Bioassay of Antibiotics in Body Fluids from Patients Receiving Cancer Chemotherapeutic Agents

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1 ANTIMICROBIL AGENTS AND CHEMOTHERAY, Mar. 10, p /80/ /06$02.00/0 Vol. 17, No. 3 Bioassay of Antibiotics in Body Fluids from Patients Receiving Cancer Chemotherapeutic Agents DONALD N. WRIGHTt* AND JOHN M. MATSEN Departments of Pathology and Pediatrics, University of Utah College of Medicine, Salt Lake City, Utah Patients receiving antitumor chemotherapy are at increased risk of developing nosocomial infections, and the antibacterial therapy of such infections is often monitored by bioassay. The effect of antitumor agents on seven bioassay procedures using strains of Sarcina, Klebsiella, Clostridium, Pseudomonas, Staphylococcus aureus, and S. epidermidis or Bacillus was evaluated. The minimum inhibitory concentrations of six antitumor drugs, cytarabine, dactinomycin, doxorubicin, 5-fluorouracil, methotrexate, and vinblastine, determined for each of the test organisms, showed that 5-fluorouracil, dactinomycin, and doxorubicin are used at blood levels sufficient to interfere with bioassay procedures. The other drugs have miniimum inhibitory concentrations as much as times the expected blood levels. Antibiotic (gentamicin, kanamycin, cephalothin, and carbenicillin) recovery experiments in the presence of therapeutic levels of antitumor agents showed no in vitro inactivation of antibiotic. However, at low cephalothin concentrations (less than 20,ug/ml) in the presence of 5-fluorouracil, bioassay results were in error by as much as %. The data indicate that bioassay procedures for the determination of antibacterial drug levels may need to be modified for those patients receiving antitumor therapy with 5-fluorouracil, doxorubicin, or dactinomycin. Due to the increased risk and presence of infections, the management of patients receiving cancer chemotherapy frequently includes the use of antimicrobial agents. Bioassay of these antimicrobial agents is often used for monitoring serum drug levels to insure the adequacy of the desired antibacterial therapy or prevent possible toxicity. Such antibacterial drug bioassays depend on a linear, first-order reaction between the drug and the test reagent (bacteria). Therefore, drugs having an antibacterial effect when used in combination with other antimicrobial agents might interfere with or bias the results of such an assay. Reports (3, 5, 8) that antitumor agents may be antagonistic to the action of several antibiotics and that certain antineoplastic chemotherapeutic agents may have antibacterial activity.(1, 3) have led us to reexamine the validity of bioassay as a monitor of antibacterial therapy in the presence of these compounds. This study was undertaken to determine the effect of six commonly used antineoplastic agents on the bioassay of antimicrobial agents frequently used in the treatment of infections in cancer patients. MATERIALS AND METHODS Bacteria. Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538p), Staphylococcus epit Present address: Department of Microbiology, Brigham Young University, Provo, UT dermidis (ATCC 27626), and Klebsiella pneumoniae strain 1296 (ATCC 277) are routinely used for the assay of gentamicin and other aminoglycosides (4, 11). Sarcina lutea (ATCC 9341) was used as the assay agent for cephalothin, Pseudomonas putida (UUMC 25707) was used to assay kanamycin, and Clostridium perfringens (UUMC 9758), maintained during the duration of the study by daily transfer in chopped-meat glucose medium containing 5% sheep erythrocytes, was used to assay carbenicillin. The aerobic bacteria were maintained by daily transfer in 4 ml of Trypticase soy broth. Antimicrobial and antitumor agents. Antitumor and antimicrobial agents were chosen for this study based on the frequency of their use at the University of Utah Hospital. The antitumor drugs dactinomycin (Cosmegan; Merck Sharp & Dohme), 5- fluorouracil (Fluorouracil; Hoffmann-LaRoche, Inc.), vinblastine sulfate (Velban; Eli Lilly & Co.), cytarabine (Cytosar; The Upjohn Co.), doxorubicin HCl (Adriamycin; Adria Labs), and methotrexate (Lederle Laboratories) were provided by each manufacturer. These drugs were prepared in aqueous solutions at 1,000 4g/ ml and, except for vinblastine, were stored in the dark at room temperature. Vinblastine was stored at 4 C. Preparations of cytarabine and vinblastine were used within 10 days, and other drugs were used within 30 days of preparation. Appropriate dilutions of these stock drugs were prepared as needed. Solutions of cephalothin (Eli Lilly & Co.), carbenicillin (Roerig), kanamycin (Bristol Laboratories), and gentamicin (Schering Corp.) were prepared in appropriate 1 M phosphate buffers as 5,000-jLg/ml stock standards. These stock solutions were frozen at -20 C 417

