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Isolation and identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis
Clinical Microbiology Newsletter March 1, 1986
Vol. 8, No. 5
i
Isolation and Identification of Thermophilic Actinomycetes Associated with Hypersensitivity Pneumonitis Gary E. Hollick, Ph.D. Director Microbiology Laboratory Rochester General Hospital Rochester, New York 14621 The thermophilic actinomycetes were among the first organisms to be implicated as sensitizing agents in the development of hypersensitivity pneumonitis. The disease can affect farm, office, factory workers, and others who come into contact with large numbers of mycelia and spores from these organisms. Acute disease is induced by repeated inhalation of mycelia and spores of fungi or actinomycetes, as well as by inhalation of various types of organic dusts. Cough, dyspnea, and fever characteristically begin within 4 to 6 hr following exposure, with resolution of symptoms within 18-24 hr. Chronic disease develops following repeated acute episodes or long-term, low-dose exposure. This chronic form of the disease is characterized by cough, dyspnea, weight loss, airway obstruction, and, ultimately, irreversible lung fibrosis. Depending upon the nature and source of the sensitin, different names have been given to the same disease (Table 1).
Diagnosis The accurate diagnosis of hypersensitivity pneumonitis depends upon a compatible clinical history, lung biopsy findings, and results of radiographic
CMNEEJ8(5)29-36,1986
and pulmonary function tests. Serologic tests have been developed but have met with several inherent problems. First, approximately 3-12% of healthy persons may possess precipitating antibody directed against the thermophilic actinomycetes (2, 9, 19). Second, Wenzel et al. (21) have found that 44.2% of patients with positive precipitin tests reacted to more than one thermophilic actinomycete. This finding has been subsequently confirmed by other investigators (1, 11). Once a diagnosis is made, however, a source should be sought because the only effective treatment is exposure avoidance. A discussion of methods for isolating and identifying thermophilic actinomycetes obtained from environmental sources follows.
report has included a large sample of environmental sources; Kurup et al. (13) were able to isolate one or more thermophilic actinomycetes from 45% of 273 samples. Sources included house dust, furnace filters, and air conditioners. To date, no reports have correlated the number of organisms present in an environmental source to the development of disease.
Isolation The suspected source of exposure determines the type of sample that should be collected. Air samples can be taken by exposure plates or by one of a variety of mechanical samplers. Water from air conditioning systems or humidifiers can be plated directly or i
Prevalence Thermophilic actinomycetes can be isolated from water (3), air (8, 17), soil and compost (3), home and industrial air conditioning systems (4, 13), house dust (13), hay (18), and bagasse (16). Reports of actual counts of these microorganisms in natural habitats have been few. Numbers have ranged from 1 × 103/g from surface peat to 3 × 108/g in baled hay (3). Air surveys, taken when moldy hay was manipulated, produced concentrations of spores approximating 1.6 × 109/m3 (17). Cigarette tobacco has been found to contain 1.5 × 104to 1 × 105 colonies of actinomycetes/g (15). Only one
Elsevier
In T h i s Issue
Thermophilic Actinomycetes . . . . The isolation and laboratory characterization of agents responsible for hypersensitivity pneumonitis
29
Characterization of Coryneforms 32 Importance of properly identifying "diphtheroid-like" organisms recovered from clinical specimens Branhamella Septicemia . . . . . . . . An uncommon complication of Branhamella pneumonia
34
0196-4399/86/$0.00 + 02.20
Table 1 Selected Diseases of the Hypersensitivity Group Disease
Source o1 sensitin
Air conditioning lung
ventilation ducts
Bagassosis
moldy sugar cane
Farmer's lung
moldy hay
Humidifier lung
humidifier water
Mushroom worker's lung
moldy compost
hnpli~'ated actinom3 cete S. viridis" T. vulgaris ~' T. candidus 7'..sacchari T. vulgaris M..[~teni' T. vulgaris T. candidus T. vulgaris T. candidus M. fiwni T. vulgaris T. candidus
Saccharomonospora. b Thermoactinomyces. '" Micropo/yspora.
