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 Legionella 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 Legionella pneumophila 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 Pseudomonas aeruginosa 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 bacteria 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.