2 4 WRIGHT AND MATSEN until they were diluted for assay. The antibiotics were received as reference standard powders from the manufacturers. Susceptibility testing. The minimum inhibitory concentration (MIC) of each antitumor agent for each of the seven assay organisms was determined by a standard procedure (6). MICs were determined by both macro- and micro-broth dilution techniques. Twofold dilutions were routinely used; however, when MICs were in a range approximating those of blood levels, dilutions of less than twofold were used to measure the MIC more accurately. Mueller-Hinton broth was used for the aerobic organisms and Schaedler broth was used for C. perfringens. Assays. Methods for the bioassay of gentamicin, cephalothin, and, carbenicillin were previously reported (4, 9, 11). Kanamycin was assayed by an agar surface streak method with P. putida as the test organism. A 25-ml amount of antibiotic medium no. 11 (Difco) in a petri dish (15 by 150 mm) was surface streaked by the Bauer-Kirby procedure (7) with a 1:10 dilution of P. putida broth culture standardized against a 0.5 McFarland reference. Antibiotic-impregnated disks for standard and test samples were then placed in duplicate on each of two agar plates which were incubated overnight, and the diameters of the zones of growth inhibition were determined. An average of the four values for each standard and test sample was used as the final determination result. This whole procedure was repeated four times to confirm the results. The concentrations of the drugs used in this study were chosen on the basis of reported blood levels and the MIC for the bacteria used in the study. The antimicrobial agents were assayed in concentrations felt to represent expected therapeutic levels as follows (1ig/ml): carbenicillin, 25; cephalothin, 10; gentamicin, 5 and 10; kanamycin, 12.5 and 20. Chemotherapeutic agents were assayed in anticipated therapeutic ranges as follows (ug/ml): cytarabine, 1 and 10; dactinomycin, 1 and 10; doxorubicin, 1 and 10; 5-fluorouracil, 20 and 30; methotrexate, 50; and vinblastine, 20. Each listed concentration of each chemotherapeutic compound was tested with eachi of the listed concentrations of the antimicrobial drugs. These agents were also tested separately in the assay system at the concentrations indicated. Dilutions of the drugs to be tested were made in single donor human serum which had been shown to have no activity against the assay organisms. Agents in combination were allowed to stand for 30 min at room temperature, before 20 pl was used to ANTIMICROB. AGENTS CHEMOTHER. impregnate a blank paper antibiotic susceptibility disk (S & S 740-E). The disks were placed on the appropriate agar surface, and the assays were completed as described (4, 9, 11). Two disks, each containing the same drug concentration, were placed on opposite sides of the agar surface. No more than 10 disks were placed in a petri dish at one time. Two 150-mm petri dishes were used for each assay series. RESULTS The antibacterial action of the chemotherapeutic agents used in this study was determined independently for each of the test organisms. The MICs of these agents are shown in Table 1 and indicate almost complete resistance of the assay organisms to methotrexate, cytabarine, and vinblastine. 5-Fluorouracil was almost uniformly antibacterial at easily achievable blood drug levels, whereas dactinomycin and doxorubicin showed mixed MICs. Both staphylococci and S. lutea, C. perfringens, and possibly B. subtilis were susceptible to dactinomycin and doxorubin at achievable blood levels, whereas K. pneumonia and P. putida were rarely susceptible. The question of whether the antineoplastic agents would react antagonistically or synergistically with antibiotics assayed with the above noted bacteria was examined by completing a bioassay experiment using the agents both in combination and individually. The results of these experiments are shown in Table 2. Results are given for only one concentration of each of the agents used; however, other concentrations were also tested without a significant change in the results. The data shown in Table 2 represent the median of from four to eight experiments, and the percent control shows the relative size of inhibition zones obtained with drug combinations as compared with those of the antimicrobial agents used alone. The data suggest that there is no apparent effect of the combinations of the agents (i.e., no additive, synergistic, or antagonistic effects), except with the combination of 5-fluorouracil and cephalothin. The fact that the percent control is often other than may reflect the additional dilution steps or ma- TABLE 1. Broth dilution MIC of antineoplastic agents for selected bacterial strains used in assay procedures Concn (,Ag/ml) Antineoplastic agent B. subtilis C. per- K. pneu- P. putida S. aureus S. epider- S. lutea firingens moniae midis Cytarabine >250 > >250 >250 >250 >250 >250 Dactinomycin Doxorubicin Fluorouracil Methotrexate >250 >500 >500 >500 >250 >250 >500 Vinblastine > >500 >500 >250 >250 >500