passed through a filter which is subsequently placed onto the agar surface. Other samples, such as pieces of furnace filter, house dust, or hay can be extracted in saline, diluted, then plated onto appropriate media. Treuhaft and Jones (20) described a method in which the sample is placed into sealed plastic bags. The bag is vigorously shaken, a corner removed, and the aerosolized dust puffed out above an open plate. This method was found to be comparable to spread plates for the isolation of Micropolyspora faeni and Saccharomonospora viridis. Optimal isolation of Thermoactinomyces candidus required the use of spread plates. Because of the multitude of other organisms present in some samples, it may be necessary to subject the sample to heat shock (100°C for 10 min) or incorporate 30 I,zg/mL of novobiocin and 400 txg/mL of cycloheximide into the plating media. Most thermophilic actinomycetes grow well on trypticase soy agar (TSA) or TSA supplemented with 1% yeast extract. Thermoactinomyces sacchari and some isolates of Thermoactinomyces vulgaris, however, grow poorly on these media, hence, half strength
nutrient agar should be included in the battery of primary isolation media. Novobiocin and cycloheximide can be added to these media to inhibit overgrowth by other organisms without deleterious effects on isolation of Thermoactinomyces species. M. faeni and S. viridis, however, will not grow on media containing these antimicrobial agents. Plates should be taped shut or placed into sealed plastic bags to avoid excessive moisture loss, and incubated at 50°C for 1 to 3 weeks.
Macroscopic and Microscopic Characteristics Colony morphology, color, growth rate, and the presence of aerial mycelia are important characteristics in the preliminary identification of thermophilic actinomycetes. Gram-stain preparations of colonies are useful for determining the size and arrangement of spores. T, candidus grows on most media within 3 - 5 days as a flat colony covered with a fine white aerial mycelium. Single spores ( 1 - 2 I~m) are borne directly from the mycelia or upon short stalks. Spores are produced on both substrate and aerial mycelia. Macroscopically and microscopically, T.
vtdgaris appears similar to T. candidus. Some isolates may require hall'strength nutrient agar for growth. T. sacchari grows poorly on most media. Colonies are buff colored and few, if any, aerial mycelia are present. When they are produced, the aerial mycelia rapidly undergo autolysis giving the colony a bacterial appearance. When present, spores are produced singly on short sporophores. S. viridis grows well on most media as a white colony that changes to blue-green after sporulation. Occasional isolates may produce a dark, diffusable pigment alter prolonged incubation. Spores are produced singly on dichotomously branched sporophores and are formed only on the aerial mycelia. M. j~teni grows slowly as a yellow, heaped colony which, after 3 - 7 days, may develop patches of fine white aerial mycelia. Chains of spores, 5 20 in number, are formed on aerial and substrate mycelia.
Biochemical Characteristics A composite of the biochemical tests and morphologic criteria needed for presumptive identification is given in Tables 2 and 3. The formulations of the media and interpretations of test resuits can be found elsewhere (7, 10, 12, 14). Thermoactinomyces species are unitbrmly resistant to heat and novobiocin. This serves to differentiate the genus from similar organisms such as Thermomonospora and Streptomyces species. T. candidus is characterized by its ability to decompose esculin and an inability to hydrolyze starch and tyrosine, whereas the morphologically similar T. vulgaris decomposes tyrosine and starch. The unique colony morphology, along with the inability to decompose tyrosine and hypoxanthine, characterize T. sacchari. M. j~leni is differentiated from similar organisms by the inability to decompose casein and the ability to liquify gelatin. Isolates of S. viridis are casein positive and most strains are able to decompose tyrosine and esculin. ii
Clinical Microbiology Newsletter (ISSN 0196-4399) is issued twice monthly in one indexed volume by Elsevier Science Publishing C o . Inc, 52 Vanderbill A'~enue, New York, NY I(KII7 Printed in the USA at 579 Colombia Turnpike, Rensselaer. NY 12144. Subscription prices per ycar: $70.00 including postage and handling in the United States. Canada, and Mexico Add $ 2 5 0 0 for postage (aimqail) in Europe and $ 2 8 0 0 in the rest of the world. Second-class postage pending Postmasler: send address changes to Clinical MicrobioIog~ Ne~sletter, Elsevier Science Publi,hing Cc~ Inc. 52 Vandcrhd[ A~enue, New Ynrk, NY 10017
30
0196-4399/86,S0.00 + 02.20
t 1986Elsevier Science PublishingCo.. lnc
Clinical MicrobiologyNe'~sletter8:5.1986
Table 2 Biochemical and Morphological Characteristics of the Thermoactinomyces Species T.
Proper~
T. candidus
intermedius
Decomposition of: casein tyrosine xanthine hypoxanthine esculin
+ . +
+ +
Hydrolysis of: starch gelatin
T.
T. vulgaris + +
.
.
T. putidus
T. sacchari
dichotomica
+
+ -
+ -
+
-
.
.
+
-+ -
+
+
+ +
+
+ +
+ +
Resistance to: heat novobiocin
+ +
+ +
+ +
+ +
+ +
+ +
Morphology: colony color
white
white
white
white
yellow
spores aerial hyphae
single +
single +
single +
single +
colorless to white single ---
Table 3 Characteristics of
+
determining the source can impact significantly on patient m a n a g e m e n t . The isolation and identification o f thermophilic a c t i n o m y c e t e s is relatively simple and within the capabilities o f most clinical laboratories.
