JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1982, p. 1061-1065 Vol. 16, No. 6 0095-1137/82/121061-05$02.00/0

Enhancement of Recovery of pneumophila from Contaminated Respiratory Tract Specimens by Heat PAUL H. EDELSTEIN,* JUDITH B. SNITZER, AND JANIS A. BRIDGE Infectious Disease Section, Research and Medical Services, Veterans Administration Wadsworth Medical Center, Los Angeles, California 90073, and University of California, Los Angeles, Los Angeles, California 90024 Received 6 July 1982/Accepted 21 September 1982

Heating of and other Legionella spp. was studied to determine whether this technique could be used as a selective technique with contaminated clinical specimens. Studies of 13 different strains of Legionella spp. showed heterogeneous heat survival; heating at 60°C for 1 to 2 min did not affect the survival of the majority of strains. Heating of four strains at 60°C for 2 min reduced bacterial counts by 98% or greater. Enterococci were heat tolerant, with virtually no inhibition under the same conditions. No inoculum effect was noted for any of the organisms tested. Heating of eight contaminated clinical specimens before plating on buffered charcoal-yeast extract medium reduced the numbers of contaminants on most plates but increased by only one the number of specimens yielding L. pneumophila. Plating the same specimens on selective media with or without heat pretreatment yielded L. pneumophila in every case. Heating of clinical specimens at 60°C for 1 to 2 min before plating may occasionally increase the recovery of L. pneumophila from contaminated specimens, but this technique should not be generally used.

Recovery of Legionella pneumophila from positive direct immunofluorescence (DFA) studies (2, clinical samples is often hindered by the pres- 9) but which were overgrown by non-Legionella bacte- ence of other in the samples. Several ria were subjected to heating and other selective selective media have now been found to be techniques to determine whether specimen heating could significantly enhance recovery of L. pneumo- useful in these situations, but a truly selective phila. medium has yet to be developed. We reasoned All Legionella spp. studied, except two, had been that, since L. pneumophila is often recovered previously frozen at -70°C in vials containing skim from hot water (4, 11, 13), the temperature of milk for periods ranging from 1 year to several weeks. which may inhibit other bacteria, heating of The vials were thawed at room temperature, and the contaminated samples might improve the recov- contents were plated on buffered charcoal-yeast ex- ery of L. pneumophila. This study was designed tract medium supplemented with 0.1% a-ketoglutarate to determine the heat sensitivity of clinical and (BCYEa medium) (6). The plates were incubated for environmental isolates of L. pneumophila, the 48 h in a 35°C humidified-air incubator. Growth was heat of some common clinical con- harvested from plates with a sterile wooden stick and sensitivity placed into sterile distilled water in a test tube contain- taminants, and the recovery of L. pneumophila ing sterile glass beads. The tube was mixed in a Vortex from contaminated clinical specimens with heat mixer, and the turbidity was adjusted to that approxi- treatment of samples. mating a one-half MacFarland barium sulfate standard by visual comparison with a standard. Serial 10-fold MATERIALS AND METHODS dilutions of the organism suspension were made with The study was performed over a 1-year period in a sterile distilled water. The dilutions, which ranged series of 33 experiments. The majority of the experi- from 10-8 to 10-3, were plated on BCYEa medium to ments were designed to determine the heat survival of determine actual colony counts. One-milliliter vol- clinical and environmental strains of Legionella spp. umes of the various dilutions were placed in a water and of clinical strains of other organisms which are not bath at specified temperatures for specified periods of inhibited by the selective medium used in this labora- time. The temperature of the water bath was checked tory (6). These studies were done to determine the during the experiment with a mercury thermometer feasibility of obtaining selective survival of Legionella which had been calibrated with a U.S. Bureau of spp. at elevated temperatures; the optimal tempera- Standards certified thermometer. ture and the effect of inoculum size were also studied. At the end of the specified heating time, the tube Clinical respiratory tract specimens, which were was plunged into an ice-water bath for 15 s and placed known to contain L. pneumophila on the basis of at room temperature (21 to 29°C) until plating. Plating 1061 1062 EDELSTEIN, SNITZER, AND BRIDGE J. CLIN. MICROBIOL. TABLE 1. Percent survival of bacteria heated in distilled water at 60°C for various times Organism and straina Inoculum size Heat exposure time (min) Orgaismandstrin'(CFU/mI xl1O)b 1 2 3 4 L. pneumophila Philadelphia 1 (C) 0.079 37 2 1 0.1 Togus 1 (C) 1.41 100 92 58 9 Bloomington 2 (E) 3.50 100 57 4 0.4 Los Angeles 1 (C) 3.50 91 77 41 22 Dallas 1E (E) 5.00 100 80 41 13 Chicago 2 (C) 1.32 70 48 6 0.4 E230a DPC (E) 2.06 37 2 <0.1 0.1 E235a DPc (E) 1.09 70 38 8 12 L. longbeachaeb (C) 0.65 8 <0.1 <0.1 <0.1 L. gormanjic (E) 0.19 40 1 0.2 <0.1 L. micdadeic (C) 1.93 70 38 13 9 L. dumoffic (C) 1.16 30 <0.1 <0..1 <0.1 L. bozemaniic (C) 2.00 72 18 1 0.1 Pseudomonas aeruginosad 14 0.82 71 0.9 <0.1 0.1 18 0.47 0.9 <0.1 <0.'.1 <0.1 19 0.19 14 2 0. .2 <0.1 24 0.67 17 <0.1 1 0.2 Pseudomonas maltophiliad 4.00 50 0.4 <0.1 <0.1 Enterococcid 5 0.44 100 75 48 <0.1 20 1.18 79 100 100 100 21 1.17 100 100 100 100 23 0.46 100 63 48 41 Proteus mirabilisd 3.60 67 0.2 <0.1 <0.1 Coagulase-negative staphylococcusd 0.44 36 2 0.9 0.1 a Letter in parentheses indicates clinical (C) or environmental (E) source of strains. b CFU, Colony-forming units. See text for identification of strain used. d Recent clinical isolates.

