their histories, apart from stating that 67 (70%) were isolated from environmental sources. It is possible that some of the isolates were related and that this may account for this high percentage of penicillin-resistant isolates.

The ability of B. anthracis to produce penicillinase was in fact recognized over half a century ago (3). Lightfoot et al. (22) demonstrated inducible ß-lactamase production in a number of strains following exposure to a subinhibitory level of flucloxacillin. Inducible ß-lactamases were again noted in relation to the anthrax events in the United States (6). The latter events led to published statements that penicillins, at least alone, are not recommended for the treatment of anthrax (5, 6). The B. anthracis genome sequence shows that this organism encodes two ß-lactamases, a penicillinase and a cephalosporinase (6, 7, 25, 31). These ß-lactamases are two examples of a significant number of genes (including those for motility, for example) that are shared with the closely related B. cereus which, though present, are not expressed as a result of a truncation in the plcR positive regulator gene (31, 34). However, the reality is that reports of naturally occurring resistance to penicillin in fresh clinical isolates are exceedingly rare and appear to number just five cases (2, 30), not all of which were well substantiated with further studies.

In this context, reports (4, 9; the latter not wholly confirmed here) that 11.5 and 12% of strains are resistant to penicillin are a little disturbing. Penicillin has long stood the test of time as the first choice for the treatment of anthrax in most parts of the world, and from the standpoint of the treatment of naturally acquired anthrax (as opposed to considerations relating to possible bioaggressive events), as it is cheap and readily available almost everywhere, it has to at least remain the basis of treatment schedules in animals and humans in developing countries. This view has been reinforced recently by others (32). Probably the fundamental principle, first stated half a century ago (14), is that adequate doses should be administered when penicillin is being used for treatment. It should be stressed, though, that there is no question that it is reasonable to add a second drug, where it is possible to do so, in cases showing signs of systemic involvement (32) or in other extreme situations such as known deliberate release exposures. That is by no means a new idea; the synergistic action of penicillin and streptomycin was recognized 40 years ago, and the recommendation was made then that both antibiotics be used at the same time in the treatment of septicemic anthrax (23).

The development of reduced susceptibility of B. anthracis to the quinolone ofloxacin but not to doxycycline following sequential subculture in subinhibitory concentrations has been demonstrated (8). The relatively low proportion of B. anthracis strains fully susceptible to erythromycin (15%) was somewhat surprising in view of the fact that this drug was regarded from the earliest days of antimicrobial chemotherapy (14) as an effective alternative to penicillin and is usually listed as such in medical microbiology texts.

B. cereus has long been associated with both food-borne illness and nongastrointestinal infections (11, 21, 24, 28, 33, 35). The latter infections are usually, but not always, opportunistic and are sometimes severe or life threatening. The incrimination of B. thuringiensis in infections is rare but has occurred, while B. mycoides appears to be totally nonpathogenic. From the many case reports of B. cereus infections, the broad picture is one of resistance to penicillin, ampicillin, cephalosporins, and trimethoprim and susceptibility to clindamycin, erythromycin, chloramphenicol, vancomycin, the aminoglycosides, and, usually, tetracycline. Ciprofloxacin was used successfully in the treatment of B. cereus wound infections (21). In a comparison of MIC methods, Andrews and Wise (1) found that, of five B. cereus strains, all were susceptible to ciprofloxacin and, with some variation between methods, to doxycycline; all were resistant to penicillin while, to tetracycline, two were susceptible, one was resistant, and two gave variable readings.

	ACKNOWLEDGMENTS

 
We are grateful to Martin Hugh-Jones and Pamala Coker, School of Veterinary Medicine, Louisiana State University, Jim McLauchlin and colleagues at the Food Safety and Microbiology Laboratory, Health Protection Agency Colindale, London, United Kingdom, and Niall Logan, Department of Biological Sciences, Glasgow Caledonian University, Glasgow, United kingdom, for making available the various cultures and their histories; to Roger Scott, Department of Microbiology, Taunton, and Somerset Hospital, Taunton TA1 5DB, United Kingdom, for technical information; and to M. Doganay, Faculty of Medicine, Erciyes University, Kayseri, Turkey, Lorraine Arntzen, National Institute for Communicable Diseases, Johannesburg, South Africa, and F. C. Tenover, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, Ga., for assistance with references.

The views, opinions, and/or findings presented are those of the authors and should not be construed as reflecting the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government.

	FOOTNOTES

 
* Corresponding author. Mailing address: BDRD NMRC, 503 Robert Grant Ave., Room 1A12, Silver Spring, MD 20910-7500. Phone: (301) 319-7515. Fax: (301) 319-7513. E-mail: turnbullp{at}nmrc.navy.mil.

Present address: Hillman Cancer Center L1.20, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213.

Present address: Department of Microbiology and Immunology, Indiana University School of Medicine, Northwest Center, Gary, IN 46408.

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