References 1. Barboriak, J. J., J. N. Fink, and G. Scribner. 1972. Immunologic cross reactions of thermophilic actinomycetes isolated from home environments. J. Allergy Clin. lmmunol. 49:81-85.
2. Chmelik, F., D. K. Flaherty, and
single +
Micropolyspora and Saccharomonospora Species
Proper~
M icropolyspora faeni caesis
Decomposition of: casein tyrosine xanthine hypoxanthine starch gelatin esculin
_ -+ + + +
Resistance to: heat novobiocin
. .
Morphology: colony color
yellow
length of spore chain
5-16
Using a c o m m e r c i a l l y available system ( A P I - Z Y M E , Analytab Products Inc., Plainview, N Y ) , we were able to generate unique e n z y m e profiles useful for p r e s u m p t i v e identification (5). In addition, we have found g a s - l i q u i d c h r o m a t o g r a p h y to be useful for separating Thermoactinomyces from Thermomonospora (6).
Clinical Microbiology Newsletter 8:5,1986
Saccharomonospora internatus viridis
+ + + + + . .
. . white to blue-green 1-2
+ + + -
+ _+ + + _+
colorless to blue-green I-6
blue-green
. .
1
The list of agents capable o f inducing hypersensitivity pneumonitis continues to expand. Isolating and identifying organisms k n o w n to induce disease, together with results of serologic tests and clinical history, can help determine a specific sensitin and, at times, a specific source. Because the only effective treatment is exposure avoidance,
© 1986 Elsevier Science Publishing Co., Inc.
C. E. Reed. 1975. Precipitating antibodies in office workers and hospitalized patients directed towards antigens causing hypersensitivity pneumonitis. Am. Rev. Respir. Dis. 111:201-205.
3. Cross, T., and D. W. Johnston. 1971. Thermoactinomyces vulgaris II. Distribution in natural habitats, pp 315-330. In A. W. Baker, G. W. Gould, and J. Wolf (eds.). Spore research. Academic Press, New York.
4. Fink, J. N., A. J. Resniek, and J. Salvaggio. 1971. Presence of thermophilic actinomycetes in residential heating systems. Appl. Microbiol. 22:730- 731. 5. HolUck, G. E. 1982. Enzymatic profiles of selected thermophilic actinomycetes. Microbios 35:187-196. 6. Hollick, G. E. 1982. Differentiation of Thermoactinomyces candidus and Thermoactinomyees vulgaris from Thermomonospora fusca by use of gas-liquid chromatography. Actinomycetes 16:113-121.
7. Hollick, G. E., and V. P. Kurup. 1983. Isolation and identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis. Lab. Med. 14:39-44. 8. Hollick, G. E. et al. 1980. Aerobiology of industrial plant air systems: fungi and related organisms. Zentralbl. Bakteriol. 8 (Suppl. B):89-95. 9. Kawai, T. et al. 1973. Precipitating antibodies against organic dust antigens in human sera by counterimmunoelectrophoresis. Chest 64:420-426. 10. Kurup, V. P. 1981. Thermophilic actinomycetes associated with hypersensitivity pneumonitis. Science-Ciencia 8:5-10. 11. Kurup, V. P. et al. 1976. Immunologic cross reactions among thermophilic actinomycetes associated with hypersensitivity pneumonitis. J. Allergy Clin. Immunol. 57:417-421. 12. Kurup, V. P°, and J. N. Fink. 1975. A scheme for the identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis. J. Clin. Microbiol. 2:55-61.
0196-4399/86/$0.00 + 02.20
31
13. Kurup, V. P., J. N. Fink, and D. M. Bauman. 1976. Thermophilic actinomycetes from the environment. Mycologia LXVIIl:662-666. 14. Kurup, V. P., G. E. Hollick, and E. Pagan. 1980. Thermoactinomyces intermedius: a new species of amylase negative thermophilic actinomycetes. Science-Ciencia 7:104- 108.
15. Kurup, V. P. et al. 1983. Allergenic fungi and actinomycetes in smoking materials and their health implications. Mycopathologia 82:61-64.
