beta-lactam antibiotics. However, for a mutant of M. fortuitum (-y 27) with virtually nonexistent P-lactamase production, the antibiotics still had relatively high MICs ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1991,
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Vol. 35, No. 9
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0066-4804/91/091760-05$02.00/0 Copyright © 1991, American Society for Microbiology
1-Lactamase of Mycobacterium fortuitum: Kinetics of Production and Relationship with Resistance to 1-Lactam Antibiotics
LANFRANCO FATTORINI,1* GIOVANNA SCARDACI,l SHAO HONG JIN,1 GIANFRANCO AMICOSANTE,2 NICOLA FRANCESCHINI,2 ARDUINO ORATORE,2 AND GRAZIELLA OREFICI' Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanitai, Viale Regina Elena 299, 00161 Rome,' and Department of Sciences and Biomedical Technology and of Biometrics, Institute of Biological Chemistry and Molecular Biology, University of L'Aquila, L'Aquila,2 Italy Received 28 January 1991/Accepted 18 June 1991 The kinetics of both intracellular and extracellular l-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacteriumfortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in ,-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of i(-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. However, for a mutant of M. fortuitum (-y 27) with virtually nonexistent P-lactamase production, the antibiotics still had relatively high MICs (for instance, benzylpenicillin and cephaloridine had MICs of 64 and 32 ,ug/ml, respectively). This suggests that, aside from P-lactamase production, other mechanisms such as cell wall permeability and/or affinity for penicillin-binding proteins could coexist in M. fortuitum and explain its natural resistance to beta-lactam antibiotics.
MATERIALS AND METHODS
Mycobacterium fortuitum has been recognized as a cause of sporadic cutaneous abscesses, pulmonary diseases, and postoperative infections (20, 21). Antibiotic therapy of these infections is often difficult because clinical isolates of M. fortuitum are usually resistant in vitro and in vivo to antimicrobial agents, including the antituberculous drugs. Amikacin, cefoxitin, and sulfonamides have partial clinical efficacy (2, 19), whereas other drugs with promising in vitro activity such as imipenem, cefmetazole, ciprofloxacin, and combinations of amoxicillin with clavulanic acid warrant further clinical investigation (3, 4, 8, 9). As in other bacteria, the high resistance of M. fortuitum to most beta-lactam antibiotics may result from different, possibly interacting mechanisms such as poor cell penetration, drug inactivation by ,B-lactamases, or low affinity for penicillin-binding proteins. For most mycobacterial species, 13-lactamases have been described as noninducible, intracellular enzymes with a broad spectrum of activity (6, 12, 13). Purified P-lactamase of M. fortuitum hydrolyzes both penicillins and cephalosporins (1). Furthermore, this enzyme was inhibited by clavulanic acid and inactivated by isoxazolylpenicillins (10). The role of ,B-lactamase in the antibiotic resistance of M. fortuitum has been little explored. In this study, we used a series of mutants of M. fortuitum selected by a multistep mutagenesis procedure, which showed a modulated production of P-lactamase. The kinetics of enzyme production and the correlation with antibiotic susceptibility allowed us some insight into the possible mechanisms of M. fortuitum resistance to beta-lactam antibiotics.
