James M. Barbaree

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-μm membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.

Viability ofLegionella pneumophila in choline-free water at elevated temperatures

Sterilization values were determined forLegionella pneumophila in chlorine-free, chlorine-demand-free water at elevated temperatures. These values were calculated from experimentally determined D values of 2500 min, 380 min, 13.93 min, 0.74 min, and 0.45 min at 45°C, 50°C, 55°C, 60°C, and 66°C, respectively. D values, Z value and temperature coefficient do not indicate unusual thermal resistance. Sterilization values, the minimum time required to eliminate an aquatic population ofL. pneumophila at a given test temperature, indicate that temperatures greater than about 65°C may not be necessary for efficient disinfection of potable quality water. These values and monitoring of time and temperature parameters can help predict the efficacy of in situ heat treatment of potable quality waters harboringL. pneumophila.

Tolerance ofLegionella species to sodium chloride

Inhibition by sodium chloride of the growth of 19 strains ofLegionella pneumophila and of 10 strains of otherLegionella spp. was studied. Results from growth in buffered α-ketoglutarate cysteine yeast extract (BAYE) broth containing 0 to 2.0% sodium chloride indicated that 15/19 laboratory strains ofL. pneumophila were capable of growing in 1.0% to 1.5% sodium chloride, whereas 4 strains ofL. pneumophila and 10 strains of 6 other species were not.L. micdadei andL. longebeachae were the most inhibited in BAYE broth, growing only in concentrations of ≤0.5% sodium chloride. These in vitro studies indicate thatL. micdadei andL. longbeachae might be differentiated from other species by their low tolerance to salt in BAYE broth, and thatL. pneumophila may be more tolerant to salt concentrations found in brackish water environments.

Studies on the thermal degradation of the H and M antigens of lyophilized histoplasmin

Lyophilized histoplasmin for the agar gel microimmunodiffusion test has been prepared as a candidate World Health Organization Biological Reference Reagent. It was subjected to elevated temperature for given periods of time and analyzed by the capillary precipitin test and the single radial immunodiffusion test to determine the stability of the H and M antigens. H antigen showed no fall in relative potency when incubated at 48 degrees C for 20 days. M antigen showed a fall in relative potency after storage at 37 degrees C and 48 degrees C, but the extent of the fall was greater in the radial immunodiffusion test than in the capillary precipitin test. Half-lives of the antigens could not be calculated from the Arrhenius equation because the response curves at each temperature followed different kinetics. However, as based on zero time data, M antigen of the lyophilized histoplasma showed a 20% drop in relative potency when stored at -20 degrees C for 2 years. Other analyses suggested that M antigen of liquid histoplasmin stored at 5 degrees C and of lyophilized histoplasma stored at -20 degrees C was degraded at equal rates.