Growth of 28 Legionella Species on Selective Culture Media: a Comparative Study

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1993, p /93/ $02.00/0 Copyright X 1993, American Society for Microbiology Vol. 31, No. 10 Growth of 28 Legionella Species on Selective Culture Media: a Comparative Study TZIELAN C. LEE,' RICHARD M. VICKERS,' VICTOR L. YU,1,2 AND MARILYN M. WAGENER2 Veterans Affairs Medical Center' and University of Pittsburgh,2 Pittsburgh, Pennsylvania Received 19 April 1993/Returned for modification 28 May 1993/Accepted 27 July 1993 We compared the growth of 28 LegioneUla spp. on four manufacturers' buffered charcoal-yeast extract (BCYE) agar media and selective BCYE media that contained polymyxin B, anisomycin, and vancomycin or cefamandole. With BCYE as a "gold standard," growth for LegioneUla pneumophila was significantly better than for the nonpneumophila species on all media tested. L. pneumophila and 24 other Legionella spp. grew on vancomycin-containing media, while L. santicrucis, L. rubrilucens, and L. erythra grew poorly. In contrast, 11 of 28 species (notably L. micdadei and L. bozemanii) did not grow on cefamandole-containing media and 8 of 28 species only grew marginally. We demonstrated that selective BCYE media that contain vancomycin or cefamandole may not support the growth of all Legionella spp. One commercial manufacturer's media were consistently suboptimal. Laboratories should not rely on a manufacturer's quality control testing in lieu of their own. Since the discovery oflegionella pneumophila, numerous other Legionella spp. have been identified in the environment and/or shown to be pathogenic for humans (6). The medium used for isolation of Legionella spp. from clinical specimens was originally developed for L. pneumophila. A comprehensive cultural survey was performed to determine the sensitivity of buffered charcoal-yeast extract (BCYE) agar and selective BCYE media for the growth of L. pneumophila and 27 other Legionella spp. We also compared the media from three commercial manufacturers for their ability to support the growth of these 28 Legionella spp. MATERIALS AND METHODS Strains. Seventeen strains of L. pneumophila and 32 strains of other Legionella spp. were evaluated for their ability to grow on Legionella media (Table 1). Media. We compared the growth of Legionella spp. on three different (BCYE and two selective BCYE) media prepared by three commercial manufacturers and the Pittsburgh Veterans Affairs Medical Center special pathogens laboratory (VAMC). Commercial BCYE and selective BCYE with polymyxin B, anisomycin, and either cefamandole (PAC) or vancomycin (PAV) were purchased from (Lenexa, Kans.), (Baltimore, Md.), and Scientific (West Warnick, R.I.) (Table 2). The Scientific PAV also contained glycine. BCYE, PAC, and PAV media were also prepared in-house (VAMC) as previously described (15). The VAMC media were tested for performance with stock strains of L. pneumophila (ATCC 33152), L. micdadei (VAMC 594), Eschenchia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), and Candida albicans (ATCC 14053) prior to use. The three commercial manufacturers' media were not performance tested prior to use. Inoculum preparation and culture procedures. The 28 Legionella spp. (49 strains) were transferred from -70 C storage onto VAMC nonselective BCYE agar plates and incubated at 35 to 37 C in air for 72 h. Colonies from the Corresponding author C culture were then subcultured to VAMC BCYE and grown to confluency at 35 to 37 C in air for 48 h. Growth samples from each 48-h incubation subculture were suspended in 2.0 ml of sterile deionized water with a turbidity that matched a 0.5 McFarland standard. Each standardized suspension was diluted 1/20,000 in sterile deionized water and then inoculated onto duplicate BCYE, PAC, and PAV agar plate media with a Spiral Plater (Model C; Spiral Systems, Inc., Bethesda, Md.). The inoculated plates were incubated at 35 to 37 C in air for 5 days, after which viable counts were determined with a Spiral Systems grid used as recommended by the manufacturer but adapted to an opaque medium. The accuracy of these measurements has been validated previously (11). Comparisons of the 28 Legionella species for all media. A 0.5 McFarland standard should produce an inoculum of approximately 1.5 x 108 CFU/ml. Growth was categorized as good, marginal, poor, or no growth. Growth of.0.75 x 108 CFU/ml (50% of the standard) was defined as good. Growth of <0.75 x 108 CFU/ml was defined as marginal. Growth was defined as poor if no growth occurred on at least one of the manufacturers' media and was <0.75 x 108 CFU/ml on the other media. Strains that did not grow on the selective PAC medium were tested for beta-lactamase activity (12, 13). Data analysis. The mean viable counts (CFU per milliliter) from duplicate plates for each Legionella strain were compared by medium (BCYE, PAC, or PAV) and manufacturer (VAMC, Scientific,, or ). Statistical analysis was done on the PROPHET system (Division of Research Resources, National Institutes of Health). A twoway analysis of variance was used to compare the mean CFU of the 17 L. pneumophila strains with respect to the four manufacturers. In addition, pairwise comparisons (VAMC versus Scientific, VAMC versus, and versus, etc.) of the four media were made by using the Newman-Keuls multiple range test. The Friedman test was used for the non-l. pneumophila strains because of the nonnormal distribution of the results. The Mann-Whitney test was used to compare the overall growth of L.