16. Lacey, J. 1971.77zermoactmomyces sacchari sp. nov., a thermophilic actinomycete causing bagassosis. J. Gen. Microbiol. 66:327-338. 17. Lacey, J., and M. E. Lacey. 1964. Spore concentrations in the air of farm buildings. Trans. Br. Mycol. Soc. 47:547-552. 18. Pepys, J. et al. 1963. Farmer's lung. Thermophilic actinomycetes as a source of farmer's lung hay antigen. Lancet ii:607-611. 19. Roberts, R. C., D. P. Zais, and D. A. Emanuel. 1976. Frequency of
precipitins to trichloroacetic acidextractable antigens from thermophilic actinomycetes in farmer's lung patients and asymptomatic farmers. Am. Rev. Respir. Dis. !14:23-28. 20. Treuhaft, M. W., and M. P. A. Jones, 1982. Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust. J. Clin. Microbiol. 16:995-999.
21. Wenzel, F, J. et al. 1974, Serologic studies in farmer's lung. Precipitins to the thermophilic actinomycetes. Am. Rev. Respir. Dis. 109:464-468.
Editorial
When, Why, and How Far Should Coryneforms be Identified? Jill E. Clarridge, Ph.D.
Chief, Microbiology Section Laboratory Service Veterans Administration Medical Center Houston, Texas 7721l Coryneforms are a group of bacteria that presumably resemble Corynebacterium diphtheriae in morphology. Most coryneforms are not associated with disease and are found as endogenous human flora. Because it is difficult to distinguish species within this group, clinical laboratories sometimes report "gram-positive pleomorphic rods," "Corynebacterium spp.," "coryneforms," or "diphtheroids" on the basis of Gram stain, colony morphology, and perhaps, a catalase test. Although we recognized the expediency of such a process, we questioned the accuracy. If coryneforms are defined as grampositive, non-acid-fast, asporogeneous, pleomorphic rods that are usually nonanaerobic and nonbranching, many genera having no taxonomic relatedness can be included in this group (1). On the other hand, if the group is limited to those with a "club-shape" during at least part of the life cycle, some of the true Corynebacteria spp. would be excluded and Cellulomonas,
Oerskovia, Arthrobacter, Brevibacterium, Microbacterium, and Curtobacterium, organisms that are generally found in soil or food, would be
32
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included (5). Mycobacterium, Nocardia, and Rhodococcus might be placed in the group if acid-fast stains are not done and branching is not evident. The strains of Actinomyces and Propionibacterium that are not strict anaerobes could also be included. In the broader definition, one would also include Listeria and Erysipelothr&. With this large number of organisms to consider, we decided to identify more completely clinical isolates that had a coryneform morphology (clubshaped, pleomorphic, and/or small regular and/or slightly branching grampositive rod) on Gram stain and that grew aerobically or in 6% CO 2 at 35°C on blood or colistin-nalidixic acid agar plates (3). Only organisms that were numerically significant were selected. For urine isolates this meant specimens with >104 organisms/mL and no more than one other species present. For other specimens, the coryneform organism had to appear on original plates as the predominant organism (ten times more than the next most common isolate) or copredominant with another species. Any isolate recovered from blood culture was considered potentially significant. Using standard methodology, identification was accomplished with the previously published criteria (4), and guides compiled by the Centers for Disease Control (17), a condensed form of which is shown in Table 1. Of 100 isolates examined, the following genera and numbers were represented: Cor>'nebacterium (40), Actinomyces (14), Lactobacillus (13), Ar-
1986 Elsevier Science Publishing Co.. l n c
canobacterium (5), Propionibacterium (4), Listeria (3), Nocardia (3), Erysipelothrix ( 1), Mycobacterium ( 1), Streptococcus (2), Gardnerella (1), unidentified (13). Although all of these organisms were included in the study by their characteristics after 24hr growth, the Listeria, Erysipelothrix, two of the Nocardia, and most of the Lactobacillus isolates were not initially thought to be coryneforms because of additional characteristics such as chaining or hemolysis. Here, and as reported elsewhere, (14, 16) the Acti-
nomyces, Arcanobacterium, Mycobacterium and Propionibacterium might have been misidentified as "diphtheroids". Most Corynebacterium isolates could not be identified to the species level. The named groups listed in decreasing order of frequency were Corynebacterium CDC group D-2, C. CDC group JK, C. minutissimus, C. xerosis, and single isolates of C.