*
Antibiotics and chemicals. Clavulanic acid was kindly supplied by Beecham Pharmaceutical, Brentford, United Kingdom; sulbactam was a gift of Pfizer Italiana, Rome, Italy; the ,-lactamase substrate 7-(thienyl-2-acetamid)-3[2-(4-N,N-dimethylaminophenylazo)pyridinium methyl]-3cephem-4-carboxylic acid (PADAC) was purchased from Calbiochem, La Jolla, Calif.; cephaloridine, cefoxitin, ampicillin, amoxicillin, and nitrosoguanidine were obtained from Sigma Chemical, St. Louis, Mo.; and imipenem was obtained from Merck Sharp & Dohme, Rome, Italy. All other chemicals used were the purest commercially available. Organism and growth conditions. M. fortuitum ATCC 19542, was grown for 3 days at 37°C under agitation (180 rpm) in the following liquid media: (i) Sauton (K2HPO4, 0.5 g/liter; MgSO4- 7H20, 0.5 g/liter; L-asparagine, 4 g/liter; citric acid, 2 g/liter; ferric ammonium citrate, 0.05 g/liter; glycerol, 40 ml/liter [final pH, 6.8]); (ii) glucose-yeast extract medium (GYM; yeast extract [Oxoid, Basingstoke, England], 10 g/fiter; glucose, 10 g/liter); and (iii) Mueller-Hinton broth (MHB; Oxoid). A bacterial suspension adjusted to a McFarland no. 1 turbidity standard was used to inoculate the media (1 ml of this inoculum for 100 ml of each medium). After centrifugation at 14,000 x g for 30 min at 4°C, the supernatants were collected and the pellets were washed and resuspended in cold 0.05 M sodium phosphate buffer (pH 7) to a final concentration of 0.1 g (wet weight)/ml. Assay of enzyme activity. The activity of the enzyme was determined by a direct spectrophotometric method with a Spectracomp 602 spectrophotometer (Carlo Erba Strumentazione, Milan, Italy), with cephaloridine as a substrate. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 ,umol of substrate per min at 30°C in
Corresponding author. 1760
VOL. 35, 1991 V-LACTAMASE OF MYCOBACTERIUM FORTUITUM
0.05 M sodium phosphate buffer (pH 7). The millimolar absorbancy difference of cephaloridine used to calculate the rate of hydrolysis was 7.96 mM-1 cm-' at 255 nm. The absorbance values were calculated as the mean of at least four independent determinations. Standard errors, usually less than 4%, were omitted. Extracellular and intracellular I-lactamase preparation. The extracellular ,3-lactamase was recovered from culture supernatants after filtration through membranes (pore size, 0.45 ,um; Millipore, Molsheim, France). The intracellular P-lactamase was prepared by disrupting bacteria in an ultrasonic disintegrator (Soniprep 150; MSE, Crawley, United Kingdom) at 4°C for 90 min at maximum output, using 5-min cycles with 5-min cooling intervals. The enzyme activity and number of viable organisms were assessed by withdrawing samples every 10 min during sonication. For colony counting, Tween 80 was added to the material to a final concentration of 4% (vol/vol) and vigorously passaged through a tuberculin needle to decrease bacterial clumping, diluted, spread on Mueller-Hinton agar (MHA) in triplicate plates, and incubated for 3 days at 37°C. Kinetics of ,-lactamase production. The time course of extracellular versus intracellular 1-lactamase production was assessed by growing microorganisms for 14 days at 37°C in flasks containing 100 ml of MHB under 180 rpm agitation. Each flask was inoculated with 1 ml of a suspension adjusted to a McFarland no. 1 turbidity standard. Immediately after inoculation and at 24-h intervals, samples were used for colony counting, as described above. After centrifugation of the culture, the supernatant was filtered while the pellet was sonicated for 20 or 40 min, depending on the strain used (see Results). Both the supernatant of the culture and that from the sonicated pellet were assayed for ,-lactamase activity as described above. Isolation of the mutants. Both high- and low-13-lactamaseproducing mutants were obtained from M. fortuitum ATCC 19542 by mutagenization with nitrosoguanidine, essentially as described by Miller (15). Briefly, parental cells were pelleted from exponentially growing MHB cultures and washed in 0.1 M sodium citrate buffer (pH 5.5) containing 1% (vol/vol) Tween 80. The cell suspension was adjusted to a McFarland no. 5 turbidity standard, treated for 30 min at 370C with 100 ,g of nitrosoguanidine per ml, washed, and resuspended in 0.05 M sodium phosphate buffer (pH 7) containing 1% Tween 80. For the selection of high-p-lactamase-producing strains, bacteria were spread on MHA plates containing twofold increasing concentrations of amoxicillin. Colonies growing at the highest concentration of antibiotic were picked with toothpicks and gridded in duplicate onto antibiotic-free plates. The ,-lactamase production was provisionally assessed by flooding the plates with the chromogenic cephalosporin PADAC (300 ,uM); colonies developing the largest hydrolysis halos were grown for 3 days on MHB to assess the amount of enzyme released. The highest-producing mutant was grown and subjected to a subsequent cycle of
mutagenic treatment.