of the dilutions of the cooled heated samples was done isolated from a BCYEa plate inoculated with sample within 20 min, using BCYEa medium. The 10-3 dilu- heated for 3 min at 60°C. tion was also plated on 5% sheep blood agar to detect Strain Los Angeles 24, isolated in this laboratory, non-Legionella contaminants. The dilution plates were was used as a representative strain of Legionella incubated at 35°C in a humidified-air incubator for 4 longbeachae. Other strains obtained from the Centers days before reading. for Disease Control were LS-13, the representative The L. pneumophila strains studied were Philadel- strain of Legionella gormanii; TATLOCK, the repre- phia 1, Togus 1, Bloomington 2, Los Angeles 1, Dallas sentative strain of Legionella micdadei; TEX-KL, the 1E, Chicago 2, E230a DP, and E235a DP. Strains Los representative strain of Legionella dumoffii; and Angeles 1, E230a DP, and E235a DP were isolated in WIGA, the representative strain of Legionella boze- this laboratory; the other isolates were obtained from manii. the Centers for Disease Control, Atlanta, Ga. Strains The non-Legionella bacteria studied were recent E230a DP and E235a DP are shower water direct human respiratory tract isolates from different patients plating isolates of L. pneumophila, serogroups 1 and 4, which had been grown on the selective BCYEa medi- respectively; these were the only Legionella spp. um supplemented with cefamandole, polymyxin B, isolates studied which had not been frozen. Strain and anisomycin used in this laboratory (BMPAa medi- E230a DP was isolated from a BCYEa plate inoculated um) (6). These included four strains of Pseudomonas with unheated sample, and strain E235a DP was aeruginosa, four strains of enterococci, and one strain VOL . 16, 1982 HEATING FOR L. PNEUMOPHILA 1063 each of coagulase-negative staphylococci, Proteus mi- the most concentrated or 10-3 dilution (about rabilis, and Pseudomonas maltophilia. These orga- 105 colony-forming units per ml). The actual nisms were passaged twice on BCYEa medium before bacterial counts used are shown in Table 1. study but were otherwise treated in the same way as On the basis of data obtained in the single- the Legionella spp.; specifically, enumeration of the it was decided to use 1-, organisms was done with BCYEa medium, as this strain survival studies, would be the medium used for plating heat-treated 2-, and 3-min exposure times at 60°C for the samples. clinical specimens. All of the clinical specimens Seven respiratory tract specimens were selected for were both culture and DFA positive for L. study with heat pretreatment before plating. Two were pneumophila; six were in serogroup 1 and one sputum specimens, and there was one each of a post- was in serogroup 4. Three were culture positive mortem lung specimen (case 2 [8]), post-mortem lung for L. pneumophila with plating on BCYEa aspirate (7), postmortem pleural fluid, transtracheal medium without the use of selective techniques; aspirate, and endotracheal aspirate. These were speci- all three of these plates contained rare L. pneu- mens which had been positive on DFA testing for L. mophila colonies mixed with other flora. It was pneumophila, serogroups 1 through 4, and which had yielded only heavy growth of non-Legionella bacteria. only with the use of a dissecting microscope that They had been frozen at -70°C for 20 to 32 months these colonies were recognized. Use of BMPAa (mean, 26.6 months) before restudy. The original medium or acid washing or both enabled recov- medium used for isolation for these samples was ery of L. pneumophila from all seven specimens charcoal-yeast extract medium (10). They were without heat treatment. Heat pretreatment did thawed at room temperature and plated in 0.1-ml increase the number of plates which had growth volumes with and without heat pretreatment on of L. pneumophila. With no heat pretreatment, BCYEa and BMPAa media with and without acid 13 of 28 plates (46%) were positive for L. wash pretreatment (1, 6). Heat pretreatment was done pneumophila. With a 1-min exposure, 15 of 28 for 1, 2, and 3 min at 60°C. The acid wash pretreatment (54%) were positive; with 2- and 3-min expo- was performed after the specimen had been heated and then cooled to room temperature. Smears for DFA sures, 17 of 28 (61%) were positive. There was examination were also performed. Two shower water no significant difference between the number of specimens were also