Is In Vitro Antibiotic Combination More Effective than Single-Drug Therapy against Anthrax?

Antibiotic combinations are used to enhance antibacterial efficacyand to prevent the development of resistance. We have testeda possible synergistic effect of several antibacterial combinationson Bacillus anthracis. The in vitro activities of antibioticcombinations against two strains of B. anthracis, strain Sterneand the Russian anthrax vaccine strain STi, were tested by thefractional inhibitory concentration (FIC) method, derived fromthe MICs of the agents in combination, and by measuring therate of bacterial killing over time by several antibiotic combinations.The FIC results showed that synergism against both B. anthracisstrains was observed only with the combination of rifampin andclindamycin. The telithromycin-amoxicillin combination showedsynergism against strain Sterne only. All other combinationswere either indifferent or antagonistic. The results of thebacterial time-kill study demonstrated indifferent effects forall combinations. These in vitro results demonstrate the difficultiesin obtaining synergistic combinations of antibiotics againstB. anthracis.

INTRODUCTION

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Introduction

Anthrax has recently been the focus of attention as a potentialbiological warfare agent. The therapy and prophylaxis of anthraxrequire vaccination combined with prolonged antibacterial therapy.It is important to examine if antibiotic combinations mightshorten the 60-day period recommended for postexposure prophylaxisby providing a more radical and rapid killing of the vegetativeanthrax forms and thus reducing the infective load (5). Of equalinterest is the question whether combinations might reduce therisk for the development of resistance by Bacillus anthracis.During the last bioterror attack in the United States, in 2001,many individuals received antibiotic combinations as therapyand as postexposure prophylaxis (6). Clindamycin was often usedbased on the assumption that it might decrease the productionof bacterial toxins and thus diminish the severity of diseasemanifestations (3).

The aim of the present study was to investigate possible interactionsamong several antibiotics potentially effective against B. anthracis.

MATERIALS AND METHODS

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Materials and Methods

Antibacterial agents. The antibiotics tested in this study were as follows: ciprofloxacinand moxifloxacin (Bayer, Leverkusen, Germany), tetracycline(Sigma, Rehovot, Israel), penicillin G (Rafa Laboratories, Jerusalem,Israel), amoxicillin (GSK, Petach Tiqva, Israel), vancomycin(Eli Lilly, Sesto Fiorentino, Italy), clarithromycin (Abbott,Promedico, Petch-Tiqva, Israel), telithromycin and quinupristin-dalfopristin(Q-D) (Aventis, Paris, France), clindamycin and linezolid [Pharmacia(Agis), Bnei Braq, Israel, and Pharmacia, Kalamazoo, Mich.],and rifampin (Sigma).

Penicillin G, vancomycin, rifampin, clindamycin, linezolid,and Q-D were received as dry laboratory powders and were dissolvedin phosphate-buffered saline (pH 7.2). Amoxicillin was dissolvedin distilled water. Clarithromycin was dissolved in analytical-gradeacetone, while telithromycin and tetracycline were dissolvedinitially in 2 drops of acetic acid and ethanol (100%), respectively,and all three of these antibiotics were subsequently dilutedin distilled water to the required concentration; at the finalconcentrations used, these solvents had no demonstrable antibacterialactivity. Prepared solutions were frozen in small aliquots andthawed before use. Antibiotic solutions were sterilized through0.45-µm-pore-size filters (Millipore S.A., Paris, France).Moxifloxacin and ciprofloxacin were obtained as injectable solutions.

Bacterial strains and growth conditions. The bacteria used in this study were two strains of B. anthracis:strain Sterne (a gift from the Colorado Serum Institute, Denver,Colo.) and the Russian strain STi, purchased in Moscow, Russia.Neither strain is a human pathogen, because both lack the plasmidnecessary to produce the capsule of the vegetative form, whichis necessary for human pathogenicity. Bacterial spores werestored in sterile 30% glycerol in phosphate-buffered salineand were spread on brain heart infusion (BHI) agar (Difco Laboratories,Detroit, Mich.) and incubated overnight at 37°C to obtainsingle colonies (vegetative form). A single colony was inoculatedinto 10 ml of BHI broth and incubated overnight at 37°C(use of four to five colonies instead of a single colony didnot reveal a clonality effect).

