Thermophilic Actinomycetes from Dust

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1982, p Vol. 16. No /82/ $2./ Copyright 1982, American Society for Microbiology Comparison of Methods for Isolation and Enumeration of Thermophilic Actinomycetes from Dust MARY W. TREUHAFT* AND MARCUS P. ARDEN JONESt Marshfield Medical Foundation, Inc., Marshfield, Wisconsin Received 3 May 1982/Accepted 5 August 1982 Thermophilic actinomycetes are the primary sensitizing agents in farmer's lung disease. We compared dilution pour-plate and spread-plate methods for tlheir usefulness in enumerating thermophilic actinomycetes in moldy silage dust and evaluated the ability of a nonquantitative gravity settling technique to recover thermophilic actinomycetes from moldy silage. Spread plates and pour plates yielded similar estimates of total thermophiles. Higher counts were observed on spread plates (P <.5) for Thermoactinomyces candidus, Micropolysporafaeni, and Saccharomonospora viridis. M. faeni and S. viridis were less efficient than T. candidus in breaking through the agar of pour plates to form colonies which could be identified. Coefficients of variability were <1% for the two methods. The relative proportion of organisms recovered by the settling method correlated well with that recovered on spread plates for M. faeni (r =.79), S. viridis (r =.88), and Thermomonospora spp. (r =.79), but not well for T. candidus (r =.28). When sophisticated air-sampling equipment is not available, dilution spread plates of dust washings provide a reproducible method for enumerating a broad range of thermophilic actinomycetes of interest. The gravity settling method is a simple, rapid alternative when isolation is all that is required. The thermophilic actinomycetes (TAs) Micropolyspora faeni, Thermoactinomyces vulgaris, T. candidus, and Saccharomonospora viridis are the major sensitizing agents of farmer's lung disease (12) and have been implicated in other forms of hypersensitivity pneumonitis (1). TAs are characteristic microflora of forage which has undergone spontaneous overheating during storage. Therefore, it is this type of forage which presents a health hazard to a previously sensitized farmer. The ability to identify and enumerate TAs in forage samples is useful not only for research studies, but also in identifying forage that presents a health hazard to a sensitized individual and in identifying the offending microorganism in forage known to have caused disease. The currently recommended method for quantitating TAs in moldy forage is to shake the forage in a wind tunnel or settling chamber and sample the liberated dust with an Andersen viable sampler (7, 8). This approach has been useful in a number of studies (1, 4-6, 9, 11). However, the equipment is expensive and not commonly available to many research and clinical laboratories. As an alternative approach, we collected dust from moldy forage and compared t Present address: Medical Research Council Unit for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School, London W.12, England. the efficiency of the pour-plate and spread-plate methods for the isolation and enumeration of TAs from serial dilutions of the dust. A nonquantitative gravity settling technique was also evaluated as a simple and rapid method to isolate TAs from dust or forage. MATERIALS AND METHODS Dust collection. Moldy hay silage samples were obtained from the tops of silos on dairy farms in central Wisconsin. Dust collection utilized clean, but not aseptic, equipment. The haylage was shaken in a plastic chamber (15 by 2 by 28 cm), and the liberated dust was drawn through two layers of polyester screen fabric, 1 XX mesh (Sax Arts and Crafts, Milwaukee, Wis.) and collected in the paper bag of a vacuum cleaner (Tornado; Breuer Electric Mfg. Corp., Chicago, Ill.). Dust was stored at 4 C until analyzed. To determine dry dust weight, samples were dried over anhydrous calcium sulfate under vacuum. Spread-plate method. Dust (2 to 1 mg) was suspended in 1 ml of quarter-strength Ringer solution (4 mm NaCl, 1.5 mm KCI, 1.1 mm CaCl,.5 mm NaHCO3,.5% gelatin, [ph 7]), an isotonic diluent, and mixed by blending in a Vortex mixer for 2 min. Triplicate.2-ml portions of serial 1-fold dilutions were pipetted onto the surface of nutrient agar (Oxoid USA, Inc., Columbia, Md.) plates containing 5,ug of cycloheximide per ml to suppress fungal growth and evenly distributed with glass spreaders. Plates were allowed to dry at room temperature for 36 to 48 h to minimize the growth of spreading bacteria before incubation at 55 C. Plates were examined at 4, 7, and Downloaded from on May 8, 218 by guest 995