pretreated and plated in the same plates positive with no heating and those posi- fashion. L. pneumophila was identified by standard tive with heating for 3 min (P > 0.20 by chi- techniques (14). Survivors of heat pretreatment, other square analysis). When growth only on BCYEa than L. pneumophila, were also identified. medium was considered, there was no difference Statistical analysis. Percent survival was calculated in plates positive for L. pneumophila with or by comparing the results of dilution plating before and without heat pretreatment. Heat pretreatment at after each experimental manipulation. One-way analy- all exposure times did increase the numbers of sis of variance was used to compare survival at each inoculum size (5). L. pneumophila on the BCYEa plates as well as decrease the numbers of contaminants. Heat RESULTS pretreatment did not prevent growth of L. pneu- The first set of experiments determined the mophila on the selective plates. Many non-L. survival of various inoculum sizes of Togus 1 (L. pneumophila organisms also survived heating of pneumophila, serogroup 2) at 50, 60, and 70°C. the clinical specimens (Table 2). There was no decrease in colony counts in Heat pretreatment of two environmental spec- suspensions heated at 50°C for up to 10 min, imens plated on BCYEa medium did not in- regardless of inoculum size, which ranged from crease the recovery of L. pneumophila over that approximately 1 x 101 to 5 x 104 colony-forming observed with plating without heat pretreatment units per ml. Survival at 60°C was also indepen- on BMPAa medium alone. It did increase the dent of inoculum size and ranged from 100 to 9% number of BCYEa and BMPAa plates having after 1- to 4-min expogure times, respectively pure growth of L. pneumophila from none with (Table 1). At 70°C, there was less than 1% no heat pretreatment to three with 3 min of heat survival for suspensions heated 1 min or longer, pretreatment. regardless of inoculum size. Because of results obtained with study of the DISCUSSION Togus strain, it was decided to use 60°C for Single-strain heat tolerance studies showed study in all further experiments. The subsequent that some of the Legionella spp. were relatively single-strain heat survival studies were done at resistant to heating at 60°C. There was consider- exposure times of 1, 2, 3, and 4 min with able strain heterogeneity observed, making it inoculum sizes of approximately 103, 104, and unlikely that heat pretreatment of specimens 105 colony-forming units per ml (Table 1). Ex- could be expected to enhance isolation of Le- amination of the inoculum effect by analysis of gionella in all cases. This variable heat tolerance variance showed no significant differences in does not appear to correlate with organism survival at the various exposure times (P = source; three of the five most resistant Legion- 0.13). For this reason, data are shown only for ella spp. were clinical isolates, and two were 1064 EDELSTEIN, SNITZER, AND BRIDGE J. CLIN. MICROBIOL. TABLE 2. Survival of non-L. pneumophila organisms in heated (60°C) clinical specimens plated on BCYEa medium Specimen Isolate Growth at following heating time (min)a no. 0 1 2 3 62 + + + + Coagulase-negative staphylococcus + + + + Yeast + - - - 73 Citrobacter sp. + + + + 154 Viridans streptococcus + Coagulase-negative staphylococcus + 392 Klebsiella sp. + + 669 E. cloacae + + + Viridans streptococcus + + + + Yeast + + Coagulase-negative staphylococcus + + + 714 + + + + Coagulase-negative staphylococcus + + + + Achromobacter sp. + + + + Corynebacterium sp. + + 731 K. pneumoniae + + + + Yeast + + a +, Moderate to heavy growth on plate after indicated exposure time; ±, light growth after indicated exposure time; -, no growth after indicated exposure time. environmental isolates. The same ratio was ob- pected non-L. pneumophila Legionella infec- served for the least heat-tolerant strains. It is tion, BMPAa medium may itself inhibit some impossible to compare these results with those Legionella spp.; this may be another indication of Muller because of major technical differences for the use of alternative selective techniques. (12). It was encouraging that all Pseudomonas Subsequent to completion of this study, we aeruginosa strains tested were significantly in- had occasion to use heat pretreatment of a DFA- hibited (<2% survival) after 2 min of exposure at positive lung specimen which was contaminated 60°C, in contrast