the other isolates produced a milky-white opacity surrounding colonies on Middlebrook 7H10 agar. This finding may facilitate the identification of N. farcinica.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1993, p. 3040-3041
Vol. 31, No. 11
0095-1137/93/113040-02$02.00/0 Copyright © 1993, American Society for Microbiology
Opacification of Middlebrook Agar as an Aid in Identification of Nocardia farcinica MARTHA FLORES AND EDWARD DESMOND*
Microbial Diseases Laboratory, California Department of Health Services, Berkeley, California 94704-1011 Received 14 May 1993/Returned for modification 8 June 1993/Accepted 22 July 1993
One hundred twenty seven human isolates of Nocardia asteroides complex were identified to the species level by drug susceptibility testing, acetamide utilization, thermotolerance studies, studies of arylsulfatase activity, and studies of acid production from rhamnose. N. asteroides complex organisms which were not identifiable as N. farcinica or N. nova were designated N. asteroides sensu stricto. All of the N. farcinica isolates and none of the other isolates produced a milky-white opacity surrounding colonies on Middlebrook 7H10 agar. This finding may facilitate the identification of N. farcinica.
The genus Nocardia has been gaining greater attention in recent years, both because it is being found more frequently as a cause of clinical illness in some areas and because of its association with infections of the central nervous system (1, 5). Within the Nocardia asteroides complex, the species N. farcinica has gained attention because of its pathogenicity, which is greater than those of other species in the complex, and because of its resistance to antibiotics (3, 7). We identified 127 human isolates of N. asteroides complex to the species level by using the methods of Wallace et al. and identification criteria as published previously (3, 6, 7). Organisms were considered to belong to the N. asteroides complex on the basis of colonial and microscopic morphologies, susceptibilities to lysozyme, and failure to hydrolyze casein, xanthine, tyrosine, hypoxanthine, adenine, or starch. N. asteroides complex organisms were identified as N. farcinica when they were arylsulfatase negative and resistant to tobramycin and erythromycin and had at least two of the following properties: growth at 45°C equal to growth at 35°C, production of acid from rhamnose, ability to use acetamide as a sole C and N source, and resistance to cefamandole. N. asteroides complex organisms were considered to be N. nova when they were unable to use acetamide as a sole C and N source and were susceptible to cefamandole and erythromycin and had at least two of the following properties: growth at 45°C less than growth at 35°C, no acid production from rhamnose, arylsulfatase positivity, and tobramycin resistance. All N. asteroides isolates not identifiable as N. farcinica or N. nova were described as N. asteroides sensu stricto. These identification tests, including the use of acetamide as a sole C and N source, production of acid from rhamnose, and susceptibility or resistance to cefamandole, etc., are specialized ones, using materials found only in large laboratories. We endeavored to find an identification method using materials found more widely in microbiology laboratories. All of the isolates were submitted for identification to the Microbial Diseases Laboratory of the California Department of Health Services between 1980 and 1993. Most of the strains had been stored in sterile water at room temperature, and after they were revived they were maintained on brain *
Corresponding author.