striatum, C. pseudodiphthericum, C. aquaticum, C. CDC group G-1 and C. CDC group F-1. As data accumulate, it is increasingly evident that the identification of these gram-positive isolates to genus and sometimes species is clinically relevant. The Nocardia, Erysipelothrix, and Mycobacterium are rarely isolated without being involved in a disease process, As recognition increases, wound infections with Mycobacterium spp. are reported more commonly (14). In contrast, all the other genera that we isolated are considered as part of the normal flora. However, the Ar-
canobacterium, Propionibacterium,
Clinical Microbiology Newsletter 8:5,1986
Vol. 8, No. 5
i
Isolation and Identification of Thermophilic Actinomycetes Associated with Hypersensitivity Pneumonitis Gary E. Hollick, Ph.D. Director Microbiology Laboratory Rochester General Hospital Rochester, New York 14621 The thermophilic actinomycetes were among the first organisms to be implicated as sensitizing agents in the development of hypersensitivity pneumonitis. The disease can affect farm, office, factory workers, and others who come into contact with large numbers of mycelia and spores from these organisms. Acute disease is induced by repeated inhalation of mycelia and spores of fungi or actinomycetes, as well as by inhalation of various types of organic dusts. Cough, dyspnea, and fever characteristically begin within 4 to 6 hr following exposure, with resolution of symptoms within 18-24 hr. Chronic disease develops following repeated acute episodes or long-term, low-dose exposure. This chronic form of the disease is characterized by cough, dyspnea, weight loss, airway obstruction, and, ultimately, irreversible lung fibrosis. Depending upon the nature and source of the sensitin, different names have been given to the same disease (Table 1).
Diagnosis The accurate diagnosis of hypersensitivity pneumonitis depends upon a compatible clinical history, lung biopsy findings, and results of radiographic
CMNEEJ8(5)29-36,1986
and pulmonary function tests. Serologic tests have been developed but have met with several inherent problems. First, approximately 3-12% of healthy persons may possess precipitating antibody directed against the thermophilic actinomycetes (2, 9, 19). Second, Wenzel et al. (21) have found that 44.2% of patients with positive precipitin tests reacted to more than one thermophilic actinomycete. This finding has been subsequently confirmed by other investigators (1, 11). Once a diagnosis is made, however, a source should be sought because the only effective treatment is exposure avoidance. A discussion of methods for isolating and identifying thermophilic actinomycetes obtained from environmental sources follows.
report has included a large sample of environmental sources; Kurup et al. (13) were able to isolate one or more thermophilic actinomycetes from 45% of 273 samples. Sources included house dust, furnace filters, and air conditioners. To date, no reports have correlated the number of organisms present in an environmental source to the development of disease.
Isolation The suspected source of exposure determines the type of sample that should be collected. Air samples can be taken by exposure plates or by one of a variety of mechanical samplers. Water from air conditioning systems or humidifiers can be plated directly or i
Prevalence Thermophilic actinomycetes can be isolated from water (3), air (8, 17), soil and compost (3), home and industrial air conditioning systems (4, 13), house dust (13), hay (18), and bagasse (16). Reports of actual counts of these microorganisms in natural habitats have been few. Numbers have ranged from 1 × 103/g from surface peat to 3 × 108/g in baled hay (3). Air surveys, taken when moldy hay was manipulated, produced concentrations of spores approximating 1.6 × 109/m3 (17). Cigarette tobacco has been found to contain 1.5 × 104to 1 × 105 colonies of actinomycetes/g (15). Only one
Elsevier
In T h i s Issue
Thermophilic Actinomycetes . . . . The isolation and laboratory characterization of agents responsible for hypersensitivity pneumonitis
29
Characterization of Coryneforms 32 Importance of properly identifying "diphtheroid-like" organisms recovered from clinical specimens Branhamella Septicemia . . . . . . . . An uncommon complication of Branhamella pneumonia
34
0196-4399/86/$0.00 + 02.20
Table 1 Selected Diseases of the Hypersensitivity Group Disease
Source o1 sensitin
Air conditioning lung
ventilation ducts
Bagassosis
moldy sugar cane
Farmer's lung
moldy hay
Humidifier lung
humidifier water
Mushroom worker's lung
moldy compost
hnpli~'ated actinom3 cete S. viridis" T. vulgaris ~' T. candidus 7'..sacchari T. vulgaris M..[~teni' T. vulgaris T. candidus T. vulgaris T. candidus M. fiwni T. vulgaris T. candidus
Saccharomonospora. b Thermoactinomyces. '" Micropo/yspora.
