Antimicrobial Susceptibilities of Diverse Bacillus anthracis Isolates

A test of 25 genetically diverse isolates of Bacillus anthraciswas conducted to determine their susceptibility to seven clinicallyrelevant antimicrobial agents. Etest strips (AB BIODISK, Solna,Sweden) were used to measure the MICs for the isolates. Usingthe National Committee for Clinical Laboratory Standards MICbreakpoints for staphylococci, three isolates were found tobe resistant to penicillin and five were found to be resistantto cefuroxime. The penicillin-resistant isolates were negativefor ß-lactamase production. Continued surveillanceof B. anthracis field isolates is recommended to monitor antimicrobialsusceptibility.

INTRODUCTION

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Introduction

The etiologic agent of anthrax, Bacillus anthracis, causes anacute disease, primarily of herbivores, that is transmissibleto humans. Susceptible animals are primarily infected by thespores formed during the vegetative state of the bacteria. Sporesmay be found in soil contaminated by diseased animals or indiseased animal products, such as hair, wool, hides, and bones.The importance of treatment of the disease in humans has beenunderscored by the bioterrorism events of October 2001 in theUnited States. Ciprofloxacin was the antimicrobial agent ofchoice for prophylactic treatment after exposure to the sporesof B. anthracis that were used in the bioterrorism events. Penicillin,traditionally the drug of choice for treatment, is still recommendedin other parts of the world despite reports of penicillin resistance(2, 3). The Centers for Disease Control recommended ciprofloxacin,penicillin, and doxycycline for the treatment of human anthraxand for use as a prophylactic measure prior to and during theOctober event (1). In the past, streptomycin, penicillin, gentamicin,and chloramphenicol have also been recommended (7).

Other articles concerning B. anthracis and antimicrobial susceptibilityhave been written: two over 10 years ago and one within thepast year. The first paper, published in 1990 by Lightfoot etal., determined the antimicrobial susceptibility of 70 isolatesof B anthracis to penicillin, amoxicillin, cefuroxime, gentamicin,streptomycin, erythromycin, tretracycline, chloramphenicol,and ciprofloxacin by agar dilution (6). Penicillin resistanceand ß-lactamase production were noted in two isolates.This paper was quickly followed in 1991 by a paper by Doganayand Aydin (2). They tested 22 B. anthracis isolates against27 antimicrobial agents by agar dilution and reported that 19isolates showed resistance to the five broad-spectrum cephalosporinstested. The isolates were shown to be sensitive to the otherantimicrobials tested. These two papers were very basic in theirapproach and reporting. The latest paper, by Mohammed et al.,tested 65 isolates with nine antimicrobial agents by using theNational Committee for Clinical Laboratory Standards (NCCLS)broth microdilution method (7). The results obtained from 50of the isolates were compared to those obtained with Etest strips.One isolate was penicillin resistant, and no statistically significantdifference was found between the broth microdilution and Etestresults.

The two earlier papers distinguished between sensitive and resistantwith regard to the MIC noted for each isolate (2, 6). Unfortunately,no basis was given for the standard they used for these designations.The paper by Mohammed et al. used the NCCLS breakpoints forStaphylococcus, which we have also chosen to use to discriminatebetween sensitive (S), intermediate (I), and resistant (R) isolates.According to the NCCLS literature, studies of Bacillus spp.are not yet adequate to develop reproducible, definitive standardsto interpret results (8). The infections caused by Staphylococcusspp. are very similar to the infections caused by B. anthracis.In addition, the distributions of the aforementioned antimicrobialdata are in line with the Staphylococcus MIC breakpoints forthe antimicrobials used in this study.

In this study, we examined 25 genetically diverse isolates ofB. anthracis to determine their susceptibilities to seven clinicallyrelevant antimicrobial agents. Etest strips (AB BIODISK, Solna,Sweden) were used to determine the MICs for the isolates.

MATERIALS AND METHODS

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Materials and Methods

A total of 25 isolates diverse in time, space, and genotypewere used in this study (Table 1). These 25 isolates are a representativesubset of 89 distinct genotypes characterized by multiple-locusvariable-number tandem repeat analysis (5).

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TABLE 1. B. anthracis isolates used by genotype and cluster

MICs of the seven antimicrobial agents penicillin, cephalexin,cefaclor, tobramycin, doxycycline, ciprofloxacin, and cefuroximewere determined by using the Etest methodology described elsewhere(4). Tryptic soy agar (TSA) plates containing 5% sheep blood(Remel) were inoculated with a swab taken from a colony suspensionequal to that of a 0.5 McFarland standard. One Etest strip wasplaced in each plate after 10 min. Quality control was assessedwith Staphylococcus aureus ATCC 29213. The plates were incubatedovernight at 37°C, and the plates were read at 16 and 24h. This procedure was repeated three times for each isolate.

The ß-lactamase activity of each isolate was determinedwith nitrocefin disks (BBL), which utilize a chromogenic cephalosporin.A drop of sterilized water was applied to the disk, and thena loop was used to remove a colony from the agar plate and itwas applied to the disk. The inoculum was taken from an overnightTSA plate for each isolate. A positive reaction was denotedby the formation of a pink or red color. The reactions wereheld for 1 h to confirm negative results.

