marinum versus the tetracyclines have failed to show in vitro susceptibility despite good in vivo .... The MICs of the antibiotics for the E. coli and S. aureus isolates ...
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Vol. 20, No. 5
Susceptibility of Mycobacterium marinum to Tetracyclines and Aminoglycosides RICHARD J. WALLACE, JR.,' 2* AND KAREN WISS' 2 Departments of Medicine' and Microbiology and Immunology,2 Baylor College of Medicine, Houston, Texas 77030 Received 26 May 1981/Accepted 10 August 1981
Current agar dilution methods for susceptibility testing of Mycobacterium marinum versus the tetracyclines have failed to show in vitro susceptibility despite good in vivo results. We found that the tetracyclines were unstable in agar and resulted in a fine haze of growth for several concentrations before complete inhibition of growth was seen. In contrast, the aminoglycosides resulted in sharp minimal inhibitory concentration endpoints, which were generally independent of the length of incubation. The problems with the tetracyclines can be lessened by shortening the incubation time to 5 days and redefining the minimal inhibitory concentration as the lowest concentration resulting in no growth or a slight haze of growth. By this methodology, 19 clinical isolates of M. marinum were tested for susceptibility to the tetracyclines and aminoglycosides. Minocycline inhibited >50% of isolates at 2.0 ,ug/ml, whereas both minocycline and doxycycline inhibited >90% of isolates at 4.0 pg/ml. These studies support the usage of the tetracyclines in the treatment of clinical diseases due to M. marinum and suggest a susceptibility method which is more predictive of clinical results.
Mycobacterium marinum is the etiological ferent susceptibility methods have usually been agent of mycobacterial diseases in both fresh- applied when these agents were tested against water and salt water fish. Infection with this bacterial isolates (3), we studied the susceptibilorganism in humans results in a cutaneous dis- ity of M. marinum to the tetracyclines and amiease often referred to as "fish tank" or "swim- noglycosides, using bacterial, rather than mycoming pool" granuloma. Although this infection bacterial, standards of inoculum size and MIC is usually self-limited, it may persist for months endpoints. to years if untreated, and the involvement of MATERIALS AND METHODS tendon sheaths and synovial membranes can lead to rapid and severe tissue destruction in the Nineteen clinical isolates of M. marinum were obabsence of therapy. tained from the Houston City Health Department, Because of the resistance of M. marinum to Houston, Tex. (Mario Saccomani); the Texas State isoniazid and the propensity to use antituber- Department of Health, Austin, Tex. (Joseph Steadculous agents in combination to prevent the ham); and the Mycobacterial Reference Section of the for Disease Control, Atlanta, Ga. (Vella Sildevelopment of resistance, therapy with rif- Centers The isolates had been identified as to species by ampin and ethambutol has most often been rec- cox). using Centers for Disease Control standards of idenommended for treatment of infections due to tification. Nine of the isolates were recent (within the this organism (10). Antibacterial agents such as preceding 3 months). Subsequent to primary isolation, trimethoprim/sulfamethoxazole and the tetra- all appeared to grow as well at 35 as at 30°C. cyclines have also been used successfully (1, 2, 5, For agar dilution susceptibility testing, stock solu6), and among dermatologists who most often tions of amikacin, kanamycin, gentamicin, tobramycin, treat this disease, the tetracyclines, especially streptomycin, doxycycline, minocycline, and tetracyminocycline, have become the treatment of cline hydrochloride were prepared from diagnostic standards. Twofold dilutions of the antibiotics were choice. added to commercial Mueller-Hinton agar suppleDespite the clinical success of the tetracy- mented with 10% Middlebrook OADC (oleic acid, alof M. marin studies vitro susceptibility clines, and catalase). No attempt to control bumin, dextrose, inum with the proportion method reported by the Ca2" or Mg2" content of the agar was made. Sanders and Wolinsky (7) and Torres et al. (8) The organisms were grown in Middlebrook 7H9 have shown minimal inhibitory concentrations broth for 3 to 5 days and then diluted to match one(MICs) that exceed the therapeutic levels that half the no. 1 McFarland standard (108 colony-forming can be achieved with these drugs. Because dif- units per ml). Organisms were further diluted 1:10 and 610