3 VOL. 17, 10 BIOASSAY OF ANTIBIOTICS IN BODY FLUIDS 419 TABLE 2. Effect on size of zones of inhibition produced by antimicrobial agents alone and in combination with antineoplastic agents by various bioassay procedures Assay system Antineoplastic agents (ANA)b and antimicrobial Cy b- Dactino- Doxoru- 5-fluor- Metho- Vinblasagentsa ytabra e mycin bicin ouracil trexate tine B. subtilis C. perfringens Carbenicillin alone Carb. + ANA K. pneumoniae P. putida Kanamycin alone Kana. + ANA S. aureus S. epidermidis S. lutea Cephalothin alone Ceph. + ANA.2a 17.8 b >40 >40 > a, average for four to eight experiments. The zone size of the combination/zone size for antimicrobial drug alone. b Values are in millimeters and are the median of four to eight experiments. nipulations necessary to complete the control tests. Variance from % was never greater than 9% for any single experiment, with the single exception just noted, and the majority of responses occurred within 2 to 3% of the control. The necessity of controlling each experiment is also apparent in Table 2, which shows variations among control experiments of up to 9%. Therefore, it appears that with two possible exceptions, the addition of these antitumor agents to antimicrobial drugs does not significantly change the expected bioassay results. When 5- fluorouracil was used in bioassay procedures including S. lutea, large zones of growth inhibition were obtained. These zones were so large as to make assay ofreasonable levels of cephalothin impossible. When dactinomycin was tested alone against B. subtilus, S. lutea, or the staphylococci, the zones of inhibition were large enough to suggest that this drug may have an effect on assay systems when the antibiotic concentration is low or the dactinomycin concentration is increased. Two questions were raised by these experi-

4 420 WRIGHT AND MATSEN ments. It would appear from the MICs that we might have expected more interferer.ce in the bioassay procedures, e.g., 5-fluorouracil interfering with gentamicin assayed on K. pneumoniae or S. aureus, than occurred when tested. Because at the concentrations of antitumor agent tested there was, in general, no inhibition of growth in the assay system, we questioned what drug levels would be necessary before we could reasonably expect to see some effect in these systems. Therefore, we decided to complete bioassays at several drug concentrations with antitumor drugs that had low MICs. Figure 1A-F shows the results obtained when various concentrations of the antineoplastic drugs were used in the assay system. In these experiments the agents were all used individually. The data in the composite Fig. 1A-D JDOXORUBICIN p ACTINOMYCIN I CARBENCILLIN 7 8 i9 0 Il 12 CEPHALOTHIN / J / t /4fLUOROURAOL B DACTINOMYCIN 'CEPHALOTHIN _-A D DACTINOMYCIN GENTAMICIN D) K E F A _ 6 E 30-9 C m 20- lo- 6- = 4-31 t ACTINOMYCIN DACTINOWCIN : 4-3 GENTAMICIN 21 i GENTAMICIN i7 9 li B ZONE OF INHIBITION(mm) FIG. 1. Bioassay of antitumor and antimicrobial drugs at various drug concentrations. (A) Dactinomycin, doxorubicin, and carbenicillin with C. perfringens; (B) dactinomycin and cephalothin with S. lutea; (C) 5-fluorouracil and cephalothin with S. lutea; (D) dactinomycin and gentamicin with B. subtilis; (E) dactinomycin and gentamicin with S. aureus; (F) dactinomycin and gentamicin with S. epidermidis. ANTIMICROB. AGENTS CHEMOTHER. indicate that the slope of the standard curve (zone size versus drug concentration) increased more rapidly with the antitumor drugs than with the other antimicrobial drugs. This suggests that, at concentrations of antibiotics less than those of the drug intersect points for the antitumor-antimicrobial drugs, the presence of selected antineoplastic agents may interfere with the determination of antimicrobial concentration by bioassay. Conversely with staphylococcal assay systems (Fig. 1E-F), the slope of the antimicrobial curve is greatest. Therefore, interference of the gentamicin assay by dactinomycin would be expected only at antineoplastic drug levels greater than those represented by the intersect points. Because the agents have, with a single exception, no in vitro synergistic or antagonistic effect, these results suggest that in general, for antimicrobial concentrations above the intersect point, the amount of antitumor drug in the circulation would need to be. very high to cause interference. For example, the data in Fig. 1A demonstrate that bioassay of carbenicillin concentrations less than 