heart infusion agar slants. Also tested were the following reference strains: N. farcinica ATCC 3318, N. asteroides ATCC 19247, and N. nova ATCC 33726; seven strains obtained from Richard Wallace at the University of Texas (one each of N. asteroides and N. farcinica and five strains of N. nova), and two strains of N. asteroides obtained from Blaine Beaman at the University of California, Davis. Pure cultures of all strains were streaked onto Middlebrook 7H10 agar and incubated at 35°C for the detection of a milky-white opacity surrounding the colonies. Middlebrook 7H10 and 7H11 agars are complex defined media with albumin, salts, glycerol, malachite green inhibitor, and enrichments of oleic acid-albumin-dextrose-catalase (OADC). They are widely used in mycobacteriology laboratories and in some laboratories that perform actinomycete work. Middlebrook 7H10 agar is one of the media of choice for the isolation of aerobic actinomycetes (2, 4). This medium is also recommended for the enhancement of the acid-fast property of Nocardia species. The Middlebrook 7H10 agar was prepared in house by using a 7H10 base and OADC enrichment from Difco Laboratories, Detroit, Mich. In addition to the comprehensive study of all N. asteroides complex strains on in-house-prepared 7H10 agar, 30 strains of N. farcinica and 10 strains each of N. nova and N. asteroides sensu stricto were tested for their abilities to produce opacity on commercially prepared Middlebrook 7H11 agar plates from BBL (Cockeysville, Md.) and PML (Tualatin, Oreg.) and on 7H10 agar slants from PML. All 36 clinical isolates identified as N. farcinica produced the milky-white opacity surrounding the colonies (Fig. 1) on the in-house-prepared 7H10 plates. The 61 isolates identified as N. asteroides and the 30 isolates identified as N. nova did not produce the opacity. Likewise, all of the reference cultures identified as N. farcinica and none of the other reference cultures showed the white opacity surrounding the colonies. Opacity surrounding the N. farcinica colonies developed within 2 to 10 days. Detection of this white opacity around colonies on 7H10 agar, reported here for the first time as a unique property of the species N. farcinica, may be a useful criterion for laboratories in the identification of this species. Although our data by this technique, using 127 clinical isolates of N. asteroides complex, indicate a sensitivity and a specificity of 100% for this technique in identifying N. farcinica, these data must be considered 3040
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FIG. 1. Photograph of 7H10 agar plates following 5 days of incubation at 350C. (A) N. asteroides; (B) N. farcinica; (C) N. nova.
preliminary until confirmed by other groups working with strains from diverse geographic regions. Tests for production of opacity on 7H10 were performed with pure cultures of N. asteroides complex organisms only; however, in our experience, cultures of N. farcinica produced opacity on 7H10 medium also when mixed with other bacteria. This study did not include a comprehensive attempt to determine what other genera or species might produce a similar opacity; however, it was our observation that cultures of species from the aerobic actinomycete genera Streptomyces, Actinomadura, Nocardiopsis, Micromonospora, and Amycolata did not produce opacity on 7H10. Likewise, several strains of Nocardia species not in the N. asteroides complex did not produce the opacity on 7H10 medium. The production of opacity on 7H10 medium by N. farcinica strains was a stable trait; this property was retained regardless of length of storage or storage conditions. In addition to our comprehensive study of opacity caused on the in-house-prepared 7H10 agar plates, we observed the production of a similar opacity on the commercially prepared 7H11 and 7H10 agar plates and tubes by all of 30 strains of N. farcinica tested and none of 10 strains each of N. nova and N. asteroides sensu stricto tested. Opacity sometimes developed more slowly on the agar slants than it did on the in-house-prepared plated media (generally with a delay of 2 to 3 days).
We thank Barbara Brown, Richard Wallace, and June Brown for their valuable comments, for confirming the identification of some troublesome isolates, and for supplying reference strains for this
study. 1.
2. 3. 4.
5. 6. 7.
REFERENCES Boiron, P., F. Provost, G. Chevrier, and B. Dupont. 1992. Review of nocardial infections in France, 1987 to 1990. Eur. J. Clin. Microbiol. Infect. Dis. 11:709-714. Campbell, M., and J. L. Stewart. 1980. The medical mycology handbook, p. 96. John Wiley & Sons, New York. Desmond, E., and M. Flores. 1993. Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates. FEMS Microbiol. Lett. 110:281-284. Haley, L. D., and C. Calloway. 1978. Laboratory methods in medical mycology. Center for Disease Control laboratory manual. U.S. Department of Health, Education, and Welfare publication no. (CDC) 79-8361, p. 141-144, 190. Center for Disease Control, Atlanta. Kohbata, S., and B. Beaman. 1991. L-Dopa-responsive movement disorder caused by Nocardia asteroides in the brains of mice. Infect. Immun. 59:181-191. Wallace, R. J., Jr., B. A. Brown, M. Tsukamura, J. M. Brown, and G. 0. Onyi. 1991. Clinical and laboratory features of Nocardia nova. J. Clin. Microbiol. 29:2407-2411. Wallace, R. J., M. Tsukamura, B. Brown, J. Brown, V. Steingrube, Y. Zhang, and D. Nash. 1990. Cefotaxime-resistant Nocardia asteroides strains are isolates of the controversial species Nocardia farcinica. J. Clin. Microbiol. 28:2726-2732.