to 5 of 13 Legionella spp. with Pseudomonas aeruginosa. Heating the strains significantly inhibited under similar con- specimen for 2 min at 60°C resulted in heavy and ditions. As expected, all of the enterococci pure growth of L. pneumophila, serogroup 4, on tested were heat tolerant; survival at 60°C is the BCYEa and BMPAa plates, whereas both characteristic of enterococci (3). plates were overgrown with Pseudomonas aeru- Heating of clinical and environmental speci- ginosa when inoculated with the untreated spec- mens before plating did not inhibit the recovery imen. Yee and Wadowsky have reported similar of L. pneumophila on selective media. In fact, success with heating under unstated conditions plate purity increased with increased heating for two contaminated clinical specimens (15). time. However, many of the non-Legionella ACKNOWLEDGMENTS bacteria were not significantly inhibited by heat- This work was supported by the Medical Research Service ing of the clinical specimens; the reason for this of the Veterans Administration. is not known in the absence of more detailed We thank Maurice White for reviewing this manuscript and studies of the heat tolerance of the individual Joyce Bullock for manuscript preparation. strains. Thus, for the specimens studied, heat LITERATURE CITED pretreatment added little to the recovery of L. 1. Bopp, C. A., J. W. Sumner, G. K. Morris, and J. G. pneumophila. This technique is therefore not Wells. 1981. Isolation of Legionella spp. from environ- one which should be performed routinely. In the mental water samples by low-pH treatment and use of a case of a specimen which is DFA positive or is selective medium. J. Clin. Microbiol. 13:714-719. from a patient with suspected Legionnaires dis- 2. Cherry, W. B., B. Pittman, P. P. Harris, G. A. Hfbert, B. M. Thomason, L. Thacker, and R. E. Weaver. 1978. ease in which contaminants overgrow BMPAa Detection of Legionnaires' disease bacteria by direct medium, both heat and acid wash pretreatment immunofluorescence staining. J. Clin. Microbiol. 8:329- should be attempted. Also, in the case of sus- 338. VOL. 16, 1982 HEATING FOR L. PNEUMOPHILA 1065

3. Cowan, S. T. 1974. Cowan and Steel's manual for the ford, J. K. Rasheed, D. C. Mackel, and W. B. Baine. 1979. identification of medical bacteria, 2nd ed., p. 51-55. Charcoal-yeast extract agar: primary isolation medium for Cambridge University Press, London. Legionella pneumophila. J. Clin. Microbiol. 10:437-441. 4. Dennis, P. J., J. A. Taylor, R. B. Fitzgeorge, C. L. R. 11. Fliermans, C. B., W. B. Cherry, L. H. Orrison, S. J. Bartlett, and G. I. Barrow. 1982. Legionella pneumophila Smith, D. L. Tlson, and D. H. Pope. 1981. Ecological in water systems. Lancet i:949-951. distribution of Legionella pneumophila. Appl. Environ. 5. Dixon, W. J., and F. J. Massey. 1969. Introduction to Microbiol. 41:9-16. statistical analysis, 3rd ed. McGraw-Hill Book Co., New 12. MOler, H. E. 1981. Die thermostabilitat von Legionella York. pneumophila. Zentralbl. Bakteriol. Parasitenkd. Infek- 6. Edelstein, P. H. 1981. Improved semiselective medium for tionskr. Hyg. Abt. 1 Orig. Reihe B 172:524-527. isolation of Legionella pneumophila from contaminated 13. Wadowsky, R. M., R. B. Yee, L. Mezmar, E. J. Wing, clinical and environmental specimens. J. Clin. Microbiol. and J. N. Dowling. 1982. Hot water systems as sources of 14:298-303. Legionella pneumophila in hospital and nonhospital 7. Edelstein, P. H., and S. M. Finegold. 1979. Isolation of plumbing fixtures. Appl. Environ. Microbiol. 43:1104- Legionella pneumophila from a transtracheal aspirate. J. 1110. Clin. Microbiol. 9:457-458. 14. Weaver, R. E., and J. C. Feeley. 1979. Cultural and bio- 8. Edelstein, P. H., and S. M. Finegold. 1979. Use of a chemical characterization of the Legionnaires' disease semiselective medium to culture Legionella pneumophila bacterium, p. 19-25. In G. L. Jones and G. A. H*bert from contaminated lung specimens. J. Clin. Microbiol. (ed.), "Legionnaires' ": the disease, the bacterium, and 10:141-143. the methodology. Centers for Disease Control, Atlanta, 9. Edelstein, P. H., R. D. Meyer, and S. M. Finegold. 1980. Ga. Laboratory diagnosis of Legionnaires' disease. Am. Rev. 15. Yee, R. B., and R. M. Wadowsky. 1982. Multiplication of Respir. Dis. 121:317-327. Legionella pneumophila in unsterilized tap water. Appl. 10. Feeley, J. C., R. J. Gibson, G. W. Gorman, N. C. Lang- Environ. Microbiol. 43:1330-1334.