passed through a filter which is subsequently placed onto the agar surface. Other samples, such as pieces of furnace filter, house dust, or hay can be extracted in saline, diluted, then plated onto appropriate media. Treuhaft and Jones (20) described a method in which the sample is placed into sealed plastic bags. The bag is vigorously shaken, a corner removed, and the aerosolized dust puffed out above an open plate. This method was found to be comparable to spread plates for the isolation of Micropolyspora faeni and Saccharomonospora viridis. Optimal isolation of Thermoactinomyces candidus required the use of spread plates. Because of the multitude of other organisms present in some samples, it may be necessary to subject the sample to heat shock (100°C for 10 min) or incorporate 30 I,zg/mL of novobiocin and 400 txg/mL of cycloheximide into the plating media. Most thermophilic actinomycetes grow well on trypticase soy agar (TSA) or TSA supplemented with 1% yeast extract. Thermoactinomyces sacchari and some isolates of Thermoactinomyces vulgaris, however, grow poorly on these media, hence, half strength
nutrient agar should be included in the battery of primary isolation media. Novobiocin and cycloheximide can be added to these media to inhibit overgrowth by other organisms without deleterious effects on isolation of Thermoactinomyces species. M. faeni and S. viridis, however, will not grow on media containing these antimicrobial agents. Plates should be taped shut or placed into sealed plastic bags to avoid excessive moisture loss, and incubated at 50°C for 1 to 3 weeks.
Macroscopic and Microscopic Characteristics Colony morphology, color, growth rate, and the presence of aerial mycelia are important characteristics in the preliminary identification of thermophilic actinomycetes. Gram-stain preparations of colonies are useful for determining the size and arrangement of spores. T, candidus grows on most media within 3 - 5 days as a flat colony covered with a fine white aerial mycelium. Single spores ( 1 - 2 I~m) are borne directly from the mycelia or upon short stalks. Spores are produced on both substrate and aerial mycelia. Macroscopically and microscopically, T.
vtdgaris appears similar to T. candidus. Some isolates may require hall'strength nutrient agar for growth. T. sacchari grows poorly on most media. Colonies are buff colored and few, if any, aerial mycelia are present. When they are produced, the aerial mycelia rapidly undergo autolysis giving the colony a bacterial appearance. When present, spores are produced singly on short sporophores. S. viridis grows well on most media as a white colony that changes to blue-green after sporulation. Occasional isolates may produce a dark, diffusable pigment alter prolonged incubation. Spores are produced singly on dichotomously branched sporophores and are formed only on the aerial mycelia. M. j~teni grows slowly as a yellow, heaped colony which, after 3 - 7 days, may develop patches of fine white aerial mycelia. Chains of spores, 5 20 in number, are formed on aerial and substrate mycelia.
Biochemical Characteristics A composite of the biochemical tests and morphologic criteria needed for presumptive identification is given in Tables 2 and 3. The formulations of the media and interpretations of test resuits can be found elsewhere (7, 10, 12, 14). Thermoactinomyces species are unitbrmly resistant to heat and novobiocin. This serves to differentiate the genus from similar organisms such as Thermomonospora and Streptomyces species. T. candidus is characterized by its ability to decompose esculin and an inability to hydrolyze starch and tyrosine, whereas the morphologically similar T. vulgaris decomposes tyrosine and starch. The unique colony morphology, along with the inability to decompose tyrosine and hypoxanthine, characterize T. sacchari. M. j~leni is differentiated from similar organisms by the inability to decompose casein and the ability to liquify gelatin. Isolates of S. viridis are casein positive and most strains are able to decompose tyrosine and esculin. ii
Clinical Microbiology Newsletter (ISSN 0196-4399) is issued twice monthly in one indexed volume by Elsevier Science Publishing C o . Inc, 52 Vanderbill A'~enue, New York, NY I(KII7 Printed in the USA at 579 Colombia Turnpike, Rensselaer. NY 12144. Subscription prices per ycar: $70.00 including postage and handling in the United States. Canada, and Mexico Add $ 2 5 0 0 for postage (aimqail) in Europe and $ 2 8 0 0 in the rest of the world. Second-class postage pending Postmasler: send address changes to Clinical MicrobioIog~ Ne~sletter, Elsevier Science Publi,hing Cc~ Inc. 52 Vandcrhd[ A~enue, New Ynrk, NY 10017
30
0196-4399/86,S0.00 + 02.20
t 1986Elsevier Science PublishingCo.. lnc
Clinical MicrobiologyNe'~sletter8:5.1986
Table 2 Biochemical and Morphological Characteristics of the Thermoactinomyces Species T.
Proper~
T. candidus
intermedius
Decomposition of: casein tyrosine xanthine hypoxanthine esculin
+ . +
+ +
Hydrolysis of: starch gelatin
T.
T. vulgaris + +
.
.
T. putidus
T. sacchari
dichotomica
+
+ -
+ -
+
-
.
.
+
-+ -
+
+
+ +
+
+ +
+ +
Resistance to: heat novobiocin
+ +
+ +
+ +
+ +
+ +
+ +
Morphology: colony color
white
white
white
white
yellow
spores aerial hyphae
single +
single +
single +
single +
colorless to white single ---
Table 3 Characteristics of
+
determining the source can impact significantly on patient m a n a g e m e n t . The isolation and identification o f thermophilic a c t i n o m y c e t e s is relatively simple and within the capabilities o f most clinical laboratories.