For the isolation of low-p-lactamase-producing mutants, colonies of mutagenized bacteria grown in MHA were gridded onto plates with or without a subinhibitory concentration of benzylpenicillin. Colonies growing in antibioticfree plates but not in those with penicillin were tested by addition of PADAC. Colonies developing the smallest hydrolysis halos were grown for 3 days on MHB to assess the amount of enzyme released. The lowest-producing mutant
1761
TABLE 1. Effect of different media on the production of P-lactamase by M. fortuitum ATCC 19542 ,-Lactamase production
Liquid medium
Total (U/g, bacterial dry wt)
% Intracellular
% Released
Sauton GYM MHB
6.2 27 72.5
33 26 33
67 74 67
was grown and mutagenized again until a strain with negligible production of enzyme was obtained. Identification procedures. Both the parent strain and its mutants were subjected to standard identification tests (18). Antimicrobial susceptibility test. MICs were determined by a twofold agar dilution technique in MHA. The following antibiotics were used: benzylpenicillin, amoxicillin, amoxicillin plus clavulanic acid (7:1), ampicillin, ampicillin plus sulbactam (1:1), and cephaloridine, at concentrations from 32,768 to 0.03 ,ug/ml, and imipenem and cefoxitin, at concentrations from 8,192 to 0.03 ,ug/ml. Bacterial suspensions, preadjusted to a McFarland no. 1 turbidity standard were inoculated onto antibiotic-containing plates to a final concentration of about 104 CFU per spot. MICs were read after incubation at 37°C for 3 days and were defined as the lowest antibiotic concentration at which no growth of the organism was observed in duplicate plates. MICs were evaluated in three independent experiments, with identical results.
RESULTS
I8-Lactamase production by M. fortuitum ATCC 19542. Of the three media tested, MHB was the best for the production of M. fortuitum ,-lactamase (Table 1). Under the above culture conditions, the yield of enzyme was 72.5 U/g (dry weight) of bacteria, which was 2.7 times higher than in GYM and 11.7 times higher than in Sauton. About 67% of the enzyme was released into MHB. The extent of cell breakage following sonication was also dependent on the growth medium: cells from MHB needed about 50 min of sonication for lysis and complete release of the enzyme, whereas the same strain, grown in Sauton, was disrupted only after 90 min of treatment (Fig. 1). In all the media tested, the enzyme activity was not changed by continued sonication once cell lysis was completed. In MHB cultures, a 48-h logarithmic phase of growth was followed by a short (24-h) stationary phase with cell lysis starting at a pH of ca. 8.7 (Fig. 2). The release of P-lactamase into the medium apparently followed the same kinetics as the exponential phase of growth (for 2 days), and then a rapid decrease in the enzyme activity was observed. The production of intracellular ,-lactamase appeared to be delayed in comparison with that of the extracellular enzyme, and appreciable amounts of the enzyme were still detected after 1 week of growth. 13-Lactamase production and resistance to beta-lactam antibiotics in mutant strains. Following multistep mutagenesis with nitrosoguanidine, three low- -lactamase-producing mutants designated oxSO, 3130, and -y27, releasing 10, 0.5, and 0.0001 U of enzyme per liter, respectively, were obtained. Five high-producing strains named A188, B180, C207, D316, and E31, releasing 31, 59, 127, 240, and 278 U/liter, respectively, were also isolated. Each mutant was tested for
ANTIMICROB. AGENTS CHEMOTHER.
FATTORINI ET AL.
