Aerobic actinomycetes and the clinical microbiology laboratory

Clinical Microbiology Newsletter

Vol. 1, No. 15

Augtlst | , 1979

Copyright © 1979 by G.K. Hall & Co.

Aerobic Actinomycetes

a n d t h e Clinical Microbiology Laboratory

i

Philip A. Tisdall, M.D. and Glenn D. Roberts, Ph.D. Mayo Clinic and Mayo Foundation Rochester, Minnesota 55901 The order Actinomycetales includes both saprophytic and pathogenic species. The latter cause a variety of diseases in humans, especially suppurative pulmonary infections and subcutaneous abscesses. Both pathogens and saprophytes may be commonly found in soil and water; thus it i~ important for the clinical microbiology laboratory not only to be able to culture actinomycetes, but also to differentiate the pathogens from the saprophytes. Identification is complicated, however, by the bewildering number and descriptions of organisms included in this order. More than 20 new genera have been described since 1950 (15). In the absence of a single, generally accepted classification, it is difficult for nonspecialists to follow the changing taxonomy. This discussion will present the clinically important genera of the Actinomycetales and give a scheme of identification that is practical for the clinical microbiology laboratory. The order Acthwmycetales includes those bacteria forming filamentous cells with a tendency towards branching. These branches often develop into a characteristic mycelium that is usually less than 1.0/zm in diameter (5). Multiplication is by means of either spores or fragmentation of hyphae into coccoid or bacillary elements. Within this descriptive framework, the order contains a markedly heterogeneous group of organisms. Mycelia vary from the rudimentary or absent buds of Mycobacteriaceae to the fungus-like aerial hyphae of Streptomycetaceae. Spores may be single, clustered, formed in sporangia, or absent altogether. Or-

ganisms may be saprophytic or parasitic, mesophilic or thermophilic, aerobic or anaerobic. While typical representatives of each family can be recognized on the basis of morphology, staining characteristics and intermediary metabolism, their distinctive features overlap because each feature varies through a spectrum. Classification attempts have been further frustrated by the variability in morphology and staining of a given strain. Changes in culture medium, temperature, pH, and light all affect morphology. Different media can alter an organism's acid-fastness (11). Some uniformity has been brought to the taxonomy of Actinomycetales with the advent of cell-wall analysis. Cell-wall amino acids, carbohydrates, and lipids appear to be stable and characteristic for many genera (9). Techniques to perform analysis are now practical at a routine laboratory level (14). The minimal analytic requirement for a laboratory is the identification of stereoisomers of the cell-wall amino acid, diaminopimelic acid (DAP). Clinical Significance Table 1 lists those aerobic members of the Actinomycetales that a routine

laboratory should be able to identify. Not included in the table is the family Actinomycetaceae, which includes, for example, the anaerobic organism, Acthzomyces israeli. Also not included are Micropolyspora, especially M. faeni; Micromonospora, and Saccharopolyspora, which are all causative agents of extrinsic allergic alveolitis. As most of these are thermophiles that grow optimally at 50°C, they are only rarely found on routine culture. The reader is referred elsewhere for further information on thermophilic actinomycetes (7,8). Mycobacteriaceae is a welldescribed family, for which the clinical significance has been recently reviewed by Wolinsky (16); thus it will not be discussed further here. The remaining pathogenic members of the Actinomycetales cause two main patterns of disease; these are 1) pulmonary infection with possible systemic dissemination and 2) subcutaneous infections, especially mycetoma. Dermatophilaceae are the etiologic agents of streptotrichosis, a zoonotic disease, occurring commonly in sheep. Humans acquire the infection by direct contact with the animals or with their hides. It is characterized by the presence of mul-

Table 1 Aerobic Actinomycetales Family

Genus

Dermatophilaceae Mycobacteriaceae Nocardiaceae

Dermatophilus Mycobacterium Noeardia

Streptomycetaceae Thermomonosporaceae

Rhodococcus Streptomyces Actinomadura Nocardiopsis

Common Species D. congolensis N. asteroides N. brasiliensis N. caviae A. madurae A. pelletieri N. (Actinomadura) dassonvillei

SOUaCE: ModifiedfromGoodfellowand Minnikin(4), and consideredto be morecurrentin nomenclature than the eighth edition of Bergey's Manital.