References 1. Barboriak, J. J., J. N. Fink, and G. Scribner. 1972. Immunologic cross reactions of thermophilic actinomycetes isolated from home environments. J. Allergy Clin. lmmunol. 49:81-85.
2. Chmelik, F., D. K. Flaherty, and
single +
Micropolyspora and Saccharomonospora Species
Proper~
M icropolyspora faeni caesis
Decomposition of: casein tyrosine xanthine hypoxanthine starch gelatin esculin
_ -+ + + +
Resistance to: heat novobiocin
. .
Morphology: colony color
yellow
length of spore chain
5-16
Using a c o m m e r c i a l l y available system ( A P I - Z Y M E , Analytab Products Inc., Plainview, N Y ) , we were able to generate unique e n z y m e profiles useful for p r e s u m p t i v e identification (5). In addition, we have found g a s - l i q u i d c h r o m a t o g r a p h y to be useful for separating Thermoactinomyces from Thermomonospora (6).
Clinical Microbiology Newsletter 8:5,1986
Saccharomonospora internatus viridis
+ + + + + . .
. . white to blue-green 1-2
+ + + -
+ _+ + + _+
colorless to blue-green I-6
blue-green
. .
1
The list of agents capable o f inducing hypersensitivity pneumonitis continues to expand. Isolating and identifying organisms k n o w n to induce disease, together with results of serologic tests and clinical history, can help determine a specific sensitin and, at times, a specific source. Because the only effective treatment is exposure avoidance,
© 1986 Elsevier Science Publishing Co., Inc.
C. E. Reed. 1975. Precipitating antibodies in office workers and hospitalized patients directed towards antigens causing hypersensitivity pneumonitis. Am. Rev. Respir. Dis. 111:201-205.
3. Cross, T., and D. W. Johnston. 1971. Thermoactinomyces vulgaris II. Distribution in natural habitats, pp 315-330. In A. W. Baker, G. W. Gould, and J. Wolf (eds.). Spore research. Academic Press, New York.
4. Fink, J. N., A. J. Resniek, and J. Salvaggio. 1971. Presence of thermophilic actinomycetes in residential heating systems. Appl. Microbiol. 22:730- 731. 5. HolUck, G. E. 1982. Enzymatic profiles of selected thermophilic actinomycetes. Microbios 35:187-196. 6. Hollick, G. E. 1982. Differentiation of Thermoactinomyces candidus and Thermoactinomyees vulgaris from Thermomonospora fusca by use of gas-liquid chromatography. Actinomycetes 16:113-121.
7. Hollick, G. E., and V. P. Kurup. 1983. Isolation and identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis. Lab. Med. 14:39-44. 8. Hollick, G. E. et al. 1980. Aerobiology of industrial plant air systems: fungi and related organisms. Zentralbl. Bakteriol. 8 (Suppl. B):89-95. 9. Kawai, T. et al. 1973. Precipitating antibodies against organic dust antigens in human sera by counterimmunoelectrophoresis. Chest 64:420-426. 10. Kurup, V. P. 1981. Thermophilic actinomycetes associated with hypersensitivity pneumonitis. Science-Ciencia 8:5-10. 11. Kurup, V. P. et al. 1976. Immunologic cross reactions among thermophilic actinomycetes associated with hypersensitivity pneumonitis. J. Allergy Clin. Immunol. 57:417-421. 12. Kurup, V. P°, and J. N. Fink. 1975. A scheme for the identification of thermophilic actinomycetes associated with hypersensitivity pneumonitis. J. Clin. Microbiol. 2:55-61.
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13. Kurup, V. P., J. N. Fink, and D. M. Bauman. 1976. Thermophilic actinomycetes from the environment. Mycologia LXVIIl:662-666. 14. Kurup, V. P., G. E. Hollick, and E. Pagan. 1980. Thermoactinomyces intermedius: a new species of amylase negative thermophilic actinomycetes. Science-Ciencia 7:104- 108.
15. Kurup, V. P. et al. 1983. Allergenic fungi and actinomycetes in smoking materials and their health implications. Mycopathologia 82:61-64.