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then inoculated with a Steers replicator for an average (4.0,ug/ml) with a very narrow range of MICs. plate inoculum of 103 to 104 colony-forming units. The isolates were all resistant to gentamicin and Escherichia coli ATCC 25922 or Staphylococcus au- tobramycin, with MICs of 16.0 ,tg/ml or greater. reus ATCC 25923 was inoculated as a control. Plates were incubated at 35°C in a moisturized 5% CO2 in- Streptomycin was somewhat intermediate in accubator. To determine whether the susceptibilities or tivity, with 16 of 18 isolates having MICs begrowth rates were temperature dependent, duplicate tween 8.0 and 16.0 ,ug/ml. Unlike the tetracysets of antibiotic plates for the tetracyclines were made clines, continued incubation of the aminoglycoand inoculated with 10 of the organisms. One set of sides for 14 days resulted in no change or only a plates was then incubated at 35°C, and the second set twofold change in the MICs. was incubated at 30°C in a room air incubator. Figure 1 shows the decline in doxycycline The MICs of the antibiotics for the E. coli and S. activity in agar at 350C. Both the high and low aureus isolates were determined at 24 h, whereas those concentrations of the drug exhibited a similar for the M. marinum isolates were determined at 5 rapid decline in activity, with a loss of more than days and, for most isolates, again at 10 or 14 days. The MIC was defined as the lowest concentration of anti- 50% activity by 48 h and more than 80% by 5 biotic resulting in complete inhibition of visible growth days. Tetracycline hydrochloride and minocyor a fine haze of growth, standards which are in cline exhibited an almost identical decline in accordance with the recommendations of the Inter- activity. national collaborative study on antimicrobial susceptibility testing (3). TABLE 1. Susceptibility of isolates of M. marinum The stability of tetracycline hydrochloride, minoto antimicrobial agents cycline, and doxycycline in agar was assayed by using Concn (,ug/ml) which Mueller-Hinton agar plates containing 2 and 16 ,ig of MICa inhibited 50 and 90% each of the drugs per ml. The plates were incubated No. of of isolates Drug strains range at 35°C. At various time intervals, measured cores of (jg/mi) agar were removed from the same plate and were MICs MIC50 added to the surface of a Mueller-Hinton agar plate 1-8 4 19 4 seeded with spores of Bacillus subtilis. The plates Doxycycline 2 19 1-4 4 containing known amounts of each tetracycline were Minocycline 19 1-16 4 16 made up fresh each time the test agar was to be Tetracycline 2 16 1-4 4 assayed, and cores of agar from these plates were used Amikacin 2 16 2-4 4 Kanamycin as reference standards. Zone diameters around the Gentamicin 16 16-32 32 32 cores of agar were measured after 18 h of incubation Tobramycin 16 16-64 64 32 and then plotted by using a semilogarithmic scale. 18 8-32 16 16 Streptomycin MIC was defined as the lowest concentration of antibiotic RESULTS resulting in complete inhibition of growth or a fine haze of The MICs for the E. coli and S. aureus stand- growth after 5 days of incubation at 35°C. ards were always within two dilutions of the published MICs for broth (4), and most were 20 identical. The MICs of the tetracyclines determined at 24 h rose an average of twofold for E. 10 _0 coli and eightfold for S. aureus when these 0 isolates were incubated for 5 days. E The MICs of the three tetracyclines were sim0 3. 0 ilar, although tetracycline hydrochloride appeared to be the least active (Table 1). Minocy2. oW 0 cine inhibited >50% of strains at 2.0 ,ig/ml, and 0 both minocycline and doxycycline inhibited C 1.0 F 0 0 >90% of strains at 4.0 ,ug/ml. (Included in these strains were six isolates of M. marinum from ._1c~ patients successfully treated with minocycline 0 or doxycycline.) Incubation of these isolates for 2 weeks (the standard incubation time for mycobacterial cultures tested by the proportion I method) resulted in an average fourfold increase 9 , 0.1 1 2 3 7 5 14 in MICs for all of the tetracyclines. Incubation of 10 isolates of M. marinum at 30°C rather than Time of Incubation (Days) 350C produced no differences in growth rates or FIG. 1. Loss of activity of two concentrations (16 in the MICs. and 2 pg/ml) of doxycycline in agar at 35°C. Almost Amikacin and kanamycin exhibited almost identical results were observed with minocycline and identical activity, inhibiting 100% of isolates at tetracycline hydrochloride. a