2 VOL. 31, 1993 TABLE 1. Legionella strains evaluated in this study Strain L. pneumophila L. pneumophila serogroup 2... L. pneumophila serogroup 3... L. pneumophila serogroup 4... L. pneumophila serogroup 5... L. pneumophila serogroup 6... L. pneumophila serogroup 7... L. pneumophila serogroup 8... L. pneumophila serogroup 9... L. pneumophila serogroup L. pneumophila serogroup L. pneumophila serogroup L. pneumophila serogroup L. pneumophila serogroup Non-L. pneumophila L. anisa... L. birminghamensis... L. brunensis... L. bozemanii serogroup 1... L. bozemanii serogroup 2... L. cherrii... L. cincinnatiensis... L. dumoffii... L. dumoffii... L. erythra... L. feeleii serogroup 1... L. feeleii serogroup 2... L. gormanii... L. hackeliae serogroup 1... L. israelensis... L. jamestowniensis... L. jordanis... L. longbeachae serogroup 1... L. longbeachae serogroup 2... L. maceachernii... L. micdadei... L. micdadei... L. moravica... L. oakridgensis... L. parisiensis... L. rubrilucens... L. sainthelensi... L. santicrucis... L. spiritensis... L. steigerwaltii... L. tucsonensis... L. wadsworthii... a, VAMC isolates. ATCC no a GROWTH OF LEGIONELLA SPP. IN CULTURE 2765 TABLE 3. Comparison of growth for 28 Legionella spp. on three Legionella media Growth on mediuma Species BCYE Selective Selective PAC PAV L. anisa Good Good Good L. birminghamensis Marginal NG Marginal L. bozemanii Good NG Good L. brunensis Good Marginal Marginal L. cherrii Marginal Marginal Marginal L. cincinnatiensis Marginal Marginal Marginal L. dumoffli Marginal Marginal Good L. erythra Good NG Poor L. feeleii Good Poor Good L. gormanii Good Poor Marginal L. hackeliae Good Marginal Good L. israelensis Good NG Good L. jamestowniensis Good Marginal Good L. jordanis Good NG Good L. Iongbeachae Marginal Marginal Marginal L. maceachemii Good NG Good L. micdadei Good NG Good L. moravica Good NG Good L. oakridgensis Good Poor Marginal L. parisiensis Good Good Good L. pneumophila Good Good Good L. rubnlucens Good NG Poor L. sainthelensi Good Poor Marginal L. santicrucis Marginal NG Poor L. spintensis Good NG Good L. steigenvaltii Marginal Marginal Marginal L. tucsonensis Good Good Good L. wadsworthii Good Good Good a Good, marginal, and poor are defined in Materials and Methods. NG, no growth. pneumophila and non-l. pneumophila for each type of medium. RESULTS Comparisons of media by Legionella spp. Table 3 summarizes the growth for all 28 Legionella spp. classified by medium type. L. pneumophila grew better than non-l. pneumophila for all the media evaluated. Overall, when comparing the medium and mean CFU, the non-l. pneumophila strains had significantly fewer CFU than L. pneumophila on all three media (Fig. 1). The mean CFU (x 108) for all manufacturers combined were as follows. BCYE had 3.54 L. pneumophila (95% confidence interval of 2.39 to 4.49) and TABLE 2. Formulation of Legionella selective media Medium contents and concna Manufacturer PAC PAV Polymyxin B Anisomycin Cefamandole Catalog Polymyxin Anisomycin Vancomycin Catalog no. (U/ml) (Gg/ml) (Gg/ml) no. B (U/ml) (FG/ml) (Gg/ml) Scientific " VAMC a BCYE medium contains the following ingredients per liter: ACES [N-(2-acetamido)-2-aminoethanesulfonic acid], 10 g; Norit, 2 g; yeast extract, 10 g; alpha-ketoglutarate, 1.0 g; and agar, 17 g. Contains glycine and is intended for environmental cultures.