2 996 TREUHAFT AND ARDEN JONES 1 days for colony formation. Mean colony counts were determined, corrected for the moisture content of the sample, and expressed as colony-forming units per milligram of dry weight of dust. Pour-plate method. Triplicate.5-mi portions of the dust suspension were pipetted into petri dishes followed by 2 ml of molten nutrient agar with swirling to effect mixing. Plates were allowed to dry at room temperature for 36 to 48 h before incubation at 55 C. Gravity settling plate method. Small plastic bags containing dust or moldy haylage were shaken in a hood. A corner was cut off the bag, and the aerosolized dust was puffed out 5 to 1 cm above open nutrient agar plates. Plates were incubated at 55 C. Between samples, air in the hood was exhausted for 3 min, and surfaces were wiped with 7% isopropyl alcohol. Microbial identification. TAs were identified by colony and microscopic features by the criteria of Cross and Goodfellow (2). T. candidus and T. vulgaris were identified, using a combination of morphological and biochemical characteristics. Cross and Unsworth (3) have reported that T. candidus colonies have a white aerial mycelium and a cream-colored reverse and that T. i'ulgaris colonies have a cream, pale-grey, or lightbrown aerial mycelium with a pale-brown to darkbrown reverse. We were able to verify this observation with our stock Thermoactinomyces cultures growing on Oxoid nutrient agar. We additionally observed that T. candiduis colonies were characteristically larger (to 15 mm) with a spreading appearance compared with the more restricted T. vulgaris colonies (<1 mm). The use of colony reverse and size to differentiate the two species was verified by routinely evaluating randomly selected T. candidus colonies from isolation plates for the ability to hydrolyze esculin and arbutin and for the inability to produce melanoid pigments from tyrosine or to hydrolyze starch. Likewise, randomly selected T. iulgaris colonies from isolation plates were regularly evaluated for the inability to hydrolyze esculin and arbutin and for the ability to produce melanin and hydrolyze starch. We wish to point out that Cross and Unsworth (3) have suggested that T. iulgaris is the appropriate name for the organism referred to here as T. candidus and that T. thalpophilius is the appropriate name for the organism we refer to here as T. vulgaris. Bacteria other than Actinomvcetales were not identified further and are referred to here simply as bacteria. Statistical analysis. Arcsine transformations were performed on percentage data, and log transformations were performed on ratio data before statistical analysis. Coefficients of variation, correlation coefficients, Student's t statistics for matched pairs, and factorial analysis of variance were calculated by standard methods (13). RESULTS TA species recovered from haylage dust. The predominant TAs isolated from these moldy haylage dust samples were T. candidus, M. faeni, S. viridis, and Thermomonospora spp. These and other TA species exhibited characteristic and reproducible morphology on the surface of agar plates and could be easily identified J. CLIN. MICROBIOL. by colony size, color of aerial mycelium, and color of colony reverse. Colonies growing within the agar of pour plates which did not break through the surface were not identified. M. faeni colonies grew as small (<3 mm in diameter), compact colonies that were initially colorless but later became yellow to orange. S. viridis colonies appeared similar but were more domed, some developing a white aerial mycelium which turned blue-green or gray-green upon maturation of the spores. A dark-green or blackish soluble pigment was sometimes liberated into the medium. T. acndidus grew as planar, spreading (to 15 mm) colonies with a white aerial mycelium extending to the edge of the colony. The reverse was colorless to cream. T. vulgaris was isolated only rarely (4/22 samples) and was never abundant. Colonies were more restricted than those of T. candidus (<1 mm) and were planar with off-white aerial mycelia and light- to dark-brown reverses. We occasionally isolated an additional Thermoactinomyces sp. which grew as a colorless to beige colony with little or no surface or aerial mycelium. Thermomonospora spp. grew as off-white to cream-colored colonies (<7 mm) with a raised center and a granular halo of speckles of sporulating aerial hyphae. Most conformed morphologically to descriptions of Thermomonospora fisca. Streptomyces thermoviolaceus colonies grew to 15 mm. The white aerial mycelium spreading from the center matured to gray, often with a lavender hue. The reverse was dark in mature colonies. White Streptomyces strains were occasionally isolated. Actinomadlura flexuiosa was isolated from several samples and appeared as yellowbrown to dark-brown, smooth, raised colonies, occasionally with a sparse whitish aerial mycelium. Occasional colorless, nonsporulating actinomycete colonies occurred which could not be further identified. Comparison of methods for recovery of TAs from dust. Although TAs grow well at 5 to 55C, moldy forage often contains spreading thermophilic Bacillus spp. which rapidly overgrow culture plates at these incubation temperatures. Our approach of examining dust, rather than washings of the forage, reduced this problem considerably. We had previously observed that incubation at 6 C inhibits the growth of these spreading bacteria. Therefore, we compared the ability of spread-plate and pour-plate methods to recover TAs at 55 and 6 C. M. faeni, S. viridis, and Thermomonospora spp. were recovered only at 55 C by both methods. T. candidus was isolated at both temperatures, but analysis of variance of five replicates (data not shown) revealed that higher numbers were recovered at 55 C than at 6 C (P <.1) and that higher numbers were recovered by the Downloaded from on May 8, 218 by guest