Determination of MICs. MICs were determined by the microdilution technique accordingto the NCCLS criteria for B. anthracis (7, 8).

The antibacterial agents to be tested were thawed and dilutedin distilled water. Twofold dilutions in 100 µl of BHIbroth were used in a concentration range from 0.015 to 1,024mg/liter, and solutions were poured into the wells of 96-wellflat-bottom microtiter plates (Nunc, Roskilde, Denmark). A 10-µlvolume of bacterial culture, which contains 10⁵ CFU, was thenadded. Following incubation of the plates for 18 h at 37°Cin ambient air, the MICs were determined. Staphylococcus aureusATCC 2119 was used as a control for antibiotic activity. MICswere recorded as the lowest concentrations that completely inhibitedvisible growth of the bacteria (8).

The effects of antibiotic combinations were determined by twomethods: the fractional inhibitory concentration (FIC) and thetime-kill method (2, 4, 11).

The time-kill method was performed as follows. An overnightculture of B. anthracis was diluted 1:1,000 with BHI broth ata final volume of 2 ml (10⁵ CFU/ml). Antibiotics at the concentrationsshown in Table 3, in a volume of 12.5 to 25 µl, were addedto the bacterial suspension. As a control, similarly dilutedbacterial inocula without antibiotics were used. Bacterial suspensionswere incubated at 37°C, and samples (10 µl) were removedat 0, 0.5, 2, 4, 6, 10, 12, and 24 h for viable bacterial counts.Bacterial counts were performed by diluting samples in sterilesaline and plating 20 to 50 µl on BHI agar. To improvedetection in samples with low bacterial counts, 100 µlof an undiluted specimen was also plated. In addition, the sporecontent at each time point and that of the inoculum were measuredby the heat shock method (65°C for 30 min.). After overnightincubation at 37°C, the number of CFU (both vegetative formsand spores) were counted. All tests were performed in triplicate,and results were averaged. Data were plotted as average log₁₀CFU per milliliter against time.

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TABLE 3. Effects of combinations of antibiotics on rates of killing of B. anthracis strains STi and Sterne

The combinations of antibiotics tested for FIC determinationare presented in Table 2. The concentration range of each antibioticin combination ranged from 1/32 the MIC to four times the MIC(4xMIC). The FIC index was calculated as [(MIC of drug A incombination)/(MIC of drug A alone)] + [(MIC of drug B in combination)/(MICof drug B alone)]. The following definitions were used: synergism,FIC index of $≤$ 0.5; indifference, FIC index of >0.5 and $≤$ 4;antagonism, FIC index of >4.

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TABLE 2. FICs of combination of antibiotics against B. anthracis strains

The time-kill method determined the rate of killing of bacteriaby each antibiotic at the concentrations of 1xMIC and 5xMIC,as well as the effects of the combination on killing at thefollowing concentrations: both drug A and drug B at 1xMIC, Aat 1xMIC and B at 5xMIC, A at 5xMIC and B at 1xMIC, and bothA and B at 5xMIC. For calculation of the killing activity, thearea under the curve of bacterial concentration over time (AUC)was calculated for each agent alone and for each combination,and the results were converted to a log₁₀ scale. If the AUCof the combination was 2 or more log units smaller than theAUC of each of the antibiotic constituents, the combinationwas considered synergistic; if the AUC of the combination wasbetween 1 log unit or less smaller and 2 log units or less greaterthan the AUC of each constituent, the combination was consideredindifferent; all other differences between the AUCs were consideredto indicate antagonism.

RESULTS

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Results

MIC. The MICs of the antibacterial agents tested against the twostrains of B. anthracis, as reported previously (2), are shownin Table 1.