RESULTS

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Results

The MIC results are summarized in Table 2. Using the StaphylococcusMIC breakpoints, all isolates were susceptible to the antimicrobials,except for cefuroxime and penicillin.

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TABLE 2. MICs for and antimicrobial susceptibilities of 25 B. anthracis strains

Only one isolate, GT 69, was sensitive. Five isolates (GT 23,28, 34, 55, and 77) were resistant, and the remaining 19 isolateswere categorized as intermediate to cefuroxime. The MIC at which90% of the isolates tested are inhibited (MIC₉₀) was 32 µg/ml,which is considered the breakpoint for resistance. The MIC rangefor cefuroxime was wide: from 6 to 48 µg/ml.

Of the three recommended antimicrobials for the treatment ofanthrax, ciprofloxacin and doxycycline exhibited potent activitiesagainst the B. anthracis isolates. Twenty-two of the isolateswere susceptible to penicillin, and 3 (GT 20, 68, and 85) wereclassified as resistant. None of the isolates examined showedany ß-lactamase activity. Ciprofloxacin, the drugof choice, had a MIC range of 0.032 to 0.38 µg/ml, witha MIC₉₀ of 0.094 µg/ml. This was the same as the MIC₅₀and was the most active agent tested. All of the isolates weresusceptible to doxycycline and the other antimicrobials: cephalexin,cefaclor, and tobramycin.

DISCUSSION

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Discussion

In this study, we have examined a spatially, temporally, andgenetically diverse group of B. anthracis isolates for susceptibilityto seven clinically relevant antimicrobials. This is the firstreport to use confirmed genetically diverse field isolates (5).Lightfoot et al. used 70 isolates, 33 of which were consideredby the authors to be unrelated (6). Doganay and Aydin used 22isolates collected from the same region in Turkey within a periodof 7 years (2). The most recent paper, by Mohammed et al., used50 historical and 15 recent isolates (7). The 50 historicalisolates, collected between 1937 and 1997, were selected asrepresentatives of temporally and spatially diverse strains.Together, these studies have utilized 182 isolates to test antimicrobialsusceptibilities. Unfortunately, the detailed isolate informationfrom previous studies could not be obtained, and, therefore,no correlations or comparisons can be made genetically, spatially,or temporally.

Cefuroxime is a broad-spectrum cephalosporin and is reportedto be effective against gram-positive bacteria. The older studiesshowed a high rate of resistance with cefuroxime, although theMIC₉₀s were lower in this study. The MIC₉₀ in the other studieswas 64 µg/ml, and for this study, it was 32 µg/ml.This could be an artifact of the methodologies or could reflecta true difference in the isolates; however, in any case, thereason for this resistance is unknown, and the mechanism ofresistance needs to be investigated further.

Penicillin and ciprofloxacin were also examined in all studies,and both agents showed good activity against the isolates ofB. anthracis. Differences in the MIC₉₀s between the reportshave been noted.

Penicillin resistance was reported in two of the other studies.Lightfoot et al. reported resistance (MIC > 0.25 µg/ml)in two isolates that originated from the same fatal case ofinfection, and Mohammed et al. reported three resistant isolates,including the same isolate used in the study by Lightfoot etal. (6, 7). Using the Staphylococcus breakpoints, our studydemonstrated three resistant isolates. These isolates were testedfor ß-lactamase production and found to be nonproducers,as were all of the isolates. Since the highest concentrationwas within a doubling dilution of 0.25 µg/ml, it is possiblethat the Staphylococcus breakpoints are not appropriate forcomparison in Bacillus species. Other explanations for thisobservation could be possible. Strains of B. licheniformis havebeen shown to produce large amounts of a ß-lactamaseand yet be sensitive to penicillin (9). In another study, astrain was shown to be highly susceptible to penicillin andstill actively produce a ß-lactamase (10). Penicillinresistance in Bacillus spp. is an area that needs more researchthan a cursory susceptibility panel every few years.

Ciprofloxacin, doxycycline, and the remaining antimicrobialagents, cefaclor, cephalexin, and tobramycin, showed that allisolates tested were highly susceptible. This was in agreementwith the findings of other studies that tested the correspondingantimicrobial agent.

Now that a reliable genotyping system is available, more genotypesshould be tested against these and other antimicrobials to verifythe trends noted here and in other studies. ß-Lactamaseproduction or nonproduction should not be considered an alternativeto testing penicillin resistance. Hopefully, this study willcontribute to the impetus for a more thorough study of ß-lactamaseproduction and Bacillus spp. Continued surveillance of B. anthracisfield isolates is recommended to monitor antimicrobial susceptibility.

ACKNOWLEDGMENTS

We express our gratitude to Eli Lilly for making this projectpossible.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathobiological Sciences, Louisiana State University School of Veterinary Medicine, Baton Rouge, LA 70803. Phone: (225) 578-9659. Fax: (225) 578-7117. E-mail: Banthracis{at}vetmed.lsu.edu.

REFERENCES

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