3 VOL. 16, 1982 spread-plate method than by the pour-plate method (P <.1). There was no significant interactive effect between temperature and method for T. candidus recovery. On the basis of these observations, 55 C was selected as the optimum temperature for the recovery of TAs from dust. The spread-plate, gravity settle-plate, and pour-plate methods were evaluated for their ability to recover TA species from multiple dust samples at 55 C (Table 1). The spread-plate and settle-plate methods were equally effective in recovering all of the predominating TA species, whereas the pour-plate method was less effective in recovering M. faeni and S. viridis. Comparison of methods for enumeration of TAs. The spread-plate and pour-plate methods were compared for their usefulness in enumerating TAs in dust. The results are presented graphically in Fig. 1. Both methods estimated similar numbers of total thermophilic microorganisms. However, the spread-plate method estimated greater numbers of T. candidus (P <.5), M. faeni (P <.1), and S. i'ridis (P <.25). This is because only an average of 5% of the colonies in pour plates broke through the agar surface to form colonies which could be identified. Coefficients of variability for the estimation of T. candidus, M. faeni, and S. i'ridis were less than 8% for both methods (n = 9 for the spread-plate method, n = 11 for the pour-plate method), indicating good reproducibility. The differences in the spread-plate and pour-plate estimates were most pronounced for M. faeni and S. viridis, which appeared to be less efficient than T. candidus in breaking through the agar surface. Thermomonospora spp. and T. vulgaris were isolated less frequently and in low numbers; therefore, no conclusions are made about enumerating these organisms. Gravity settling method. Because the gravity settling method was as effective as the spreadplate method in recovering a broad range of TA species from dust, we were interested in determining whether the populations of organisms TABLE 1. Recovery rates of TAs from dust samples by spread-plate, settle-plate, and pour-plate methods at 55 C T. M. S. Thermocandidus faeni viridis monospora Spread plate looa (n = 22) Settle plate (n = 22) Pour plate (n = 1) a Percentage of samples examined showing growth. ISOLATION AND ENUMERATION OF TA FROM DUST 997 recovered by the two methods were similar. Each species was counted separately, and its ) D LL 17 Total Thermophilic Microorganisms - I L 17 Thermoactinomyces candidus relative abundance was determined as a percentage of the total number of thermophiles recov o io 14 r Micropolyspora IL faeni idis 17 Sa( I II I T.hermomonospora Thermomonospora p Iii X CK AR BJ AH BW CB BS AD B Sample FIG. 1. Comparison of spread-plate and pour-plate methods for enumeration of TAs in dust. The solid line represents results from spread plates. The hatched line represents results from pour plates. CFU, Colonyforming units. Downloaded from on May 8, 218 by guest

4 998 TREUHAFT AND ARDEN JONES TABLE 2. Comparison of spread-plate and settleplate methods for characterizing relative abundance of TA species as percentage of total thermophiles recovered Correlation Increase by Organism n and settle-plate ad method o) significance M. faeni ± 7.6 P <.1 P <.25 T. candidus NSb S. viridis ± 1.8 P <.1 P <.5 Thermomonospora ± 1.2 spp. P <.1 P <.5 Bacteria 15.3 NS D from matched-pair Student t test ± standard deviation. b NS, Not significant, P <.5. ered. Correlation coefficients for the relative percentages offrequently isolated species recovered by the two methods are presented in Table 2. Good agreement and correlation were observed for estimating the percentages of total thermophiles which were M. faeni, S. viridis, and Thermomonospora spp. Poor correlations were observed for T. candidus and thermophilic bacteria. Diagrammatic presentation of the relative percentages of M. faeni and S. viridis recovered by the two methods is presented in Fig. 2. Coefficients of variability varied from sample to sample for both the spread-plate method (n = 4, 4, 3) and the settle-plate method (n = 5, 4), but were similar for both methods and ranged from 17 to 3% for T. candidus, 5 to 37% for M. faeni, 