1762
100 alO" ON
0
10
20
30
40
50
60
70
80
90
TIME (mn /
SONICA TION
FIG. 1. Relationship between lysis of M. fortuitum ATCC 19542 and release of the enzyme from the cells obtained by sonication. Shown are percent viable organisms in Sauton (0), GYM (O), and MHB (A) and P-lactamase activity in Sauton (0), GYM (M), and MHB (A).
susceptibility to benzylpenicillin, cephaloridine, imipenem, and cefoxitin (Fig. 3A), as well to amoxicillin, amoxicillin plus clavulanic acid, ampicillin, and ampicillin plus sulbactam (Fig.. 3B). The strains with the lowest production of P-lactamase (-y27 and p136) wefe the most susceptible. In all other mutants, resistance to benzylpenicillin, cephaloridine, ampicillin, and arnoxicillin progressively increased with increased enzyme production. In particular, for the highest ,B-lactamase producer (strain E31), the MICs of amoxicillin, ampicillin (or benzylpeniciilin), and cephaloridine were 2,048-, 512-, and 32-fold higher, respectively, than their MICs for y27. The MICs of imipenem and cefoxitin were the same (2 and 16 ,ug/ml, respectively) for the mutants as for the parental strain. Figure 3B shows that the addition of a P-lactamase inhibitor (clavulanic acid or sulbactam) causes a sufficient increase in antibiotic susceptibility to render the MICs for the strains that produced up to 31 U of ,-lactamase per liter identical to those for the strains that did not produce
N
10
r75
-
-50
9-
appreciable amounts of the enzyme. For the mutants which released more than 31 U of enzyme per liter (B180, C207, D316, and E31), the MICs of amoxicillin were reduced up to 32-fold by clavulanic acid, whereas the MICs of ampicillin were lowered 8-fold by sulbactam. All mutants described were very stable in their capacity for producing large or small amounts of enzyme, as shown by repeated subcultures in MHB (data not shown). Biochemical properties and growth kidetics of M. for-tuitum mutant strains.Because mutagenization with nitrosoguanidine could produce such pleiotrcipic effects as tq alter the basic biochemistry and growth of the mutants; thus nonspecifically influedcing their susceptibility to antibiotics, we evaluated some physiological, parameters and, in particular, the kinetics of growth of the different mutants. All basic biochemical tests for identification of mycobacteria were unchanged in the mutants With the exception of a small decrease in nitrate reductase. Moreover, the cells of mutant D316 required less time (ahout 20 min) for disruption and P-lactamase release than did the parental strain. The growth kinetics of the P-lactamase-producing strain D316 are shown in Fig. 4. The mutant grew more slowly than the parental strain (compare with Fig. 2). The stationary phase of growth had a longer duration (about 4 days), and cell lysis took place at ca. pH 9. Both intracellular and extracellular enzymnatic activities were detected in the middle of the stationary phase. However, the ratio of extracellular to intracellular enzyme was lower in the mutant than in the parental strain (Fig. 2 and 4).
c:>
8-
k -25 "a
8-
.I
6-
II
7 - 4-
Days FIG. 2. Kinetics of P-lactamase production iri M. fortuitum ATCC 19542. Samples were taken every day, and pH (0), CFU per milliliter (A), and extracellular (0) and intracellular (A) P-lactamase activities were determined.
DISCUSSION
1-Lactamases of mycobacteria have beeui reported to be constitutive intracellular enzymes which are released into the medium mainiy in aged culturds, following cellular lysis (13). Kasik and Peacham (14) found that the 131actainase of M. fortuitum was mainly cell bouhd when the micfoorganism was grown for 14 io 16 days on Sauton.,Other authors (16, 17) recovered the enzyme frorn both culture superhatants and extracts of cells grown in submerged cultures. Under our culture conditions, wheh M. fortuitum ATCC 19542 was grown in shaken cultures, MHB was the best medium for the production of both extracellular and intracellular P-lactamases, whereas cells grown in Sauton pro-
32 768 16 384 8 192
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P-LACTAMASE OF MYCOBACTERIUM FORTUITUM
-300 0
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o Qi
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STRAINS
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BETA-LACTAMASE PRODUCTION (U,/Il
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VOL. 35, 1991
...