3 2766 LEE ET AL. J. CLIN. MICROBIOL mean CFU, 10 I- ii Media BCYE VAMC V I o- 0 BCYE PAC PAV Media FIG. 1. L. pneumophila (_) (17 strains) grew better on BCYE and PAV media compared with the 27 other Legionella spp. ( (32 strains). Note that PAC did not readily support the growth of the non-l. pneumophila species. PAV was about equal to BCYE. The combined mean CFU for all manufacturers was used to compare each medium type non-l. pneumophila spp. (95% confidence interval of 1.33 to 2.73) (P < 0.01 on the Mann-Whitney test). PAC had 2.66 L. pneumophila (95% confidence interval of 1.92 to 3.40) and 0.32 non-l. pneumophila (95% confidence interval of 0.09 to 0.55) spp. (P < 0.01 on the Mann-Whitney test). PAV had 3.41 L. pneumophila (95% confidence interval of 2.32 to 4.5) and 1.72 non-l. pneumophila (95% confidence interval of 1.02 to 2.42) spp. (P < 0.01 on the Mann-Whitney test). Six Legionella spp. (L. birminghamensis, L. cherrii, L. cincinnatiensis, L. santicrucis, L. steigerwaltii, and L. longbeachae) failed to show good growth for any medium. Comparisons of media by medium type. (i) BCYE. Growth on BCYE was good for all L. pneumophila strains but varied from marginal to good for the non-l. pneumophila species (Table 3 and Fig. 1). (ii) PAC. The growth of non-l. pneumophila species was poor on PAC when compared with L. pneumophila (Table 3 and Fig. 1). Eleven species (L. birminghamensis, L. bozemanii (serogroup 1 and 2), L. erythra, L. israelensis, L. jordanis, L. maceachernii, L. micdadei, L. moravica, L. rubrilucens, L. santicrucis, and L. spiritensis) failed to grow on the PAC media. Failure of these strains to grow in the presence of cefamandole may have been due to an absence of beta-lactamases. No beta-lactamase activity was found for L. maceachernii, L. micdadei, L. israelensis, and L. spiritensis by the recommended method at room temperature (12, 13). However, L. israelensis and L. spinitensis readily demonstrated beta-lactamase activity when the reaction temperature was increased to 50 to 52 C, whereas L. maceachernii and L. micdadei still tested negative. A temperature-related enhancement of beta-lactamase activity was confirmed by using a different method. Drops of the cephalosporin solution were placed onto Whatman no. 1 filter paper, and growth from BCYE agar of both isolates was rubbed into the moist areas on the filter paper. A red color characteristic of a positive reaction was readily seen within 5 min on the filter paper that was incubated at 35 to 37 C, but the reaction was negative when tested at room temperature (data not shown). (iii) PAV. Growth on PAV was generally good for all L. pneumophila strains but varied from marginal to good for the non-l. pneumophila species (Table 3 and Fig. 1). PAC VAMC PAV VAMC _~~~~~~~~~~~~~~~~ _~~~ l l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ =~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ - - l~~~~~~~~~~~~~~~~~~~~~~~~~~ O 1 2 3m4 5 mean CFU, 10 a FIG. 2. Performance of three types of Legionella media manufactured in-house and by three commercial firms. The performance of the media differed among the manufacturers. Note that selective PAC, which contains cefamandole, was less sensitive than BCYE or selective PAV, regardless of the manufacturer. _, L. pneumophila; M, non-l. pneumophila. Comparisons of media by manufacturer. (i) BCYE media. The means of CFU of L. pneumophila (17 strains) were not significantly different among the VAMC and the three manufacturers (Fig. 2). However, the growth of non-l. pneumophila species (32 strains) was significantly poorer for media manufactured by Scientific (mean CFU, 1.51 x 108) compared with VAMC (mean CFU, 2.21 x 108), (2.14 x 108), and (2.23 x 106) (P < 0.01 by Friedman's test). (ii) PAC media. VAMC (mean CFU, 3.11 x 108) and (3.46 x 108) media gave significantly better yields for L. pneumophila than Scientific (2.19 x 108) and (1.90 x 10 ) (P < 0.01 by analysis of variance [ANOVA]). Non-L. pneumophila strains grew poorly on all PAC media (Fig. 2). Although there were significant differences for the four media by the rank sum test (by Friedman's statistic, P < 0.05), no significant differences in the pairwise comparisons were found. (iii) PAV media. media yielded a higher mean CFU count (4.33 x 108) than (2.92 x 108), Scientific (3.01 x 108), or VAMC (2.72 x 108) media (P < 0.01 by ANOVA) for the L. pneumophila. However, for the non-l. pneumophila strains, PAV yielded a lower mean CFU count (P < 0.01) (Fig. 2). The colony counts showed variation from 0.5 to 1.0 log on all manufactured BCYE media, even though the inoculated suspensions were matched to a 0.5 McFarland standard (data not shown). These discrepancies were possibly attributable to morphological characteristics of the bacteria. Several species, notably, L. hackeliae, L. birminghamensis, L. gormanii, L. santicrucis, L. moravica, and L. sainthelensi, had numerous filamentous structures. These filamentous structures were larger than the usual smaller bacilli of Legionella spp.; therefore, the use of turbidity to standardize the inocula might have been suboptimal.