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TABLE 1. MICs of antibiotics against B. anthracis strains STi and Sterne

Table 2 shows the FIC indices of the antibiotic combinations.Ciprofloxacin-tetracycline, ciprofloxacin-penicillin, ciprofloxacin-clarithromycin,ciprofloxacin-clindamycin, ciprofloxacin-Q-D, ciprofloxacin-rifampin,ciprofloxacin-linezolid, tetracycline-clindamycin, tetracycline-rifampin,tetracycline-moxifloxacin, tetracycline-telithromycin, rifampin-Q-D,rifampin-linezolid, rifampin-clarithromycin, rifampin-telithromycin,telithromycin-moxifloxacin, linezolid-clarithromycin, and penicillin-tetracyclineshowed indifferent effects against both B. anthracis strains.

The tetracycline-linezolid, penicillin-rifampin, penicillin-clindamycin,penicillin-vancomycin, and penicillin-telithromycin combinationsshowed indifferent activities against strain Sterne and antagonisticeffects against strain STi.

Antagonistic effects against both strains were demonstratedfor the linezolid-clindamycin, tetracycline-Q-D, penicillin-Q-D,and penicillin-linezolid combinations. Synergistic activitieswere found for rifampin-clindamycin against both strains andfor telithromycin-amoxicillin against strain Sterne only.

The time-kill method revealed indifferent effects for all combinationsat all concentrations used (Table 3).

DISCUSSION

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Discussion

Clear synergism was observed only for the combination of rifampinand clindamycin by the FIC method; indifference was observedfor all combinations by the time-kill method. Thus, the inclusionof clindamycin, while possibly reducing B. anthracis toxin production(9), does not contribute to more-rapid killing of the organismwhen it is used with conventional antianthrax agents such aspenicillin, ciprofloxacin, or doxycycline. Resolution of thesecontradictory findings for clindamycin, with implications forits contributions to recommended therapeutic regimens, awaitsanimal studies using the toxin-producing B. anthracis strains.

There are no well-established methods for assessing the effectsof antibiotic combinations on killing. Furthermore, no systematiccomparison between the assay methods is available. Therefore,our results cannot be compared to a "gold standard."

A shortcoming of this study is that we did not investigate whetherthe B. anthracis strains developed resistance during exposureto the combinations studied. Nevertheless, previous observationswith these and other strains suggest that derepression of ß-lactamasesof the two types, penicillinase and cephalosporinase, may occurafter a short exposure to these antibiotic classes (10). Inour previous studies, resistance to other commonly used agents(fluoroquinolones, tetracyclines, clindamycin, etc.) occurredonly at passage $≥$ 9 and required prolonged exposure to the antibiotic(1). An additional shortcoming of the present study is thatwe have not measured the effects of higher antibiotic concentrationswhich may be clinically achieved in serum following conventionaldoses of, e.g., ciprofloxacin and rifampin. It is possible thatdifferent effects on B. anthracis may be observed at such highconcentrations.

Postexposure prophylaxis and therapy of anthrax are prolonged( $≥$ 60 days of therapy) because of surviving spores within themacrophages that can germinate and turn into the toxin-producingvegetative forms. In addition, all the currently recommendedagents are inactive against spores (2). Our experience suggeststhat combinations of the antibiotics currently recommended arenot likely to shorten the treatment period, due to the lackof synergistic effect. A synergistic combination might havecaused rapid and radical bacterial killing, so that the organismgave rise to a smaller number of spores. The only exceptionwas the combination of rifampin and clindamycin, which showedsynergism by the FIC method. Additional studies on differentB. anthracis strains need to be performed in order to substantiatethese preliminary observations.

FOOTNOTES

* Corresponding author. Mailing address: Infectious Diseases Unit, Sheba Medical Center, Tel Aviv University School of Medicine, Tel Hashomer 52621, Israel. Phone: 972 3 5345 389. Fax: 972 3 5303501. E-mail: erubins{at}yahoo.com.

REFERENCES

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