tiple, non-painful pustules or a desquamative dermatitis appearing approximately one week after exposure (2). Infections with Nocardia are now described with increasing frequency in debilitated hosts (13). Nocardiosis is associated with malignancy, antineoplastic therapy, and steroid therapy (14). The primary infection is pulmonary, with uncommon instances of systemic spread. Infections caused by Rhodococcus, the rhodoehrous complex, have been described only recently (6). All three patients described were immunocompromised; two infections were pulmonary, and one was systemic. Streptomyeetaceae comprise the largest number of species among all the genera of Actinomycetales. Over 700 species have been described, differing widely in their morphology and biochemical characteristics. While they have rarely been described as causing mycetoma, they are generally regarded as saprophytes (13) and are seen in the laboratory as common contaminants. The family Thermomonosporaceae contains the most common etiologic agents of actinomycotic mycetoma. Originally classified as Nocardia, they have been separated and subdivided on

the basis of their cell-wall carbohydrates. Members of this group have few similarities (12) and are grouped together for convenience. Examination and Culturing of

Specimens All specimens submitted for culture should be examined macroscopically for the presence of granules, and an unstained wet mount should be examined microscopically for branching filaments 1.0/.trn or less in diameter. Morphology of the filaments may have characteristic features. Dermatophilaceae, for example, have a diagnostic longitudinal and transverse divisions of their filaments (13). All species of Actinomycetales are gram-positive. Intact granules, if present, should be crushed to demonstrate mycelia, which would rule out staphylococcal botryomycosis. While mycobacteria are strongly acid-fast, Nocardia species vary greatly in their acid-fast characteristics; some cells stain well while others do not. Since the nocardiae are easily overdecolorized, it is recommended that 0.5% H2SO4 be used as the decolorizing agent when an acid-fast stain is performed. In tissues,

all Actinomycetales stain well with silver-impregnation techniques. Most aerobic actinomycetes grow well on conventional bacteriologic and fungal culture media. Brain heart infusion agar with and without antibiotics is recommended (3). Antibiotics, however, are often inhibitory for many aerobic actinomycetes. Cultures should be incubated at 35°C and kept for at least three weeks. Nocardia asteroides survives mycobacterial isolation procedures and its growth is stimulated by 5% COz; consequently, it is most frequently recovered on Lowenstein-Jensen medium. Identification

The presence of aerobic actinomycetes may be suspected when small, heaped, and folded colonies grow on primary isolation media. They have a moist, glabrous surface or are covered by chalky aerial mycelia. There may be diffusion of pigment around the colony into the medium. Microscopic examination will demonstrate the presence of branching filaments 1.0 p.m in diameter. Evaluations of current methods for identifying aerobic actinomycetes have

Typical colonial morphology Not strongly acid-fast No spores formed in chains

! J, Casein Xanthine + Urease +

!

I

N. caviae

Non-acid-fast

Acid-fast

1

N. asteroides

Casein + Xanthine + Urea + or Non-acid-fast

Casein + Xanthine -

Casein Xanthine Urease + or -

1

Probable N. asteroides

Refer for further testing.

Urease + Acid-fast

Urease Non-acid-fast

L-DAP

Streptomyces sp.

1

Meso-DAP

A. dassonvillei

N. brasiliensis Uncertain: A. tltadttrae Streptomyces S. somaliensis A. pelletierii Refer for further testing.

Fig. 1 Biochemical identification of aerobic acfinomycetes (adapted with modificationfrom Berd (1)).

Heaped, glabrous colony Typical microscopic morphology L-DAP~,

[

~Meso.DAP

Cell-wall carbohydrate analysis

Streptomyces

Arabinose, xylose

Arabinose, galactose

Madurose

3.

None 4.

Mesophilic Micromonospora

Nocardia Rhodococcus Mycobacterium

Weak o r - ~ ,

Acthzomadura: .4. madurae .4. pelletieri *

I

Nocardiopsis: N. dassonvillei

j.Strong

Nocardia Rhodococcus

Mycobact erium

~l' Ethylene glycol degradation

Acid-fast stain

6.

7.

I

~+

Rhodococcus

Nocardia

N. asteroides N. brasiliensis iV. caviae

5.

Casein

Tyrosine

Xanthine

+ -

+ -

+ +

8.

9.