4 VOL. 31, 1993 Variation in the colony size was observed. BCYE produced the largest colonies. The diameter of L. pneumophila colonies ranged from 3 to 5 mm on all media, while the non-l. pneumophila colonies ranged from 1 to 3 mm. In the marginal and poor categories, colonies of Legionella spp. such as L. feeleii and L. gormanii had diameters of about 1 mm. DISCUSSION The growth of Legionella spp. from clinical specimens requires the use of specialized media which contain nutritional supplements and charcoal. Because L. pneumophila competes poorly with oropharyngeal flora in culture, BCYE agar medium is supplemented with antimicrobial agents to suppress competing flora. The original formulations of media were designed to optimize the growth of L. pneumophila. Numerous species of Legionella have been discovered, and little is known about their ability to grow on the selective BCYE formulations. Therefore, we evaluated the Legionella media prepared by three commercial manufacturers and our laboratory for their ability to support the growth of 27 Legionella spp. other than L. pneumophila. Three types of media were studied. BCYE is a medium that readily supports the growth of all Legionella spp. PAV is a selective BCYE medium supplemented with polymyxin B, anisomycin (a yeast inhibitor), and vancomycin that supports the growth of L. pneumophila and L. micdadei (3). Dyes which allow differentiation of L. micdadei or L. maceachemii (blue colonies) from the other Legionella spp. (green colonies) are also added to PAV (6). PAC is a selective BCYE medium supplemented with polymyxin B, anisomycin, and cefamandole (4). PAC supports the growth of L. pneumophila, but there is little information about growth for the other Legionella spp. As expected, the BCYE medium without antimicrobial agents produced the highest-yield CFUs for L. pneumophila and for the other 27 non-l. pneumophila species. However, six Legionella spp. (L. birminghamensis, L. cherrii, L. cincinnatiensis, L. santicrucis, L. steigerwaltii, and L. longbeachae) failed to achieve good growth (>0.75 x 108 CFU/ ml) on BCYE, PAC, or PAV for any of the manufacturers (Table 3 and Fig. 1). BCYE medium appears to be more suitable for the growth of L. pneumophila than for other Legionella spp. Whether the agar type used affects colony size and numbers (5) or whether the addition of additives such as albumin (9) or CO2 incubation at a concentration of 2.5 to 10% would better support the growth of the non-l. pneumophila spp. warrants further investigation. Although PAV was comparable to BCYE, the PAC media were clearly inferior to BCYE and PAV (Fig. 1 and 2). Eleven Legionella spp. (L. birminghamensis, L. bozemanii, L. erythra, L. israelensis, L. jordanis, L. santicrucis, L. micdadei, L. moravica, L. spiritensis, L. maceachemii, and L. rubrilucens) that grew on PAV failed to grow on PAC, regardless of the manufacturer. Failure to grow on PAC media by a small number of L. pneumophila strains was initially attributed to failure of these strains to secrete beta-lactamase (14); however, 9 of 11 Legionella spp. that did not grow on the PAC medium in the present study demonstrated the presence of beta-lactamase. It was interesting that two of the nine beta-lactamase-producing strains previously reported to be positive with room temperature incubation (1, 2) were negative in our laboratory at room temperature but tested positive at a higher temperature. GROWTH OF LEGIONELLA SPP. IN CULTURE 2767 Testing Legionella strains at 50 to 52 C to detect low-level beta-lactamase activity warrants further investigation. Selective BCYE media containing antimicrobial agents (PAC and PAV) are used to suppress competing bacteria and yeasts contained in clinical samples which could inhibit or mask the presence of Legionella spp. However, cefamandole in PAC inhibited some non-l. pneumophila strains in our study. Thus, laboratories should not use PAC as their sole medium to screen for Legionella spp. On the other hand, we have encountered several patients for whom L. pneumophila was isolated only on PAC media; coexisting Proteus, Providencia, and Serratia spp. were suppressed by cefamandole but grew on BCYE and PAV media. Therefore, we