19 542
1000 100 BETA-LACTAMASE PRODUCTION (U/I) FIG. 3.:Relationship, between resistance to beta-lactam antibiotics and prc duction of extracellular 1-lactamase in M. fortuitum ATCC 195412 and in its mutants obtained by treatment with nitrosoguanidine. ((A) Resistance of benzylpenicillin (0), cephaloridine (O), cefoxitin (* ), and imipenem (A). (B) Resistance of amoxicillin (0), amoxicillin plus clavulanic acid (7:1) (0), ampicillin (A), and ampicillin plus s;ulbactam (1:1) (A). Concentrations reported in panel B refer only t,:oamoxicillin ,and ampicillin, respectively. 10
duced negligible amounts of the enzyme. Thus, synthesis of the ,B-lactamase may be nonspecifically induced or up' regulated by specific constituents of the medium, as'is the case with ,B-lactamase of Enterobacter'cloacae (7). Kinetics of enzyme production by the parental strain grown in ]MHB showed that ,B-lactamase wa's released into culture me-dium during the active phase of growth, when the level of iritracellular enzyme'was still low. This was also observed iin shaken cultures of M. smegmatis (5) and might be explain ed by an early lytic phenomenon or early secretion of the en;zyme connected with more rapid growth under agitation. Some support for this hypothes'is comes from the
-0 0
2
4
6
8
10
12
14
Days
FIG. 4. Kinetics of P-lactamase production in M. fortuitum D316. Samples were taken every day, and pH (0), CFU per milliliter (A), and extracellular (0) and intracellular (A) P-lactamase activites were cetermined.
behavior of strain D316, which had a stationary phase longer than that of the parental strain, with a lower percentage of extracellular enzyme than that found in the parent itself. To obtain some insight into the role of -lactamase in the .°in vitro resistance of M. fortuitum to beta-lactam antibiotics, we isolated mutants either with negligible P-lactamase pron (strain with ductio or ) about 15-fpld higher production Ey27 than the parent strain (strain E31). T hesemutants were very stable and showed only small changes in their phenotype and growth characteristics. The susceptibility to 3-lactamaselabile beta-lactam antibiotics such as benzylpenicillin, ampicillin, amoxicillin, and cephaloridine correlated well with the amount of 3-lactamase released into the medium, suggesting that the enzyme played some role in determining the high resistance of M. fortuitum to beta-lactam antibiotics. This is strengthened by our findings that the addition of clavulanic acid to 'amoxicillin reduced the MICs for the mutants, probably as a result of a protective effect exerted by clavulanic acid on amoxicillin (9). The lower capacity of sulbactam, as compared with clavulanic acid, to decrease the MICs of ampicillin might be explained by a slow hydrolytic effect of mycobacterial ,-lactamase on this inhibitor or by a lower-affinity binding (10). Interestingly, the MICs of imipenem and cefoxitin, which are poorly hydrolyzable by M. fortuitum 3-lactamase (unpublished data), were not affected either by enzyme overproduction or by modification of the rate of autolysis, as observed especially for strain D316. Altogether, the data clearly substantiate a correlation between P-lactamase production and resistance to beta-lactam antibiotics in M. fortuitum. However, mutants with a negligible production of 3-lactamase such as strain -y27 were still relatively resistant to the beta-lactam antibiotics tested. This phenomenon seems to indicate that, as observed in M. chelonei (11), there is a threshold level of drug resistance in M. fortuitum which probably depends on mechanisms other than 1-lactamase production, such as cell wall permeability and affinity for penicillin-binding proteins.
1764
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FATTORINI ET AL.
ACKNOWLEDGMENTS We thank Antonio Cassone, Istituto Superiore di Sanita, Rome, Italy, for help in reviewing the manuscript. This work was supported in part by the Consiglio Nazionale delle Ricerche, Italy, Project FATMA, grant 91.00276.41, and in part by the Italian AIDS Project, Istituto Superiore di Sanita, Ministero della Sanita, grant 5203-032.
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