14,ug/ml cannot be accurately measured in sera containing dactinomycin concentrations of 14 Ag or more per ml. Further, as the carbenicillin concentratioi increases to values greater than 14,tg/ml, dactinomycin will not interfere with the assay unless there is a concomitantly greater increase in the dactinomycin concentration. Figure 1C shows that even low concentrations of 5-fluorouracil produce zone sizes so large that the assay of cephalothin in this system would be impractical at concentrations of less than 40,ug/ ml and may be difficult at any concentration. This type of experiment was also performed with 5-fluorouracil by the C. perfringens and Klebsiella assays, and with dactinomycin by the Staphylococcus, Bacillus, and Klebsiella assays. Instances where no zones of inhibition occurred when drug concentrations were as high as 80,ug/ml showed a lack of congruity between broth MICs, which suggested moderate susceptibility of Klebsiella to both 5-fluorouracil and dactinomycin as well as susceptibility of Clostridium and Staphylococcus to 5-fluorouracil, and which also suggested low levels of susceptibility by disk diffusion techniques. These results led us to determine the effect of the medium on the disk diffusion procedure in the Klebsiella and Sarcina assay systems (Table 3). These data suggest that ph has little or no effect on the expected activity of the antineoplastic agents tested, whereas media may have a marked effect resulting in a reduction of bacterial growth inhibition in the presence of some antineoplastic agents.

5 VOL. 17, 10 DISCUSSION The importance of monitoring antimicrobial levels in patients receiving certain antibiotics is well established (2). Such procedures are beneficial to patients by insuring adequate antibacterial therapy while limiting risks due to the accumulation of compounds to toxic levels. Among patients who frequently receive extensive, and therefore potentially toxic, antimicrobial therapy are those who derive their infections secondary to. neoplastic presence and antineoplastic therapy. Antineoplastic therapy often reduces host antimicrobial defenses to a point of considerable vulnerability to serious infections. Although recent reports have shown that some antibacterial benefits may be derived from the use of certain tumor chemotherapeutic agents (1, 3), no one, to our knowledge, has investigated the effect that these agents may have on bioassays of concomitantly administered antibiotics. The selection of antitumor agents used in this study was based on the extent of their use within our institution. The mechanisms of action of these drugs are representative of those of antitumor drugs in general with two notable excep- TABLE 3. Inhibition of bacteria ued for bioassay by antineoplastic agents at various ph levels Inhibition' at indicated ph on: Antineoplastic agents for Mueller-Hinton Antibiotic inhibition of: broth no. 11 medium ph 7.4 ph 7.9 ph 6.8 ph 7.8 Klebsiella Dactinomycin NI SI SI SI 5-Fluorouracil I I NI NI Sarcina Dactinomycin NI SI NI NI 5-Fluorouracil I I NI NI a I, Inhibition; NI, no inhibition; SI, slight inhibition. Drug concentrations were between 20 and,ug/ml. TABLE 4. BIOASSAY OF ANTIBIOTICS IN BODY FLUIDS 421 tions. We did not include alkylating agents such as cyclophosphamide because of the low expectancy of use or steroid compounds because of the difficulty in discovering therapeutic blood levels. However, both antimetabolites, e.g., methotrexate, and antibiotics, e.g., dactinomycin, and some miscellaneous drugs, e.g., vinblastine, were studied Ȧlthough there are a variety of bioassay procedures for measuring concentrations of various antimicrobial agents, the procedures selected for this study are not only commonly used but they are representative procedures for the assay of the majority of the commonly used antimicrobial agents (Table 4). The therapeutic antimicrobial agents actually used in this study are representative of several classes of antimicrobial compounds that might be assayed by the procedures employed. We selected those antimicrobial drugs which are most commonly used and therefore seemed to have the greatest practicality associated with the study. The data reported here suggest that some caution should be exercised when performing antimicrobial bioassays on sera of patients receiving antitumor therapy. This is particularly true for patients receiving 5-fluorouracil as it has a fairly low MIC for several of the assay organisms and also reaches blood levels as high as 40,ug/ml (10). It is also possible that under some circumstances both dactinomycin and doxorubicin could be present in the blood in concentrations sufficient to interfere with assay of some antibiotics (Fig. 1). This is particularly true with respect to the assay of carbenicillin or other drugs