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DISCUSSION In this study, the MICs of the tetracyclines (especially minocycline and doxycycline) to isolates of M. marinum were within achievable serum levels for almost all isolates and were much lower than those previously reported with different methodologies (7, 8). The instability of tetracyclines in agar, which may allow organisms to regrow as the bacteriostatic (or mycobacteriostatic) tetracyclines break down, and the fine haze of growth frequently present for several drug concentrations before complete inhibition of growth occurred are probably responsible for these differences. (A fine haze of growth is ignored when reading MICs by antibacterical standards, but is considered a significant growth by the proportion method.) Clinical success with doxycycline and minocycline in the treatment of infections due to M. marinum has been almost uniform to date, and six of the current isolates came from patients successfully treated with these agents. In vitro susceptibility tests should reflect in vivo results, suggesting that modifications of the current methods such as those suggested here should be used for future determinations of MICs of the tetracyclines for isolates of M. marinum. All of these isolates of M. marinum were highly susceptible to amikacin and kanamycin, but resistant to gentamicin and tobramycin. Similar susceptibilities to gentamicin, kanamycin, and amikacin for 16 strains of M. marinum were reported by Sanders and Wolinsky (7), who used the proportion method and 14 days of incubation. They reported that all of their isolates were inhibited by 5 ,tg of amikacin per ml, and all but one isolate were inhibited by 5 ,ug of kanamycin per ml, whereas all isolates were resistant to the same concentration of gentamicin. These comparable results, despite different methods, probably reflect the stability of the aminoglycosides (and hence little deterioration with a longer incubation time) and the sharp MIC endpoints usually seen with these agents. Previous studies with the rapidly growing mycobacteria (M. fortuitum and M. chelonei) have also shown that amikacin and kanamycin are the most active of the aminoglycosides (9).
Isolates of M. marinum generally grow better at 28 to 30°C than at 35°C, especially on primary isolation. All of these clinical isolates, including the recent ones, grew as well at 35 as at 30°C. Similar results in MIC at these two temperatures suggest that isolates with a low thermal range could be tested at the lower temperature, although the bacterial control strains used in this study grew too poorly at this temperature to be used reliably. The narrow range of MICs observed for most of these agents tested in this study suggest that routine susceptibility testing of M. marinum to the antibacterial agents may not be necessary. ACKNOWLEDGMENT Research support was provided by the Alma Wright Fund for Mycobacterial Research. LITERATURE CITED 1. Black, M. M., and S. J. Kykyn. 1977. The successful treatment of tropical fish tank granuloma (Mycobacterium marinum) with co-trimoxazole. Br. J. Dermatol. 97:689-692.
2. Contorer, P., and R. N. Jones. 1979. Minocycline therapy of aquarium granuloma. Cutis 23:864-868. 3. Ericsson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B Suppl. 217. 4. Fass, R. J., and J. Barnishan. 1979. Minimal inhibitory concentrations of 34 antimicrobial agents for control strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Antimicrob. Agents Chemother. 16:622-624. 5. Kirk, J., and G. W. Kaminski. 1976. Mycobacterium marinum infection. Aust. J. Dermatol. 17:111-116. 6. Loria, P. R. 1976. Minocycline hydrochloride treatment for atypical acid-fast infection. Arch. Dermatol. 112: 517-519. 7. Sanders, W. J., and E. Wolinsky. 1980. In vitro susceptibility of Mycobacterium marinum to eight antimicrobial agents. Antimicrob. Agents Chemother. 18:529531. 8. Torres, J. R., M. Sands, and C. V. Sanders. 1978. In vitro sensitivity of Mycobacterium marinum to minocycline and doxycycline. Tubercle 59:193-195. 9. Wallace, R. J., Jr., J. R. Dalovisio, and G. A. Pankey. 1979. Disk diffusion testing of susceptibility of Mycobacterium fortuitum and Mycobacterium chelonei to antibacterial agents. Antimicrob. Agents Chemother. 16:611-614. 10. Wolinsky, E., F. Gomez, and F. Zimpfer. 1972. Sporotrichoid Mycobacterium marinum infection treated with rifampin-ethambutol. Am. Rev. Respir. Dis. 105: 964-967.