recommend the use of three media, including BCYE without antimicrobial agents as well as PAC and PAV, to provide maximal sensitivity for isolation of L. pneumophila and related species from clinical samples. The performance of the media varied among manufacturers (Fig. 2). When the commercial media were tested without any quality control testing other than that of the manufacturer, it was discovered that the BCYE media were not equally efficacious in supporting bacterial growth. One manufacturer provided two different lots of BCYE that during testing failed to support growth of the L. pneumophila control or other Legionella spp. This same manufacturer had starch and peptones which are not recommended additives to BCYE media listed on the label as proprietary ingredients (4, 7). Although an authoritative source suggests that some commercial media need not be quality controlled in-house (8), our findings and those of others (10) show that all commercial Legionella media must be quality controlled upon receipt by the laboratory prior to use. Given our findings, the efficacy of selective BCYE as the primary isolation medium for non-l. pneumophila spp. requires continued evaluation, especially in the clinical setting. ACKNOWLEDGMENTS We thank Janet E. Stout and John D. Rihs for their critique of the manuscript and Shirley Brinker and Linda Szalla for secretarial assistance. REFERENCES 1. Bercovier, H., A. G. Steigerwalt, M. Derhi-Cochin, C. W. Moss, H. W. Wilkinson, R. F. Benson, and D. J. Brenner Isolation of legionellae from oxidation ponds and fish ponds in Israel and description of Legionella israelensis sp. nov. Int. J. Syst. Bacteriol. 36: Brenner, D. J., A. G. Steigerwalt, G. W. Gorman, H. W. Wilkinson, W. F. Bibb, M. Hackel, R L. Tyndall, J. Campbell, J. C. Feeley, W. L. Thacker, P. Skalt, W. T. Martin, B. J. Brake, B. S. Fields, H. V. McEachern, and L. K. Corcoran Ten new species of Legionella. Int. J. Syst. Bacteriol. 35: Brown, A., J. W. Shonnard, S. J. Geyer, J. Rihs, E. Elder, J. Stout, R. Vickers, and V. L. Yu Coincident infection with Legionnaires' disease bacterium and Pittsburgh pneumonia agent. Lancet ii:1041. (Letter.) 4. Edelstein, P. H Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 23: Edelstein, P. H., and M. A. C. Edelstein Comparison of different agars used in the formulation of buffered charcoal yeast extract medium. J. Clin. Microbiol. 29: Fang, G. D., V. L. Yu, and R. M. Vickers Disease due to Legionellaceae (other than Legionella pneumophila): historical, microbiological, clinical, and epidemiological review. Medicine (Baltimore) 68: Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Langford,

5 2768 LEE ET AL. J. CLIN. MICROBIOL. J. K. Rasheed, D. C. Mackel, and W. B. Baine Charcoalyeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol. 10: Miller, J. M Quality control of media, reagents, and stains, p In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 9. Morrill, W. E., J. M. Barbaree, B. S. Fields, G. N. Sanders, and W. T. Martin Increased recovery of Legionella micdadei and Legionella bozemanii on buffered charcoal yeast extract agar supplemented with albumin. J. Clin. Microbiol. 28: Rodgers, F. G., and A. W. Pasculle Legionella, p In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 11. Stout, J. E., M. G. Best, and V. L. Yu Susceptibility of members of the family Legionellaceae to thermal stress: implications for heat eradication methods in water distribution systems. Appl. Environ. Microbiol. 52: Thornsberry, C., I. L. Gavan, and E. H. Gerlach Cumitech 6, New developments in antimicrobial agent susceptibility testing. Coordinating ed., J. C. Sherris. American Society for Microbiology, Washington, D.C. 13. Thornsberry, C., and L. A. Kiven Beta-lactamase of the Legionnaires' bacterium. Curr. Microbiol. 1: Vickers, R. M., J. E. Stout, L. S. Tompkins, N. J. Troup, and V. L. Yu Cefamandole-susceptible strains of Legionella pneumophila serogroup 1: implications for diagnosis and utility as an epidemiological marker. J. Clin. Microbiol. 30: Vickers, R. M., J. E. Stout, V. L. Yu, and J. D. Rihs Manual of culture methodology for Legionella. Semin. Respir. Infect. 2: Downloaded from on January 9, 2019 by guest