requiring use of staphylococci, C. perfringens, or S. lutea in the assay system. The data in Table 2 suggest that for patients receiving dactinomycin, the Klebsiella assay system would likely be most useful. The level at which these antitumor drugs are expected to appear in patient sera is not entirely clear. However, in general, it is expected that Antibiotics commonly assayed by bioassay procedures employing specific bacterial species Antibiotics assayed using: B. subtilis C. per- K. pneu- P. put epider- S. Iutea fringens moniae midis Gentamicin Ampicillin Amikacin Kanamycin Gentamicin Amikacin Amoxicilhin Carbenicillin Genta- Gentamicin Cephalosporins Chloramphen- micin Tobramycin Cloxacillin icol Dicloxacillin Clindamycin Erythromycin Penicllin Nafcillin Rifampin Tobra- Vancomycin mycin

6 422 WRIGHT AND MATSEN appropriate therapeutic levels would be low enough that little interference with antibacterial assay should be expected. Certainly with dactinomycin, doxorubicin, vinblastine, and cytarabine, concentrations higher than 10,ug/ml would be unusual; yet much higher levels of methotrexate and 5-fluorouracil might be expected. The relatively low MIC of doxorubicin for S. leutea and C. perfringens was not carried over into the assay systems, and, except for unusual circumstances, this drug should not interfere with bioassay using these bacteria. The fact that Klebsiella was inhibited by 5- fluorouracil when assayed on Mueller-Hinton but not on antibiotic no. 11 agar (Table 3) suggests that the use of the recommended assay media seems most appropriate. There appears to be either an inactivation of the drug by the components of some media or a reversal of the antimetabolite effect due to the nutrient content of the medium involved. Additional study suggested that ph inactivation of the drug was not a factor on growth over the ph range studied. Vinblastine, methotrexate, and cytarabine were found to have very high MICs and did not interfere with the various disk bioassay systems. In light of the report by Jacobs et al. (3) that synergy and antagonism between antitumor and antibacterial drugs were observed, it was somewhat surprising that neither activity was observed in our study for any drug combination at any concentration tested. However, the drug concentrations used in their study were as much as 10 times those used by us. We feel that the value of further study of possible interactions at much higher drug levels is not indicated because of the low blood level concentrations of antitumor agents obtained in normal use. Further study of possible chemotherapeutic antibiotic interaction seems to be suggested either as new agents are developed for therapy ANTIMICROB. AGENTS CHEMOTHER. or as alternative assay systems are developed and applied in the clinical laboratory. LITERATURE CITED 1. Goldschmidt, M. C., and G. P. Bodey Effect of chemotherapeutic agents upon microorganisms isolated from cancer patients. Antimicrob. Agents Chemother. 1: Hoeprich, P. D Antimicrobics and antihelmintics for systemic therapy, p In P. D. Hoeprich (ed.), Infectious diseases, 2nd ed. Harper and Row, Hagerstown, Md. 3. Jacobs, J. Y., J. Michel, and T. Sacks Bactericidal effect of combinations of antimicrobial drugs and antineoplastic antibiotics against Staphylococcus aureus. Antimicrob. Agents Chemother. 15: Lund, M. E., D. J. Blazevic, and J. M. Matsen Rapid gentamicin bioassay using a multiple-antibioticresistant strain of Kkbsiella pneumoniae. Antimicrob. Agents Chemother. 4: Manten, A., and J. I. Terra Some observations of antagonism between penicillin and antineoplastic antibiotics. Acta Physiol. Pharmacol. Neerl. 14: Matswn, J. M Antimicrobial susceptibility tests, p In J. B. Henry (ed.), Clinical diagnosis and management of laboratory methods. W. B. Saunders Co., Philadelphia. 7. Matsen, J. M., and A. L. Barry Susceptibility testing: diffusion test procedures, p In E. H. Lennette (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 8. Moody, M. R., M. J. Morris, V. M. Young, L A. Moye, S. C. Schimpff, and P. H. Wiernick Effect of two cancer chemotherapeutic agents on the antibacterial activity of three antimicrobial agents. Antimicrob. Agents Chemother. 14: Sabath, L. D., and I. Toftegaard Rapid microassays for clindamycin and gentamicin when present together and the effect of ph and of each on the antibacterial activity of the other. Antimicrob. Agents Chemother. 6: Sadee, W., and C. G. Wong Pharmacokinetics of 5-fluorouracil: interrelationship with biochemical kinetics in monitoring therapy. Clin. Pharmacokinetics 2: Washington, J. A. II, E. Warren, C. T. Dolan, and A. G. Karlson Tests to determine the activity of antimicrobial agents, p In J. A. Washington II (ed.), Laboratory procedures in clinical microbiology. Little, Brown & Co., Boston.