24 to 27% for S. viridis, and 22% for Thermomonospora spp. DISCUSSION The purpose of this investigation was to evaluate methods for the recovery and enumeration of TAs from forage which do not require expensive air-sampling equipment and which can be easily performed in most microbiology laboratories. A summary of our findings is presented in Table 3. We found the use of dilution spread plates of dust washings to be a reproducible method for enumerating a broad range of TAs. Spread plates recovered M. faeni and S. viridis more frequently than did pour plates. In addition, spread plates yielded higher numbers of identifiable colonies of T. candidus, M. faeni, and S. viridis. The gravity settle-plate method is a simple, rapid alternative when isolation and identification are all that is required. It was as effective as the spread-plate method in recovering TAs from a Saccharomonospora viridls r =.88 ~~~~P<.1. 1oo Ib Settle - Percent FIG. 2. Correlation between estimates of relative abundance of M. faeni and S. viridis obtained by spread-plate and settle-plate methods. dust samples and yielded similar proportions of M. faeni and S. viridis, although not of T. candidus. T. candidus, M. faeni and S. viridis were the predominant organisms isolated from the dust of the moldy haylage samples examined in this study. Although Thermomonospora spp. and T. vulgaris were isolated less frequently and in lower numbers, we predict that the spread- and settle-plate methods will also be useful in characterizing dusts in which these organisms predominate. In this study, we found the dilution pour-plate TABLE 3. J. CLIN. MICROBIOL. Comparison of methods for evaluating TAs in dust No. of Method Recovery Quantiof species tation Set-up time plates per sample Spread Good + Intermediate Settle Good - Shortest 1-3 Pour Fair + Longest I Downloaded from on May 8, 218 by guest

5 VOL. 16, 1982 ISOLATION AND ENUMERATION OF TA FROM DUST 999 method to be useful in characterizing the TA microflora of moldy forage dust. This method, in combination with portable, personal air-sampling pumps fitted with dust-collecting filters, may provide an inexpensive and practical approach to determining levels of airborne TAs. The viable air-sampling equipment currently available has the disadvantages of being expensive and easily overloaded so that sampling for only a fraction of the total exposure period is practical. In contrast, personal air-sampling pumps with conventional 5-[Lm nuisance dust filters are less expensive, are readily available to industrial hygienists, and can be used to sample dust over the entire work or exposure period. Dilution spread plates of dust washed from the filters should give reproducible, relative estimates of levels of the TAs and fungi which are known to cause hypersensitivity diseases. ACKNOWLEDGMENTS This work was supported by grant HL from the Wisconsin Pulmonary Specialized Center of Research and by the Marshfield Medical Foundation, Inc. We thank John Edwards for originally suggesting the gravity settling or "puff plate" method to us. We also thank Joleen Soukup for excellent technical assistance and Doreen Luepke for assistance in preparing this manuscript. LITERATURE CITED 1. Corbaz, R., P. H. Gregory, and M. E. Lacey Thermophilic and mesophilic actinomycetes in mouldy hay. J. Gen. Microbiol. 32: Cross, T., and M. Goodfellow Taxonomy and classification of the actinomycetes, p In G. Sykes and F. A. Skinner (ed.), Actinomycetales: characteristics and practical importance. The Society for Applied Bacteriology Symposium Series No. 2, Academic Press, Inc., London. 3. Cross, T., and B. A. Unsworth List of actinomycete names: alternative proposals for the genus Thermoac tinomvces, actinomycetes and related organisms. Actinomycetes 12: Gregory, P. H., and M. E. Lacey Mycological examination of dust from mouldy hay associated with farmer's lung disease. J. Gen. Microbiol. 3: Lacey, J The microflora of fodders associated with bovine respiratory disease. J. Gen. Microbiol. 51: Lacey, J The microbiology of moist barley storage in unsealed silos. Ann. Appl. Biol. 69: Lacey, J., and J. Dutkiewicz Methods for examining the microflora of mouldy hay. J. AppI. Bacteriol. 41: Lacey, J., and J. Dutkiewicz Isolation of actinomycetes and fungi from mouldy hay using a sedimentation chamber. J. Appl. Bacteriol. 41: Lacey, J., and M. E. Lacey Spore concentrations in the air of farm buildings. Trans. Br. Mycol. Soc. 47: Roberts, R. C., and V. L. Moore Immunopathogenesis of hypersensitivity pneumonitis. Am. Rev. Respir. Dis. 116: Terho, E. O., and J. Lacey Microbiological and serological studies of farmers' lung in Finland. Clin. Allergy 9: Wenzel, F. J., R. L. Gray, R. C. Roberts, and D. A. Emanuel Serologic studies in farmer's lung. Am. Rev. Respir. Dis. 19: Zar, J. H Biostatistical analysis. Prentice-Hall, Inc., Englewood Cliffs, N.J. Downloaded from on May 8, 218 by guest