16. Lacey, J. 1971.77zermoactmomyces sacchari sp. nov., a thermophilic actinomycete causing bagassosis. J. Gen. Microbiol. 66:327-338. 17. Lacey, J., and M. E. Lacey. 1964. Spore concentrations in the air of farm buildings. Trans. Br. Mycol. Soc. 47:547-552. 18. Pepys, J. et al. 1963. Farmer's lung. Thermophilic actinomycetes as a source of farmer's lung hay antigen. Lancet ii:607-611. 19. Roberts, R. C., D. P. Zais, and D. A. Emanuel. 1976. Frequency of
precipitins to trichloroacetic acidextractable antigens from thermophilic actinomycetes in farmer's lung patients and asymptomatic farmers. Am. Rev. Respir. Dis. !14:23-28. 20. Treuhaft, M. W., and M. P. A. Jones, 1982. Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust. J. Clin. Microbiol. 16:995-999.
21. Wenzel, F, J. et al. 1974, Serologic studies in farmer's lung. Precipitins to the thermophilic actinomycetes. Am. Rev. Respir. Dis. 109:464-468.
Editorial
When, Why, and How Far Should Coryneforms be Identified? Jill E. Clarridge, Ph.D.
Chief, Microbiology Section Laboratory Service Veterans Administration Medical Center Houston, Texas 7721l Coryneforms are a group of bacteria that presumably resemble Corynebacterium diphtheriae in morphology. Most coryneforms are not associated with disease and are found as endogenous human flora. Because it is difficult to distinguish species within this group, clinical laboratories sometimes report "gram-positive pleomorphic rods," "Corynebacterium spp.," "coryneforms," or "diphtheroids" on the basis of Gram stain, colony morphology, and perhaps, a catalase test. Although we recognized the expediency of such a process, we questioned the accuracy. If coryneforms are defined as grampositive, non-acid-fast, asporogeneous, pleomorphic rods that are usually nonanaerobic and nonbranching, many genera having no taxonomic relatedness can be included in this group (1). On the other hand, if the group is limited to those with a "club-shape" during at least part of the life cycle, some of the true Corynebacteria spp. would be excluded and Cellulomonas,
Oerskovia, Arthrobacter, Brevibacterium, Microbacterium, and Curtobacterium, organisms that are generally found in soil or food, would be
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included (5). Mycobacterium, Nocardia, and Rhodococcus might be placed in the group if acid-fast stains are not done and branching is not evident. The strains of Actinomyces and Propionibacterium that are not strict anaerobes could also be included. In the broader definition, one would also include Listeria and Erysipelothr&. With this large number of organisms to consider, we decided to identify more completely clinical isolates that had a coryneform morphology (clubshaped, pleomorphic, and/or small regular and/or slightly branching grampositive rod) on Gram stain and that grew aerobically or in 6% CO 2 at 35°C on blood or colistin-nalidixic acid agar plates (3). Only organisms that were numerically significant were selected. For urine isolates this meant specimens with >104 organisms/mL and no more than one other species present. For other specimens, the coryneform organism had to appear on original plates as the predominant organism (ten times more than the next most common isolate) or copredominant with another species. Any isolate recovered from blood culture was considered potentially significant. Using standard methodology, identification was accomplished with the previously published criteria (4), and guides compiled by the Centers for Disease Control (17), a condensed form of which is shown in Table 1. Of 100 isolates examined, the following genera and numbers were represented: Cor>'nebacterium (40), Actinomyces (14), Lactobacillus (13), Ar-
1986 Elsevier Science Publishing Co.. l n c
canobacterium (5), Propionibacterium (4), Listeria (3), Nocardia (3), Erysipelothrix ( 1), Mycobacterium ( 1), Streptococcus (2), Gardnerella (1), unidentified (13). Although all of these organisms were included in the study by their characteristics after 24hr growth, the Listeria, Erysipelothrix, two of the Nocardia, and most of the Lactobacillus isolates were not initially thought to be coryneforms because of additional characteristics such as chaining or hemolysis. Here, and as reported elsewhere, (14, 16) the Acti-
nomyces, Arcanobacterium, Mycobacterium and Propionibacterium might have been misidentified as "diphtheroids". Most Corynebacterium isolates could not be identified to the species level. The named groups listed in decreasing order of frequency were Corynebacterium CDC group D-2, C. CDC group JK, C. minutissimus, C. xerosis, and single isolates of C.
striatum, C. pseudodiphthericum, C. aquaticum, C. CDC group G-1 and C. CDC group F-1. As data accumulate, it is increasingly evident that the identification of these gram-positive isolates to genus and sometimes species is clinically relevant. The Nocardia, Erysipelothrix, and Mycobacterium are rarely isolated without being involved in a disease process, As recognition increases, wound infections with Mycobacterium spp. are reported more commonly (14). In contrast, all the other genera that we isolated are considered as part of the normal flora. However, the Ar-
canobacterium, Propionibacterium,
Clinical Microbiology Newsletter 8:5,1986