*May be distinguished from A. madurae by its dark ted pigment. Fig. 2 Modified Mayo Clinic scheme (14) for biochemical identification of aerobic actinomycetes.

been reviewed recently by Berd (1) and Staneck and Roberts (14). Figures 1 and 2 present two different schemes for identifying isolates. The first is a modification of Berd's simplified scheme. The use of this scheme, along with careful morphologic examination, should permit identification of most isolates. The second decision tree is a modification of the method used at the Mayo Clinic. Morphologic examination is followed by analysis for isomers of DAP. Nocardia species, however, are the most common pathogens recovered in a clinical laboratory. These could be identified to the species level using only the acidfast stain and the hydrolysis of casein, xanthine, and tyrosine (see Fig. 1). A laboratory of limited means could identify Nocardia and refer all other isolates for final identification. Neither of the schemes presented here should be interpreted beyond its clinical limits. Authors with extensive experience and who use a large battery of tests

report that strains exist that fit only loosely into these identification systems (3,10). Any isolates not conforming to the system should be sent to a reference laboratory. The current tools available for analysis of aerobic actinomycetes should make their identification possible at a routine level and significantly decrease the time of reporting results. Laboratory data then will be of active rather than historical interest to the clinician. The present complexities of taxonomy and nomenclature are gradually improving. Until such time as there is a single, accepted classification of actinomycetes, the routine laboratory should do its best to keep abreast of taxonomic developments. References 1. Berd, D. 1973. Laboratory identification of clinically important aerobic actinomycetes. Appl. Microbiol. 25(4):665-681. 2. Dean, D. J., M. A. Gordon, C. W.

10.

I I.

12. 13.

14.

15.

16.

Severinghaus, E. T. Kroll, and J. R. Reiily. 1961. Streptothricosis: A new zoonotic disease. N.Y. State J. Med. 61:1283-1287. Georg, L. K. Nocardia species as opportunists, and current methods for their identification. U.S. Department of Health, Education and Welfare, Center for Disease Control, Atlanta, GA. Goodfellow, M., and D. E. Minnikin. 1977. Nocardioform Bacteria. Ann. Rev. Microbiol. M. P. Start, ed. 31:159-180. Gottlieb, D. 1974. Order I. Actinomycetales Buchanan 1917, 162, p. 657-681. In Bergey's manual of determinative bacteriology. R. E. Buchanan and N. E. Gibbons, eds. Williams and Wilkins, Baltimore, MD. Haburchak, D. R., Betty Jeffery, J. W. Higbee, and E. D. Everett. 1978. Infections caused by Rhodochrous. Am. J. Med. 65:298-302. Lacey, J. 1978. Thermophilic actinomycetes: Characteristics and identification. J. Allergy Ciin. Immunol. 61:231-232. Laeey, J., and M. Goodfeliow. 1975. A novel actinomycete from sugar-cane bagasse: Saccharopolyspora hirsuta gen. et sp. nov. J. Gen. Microbiol. 88:75-85. Lechevalier, M. P. 1976. The taxonomy of the genus Nocardia: Some light at the end of the tunnel? In the Biology of the Nocardia. M. Goodfellow, G. H. Brownell, and J. A. Serrano, eds. Academic Press, New York, N.Y. Leehevalier, H. A,, and M. P. Lechevalier. 1970. Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol. 20:435-443. Mariat, F. 1965. Etude comparative de souches de Nocardia isol6es de myc6tomes. Ann. Inst. Pasteur (Paris). 109:90. Meyer, J. 1978. Nocardiopsis, a new genus of the orderActinomycetales. Int. J. Syst. Bacteriol. 26(4):487-493. Rippon, J. W. Medical mycology: the pathogenic fungi and the pathogenic actinomycetes. 1974. W. B. Saunders Co. Philadelphia, PA. Staneck, J. L., and G. D. Roberts. 1974. Simplified approach to identification of aerobic actinomycetes by thinlayer chromatography. Appl. Microbiol. 28:226-231. Williams, S. T., F. L. Davies, and T. Cross. 1968. Identification of genera of the Actinomycetales. hz Identification methods for microbiologists. B. M. Gibbs and D. A. Shapton, eds. Academic Press, New York, N.Y. Wolinsky, E. 1979. Nontuberculous mycobacteria and associated diseases. Am. Rev. Resp. Dis. 119:107-159.