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We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.

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Hartmannella vermiformis, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of Legionella pneumophila and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of H. vermiformis. H. vermiformis cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with L. pneumophila. Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60 degrees C for 30 min and alternatively to 50 degrees C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55 degrees C, and there were no survivors at 60 degrees C. Pretreatment at 50 degrees C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.

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A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.

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Photosynthetic cyanobacteria, heterotrophic bacteria, free-living amoebae, and ciliated protozoa may support growth of Legionella pneumophila. Studies were done with two tap water cultures (WS1 and WS2) containing L. pneumophila and associated microbiota to characterize growth-supporting activity and assess the relative importance of the microbiota in supporting multiplication of L. pneumophila. The water cultures were incubated in the dark at 35 degrees C. The growth-supporting factor(s) was separated from each culture by filtration through 1-micron-pore-size membrane filters. The retentate was then suspended in sterile tap water. Multiplication of L. pneumophila occurred when both the retentate suspension and the filtrate from either culture were inoculated into sterile tap water. L. pneumophila did not multiply in tap water inoculated with only the filtrate, even though filtration did not reduce the concentration of L. pneumophila or heterotrophic bacteria in either culture. Growth-supporting activity of the retentate suspension from WS1 was inactivated at 60 degrees C but unaffected at 0, 25, and 45 degrees C after 30-min incubations. Filtration experiments indicated that the growth-supporting factor(s) in WS1 was 2 to 5 micron in diameter. Ciliated protozoa were not detected in either culture. Hartmannellid amoebae were conclusively demonstrated in WS2 but not in WS1. L. pneumophila multiplied in tap water inoculated with the amoebae (10(3)/ml) and the 1-micron filtrate of WS2. No multiplication occurred in tap water inoculated with the filtrate only. Growth-supporting activity for L. pneumophila may be present in plumbing systems; hartmannellid amoebae appear to be important determinants of multiplication of L. pneumophila in some tap water cultures.

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Earlier field and laboratory studies have shown that Legionella species survive and multiply in the pH range 5.5 to 9.2. Additionally, the technical feasibility of operating cooling towers at elevated alkalinities and pH has previously been documented by published guidelines. The guidelines indicate that these conditions facilitate corrosion control and favor chlorine persistence which enhances the effectiveness of continuous chlorination in biofouling control. This information suggests that control of Legionella species in cooling towers can be accomplished by operating the towers under alkaline conditions. To test this possibility, we collected water samples over a period of months from a hospital cooling tower. The samples were analyzed for a variety of chemical parameters. Subsamples were pasteurized and inoculated with non-agar-passaged Legionella pneumophila which had been maintained in tap water. Correlation of subsequent Legionella growth with corresponding pH and alkalinity values revealed statistically significant inverse associations. These data support the hypothesis that operating cooling towers outside of the optimal conditions for Legionella growth (e.g., at elevated alkalinities and a pH greater than 9) may be a useful approach to controlling growth in this habitat.

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An investigation of the chemical environment and growth of Legionella pneumophila in plumbing systems was conducted to gain a better understanding of its ecology in this habitat. Water samples were collected from hospital and institutional hot-water tanks known to have supported L. pneumophila and were analyzed for 23 chemical parameters. The chemical environment of these tanks was found to vary extensively, with the concentrations of certain metals reaching relatively high levels due to corrosion. The effect of various chemical conditions on L. pneumophila growth was then examined by observing its multiplication in the chemically analyzed hot-water tank samples after sterilization and reinoculation with L. pneumophila. L. pneumophila and associated microbiota used in these experiments were obtained from a hot-water tank. These stains were maintained in tap water and had never been passaged on agar. The results of the growth studies indicate that although elevated concentrations of a number of metals are toxic, lower levels of certain metals such as iron, zinc, and potassium enhance growth of naturally occurring L. pneumophila. Parallel observations on accompanying non-Legionellaceae bacteria failed to show the same relationship. These findings suggest that metal plumbing components and associated corrosion products are important factors in the survival and growth of L. pneumophila in plumbing systems and may also be important in related habitats such as cooling towers and air-conditioning systems.

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Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.

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A naturally occurring suspension of Legionella pneumophila and associated microbiota contained three unidentified non-Legionellaceae bacteria which supported satellite growth of a subculture of L. pneumophila on an L-cysteine-deficient medium and another bacterium which did not support growth of the subculture. Washed suspensions containing 10(3), 10(5), 10(7), or 10(8) CFU of a mixture of isolates of these non-Legionellaceae bacteria failed to support the multiplication of an isolate of agar-grown L. pneumophila which had been washed and seeded into the suspensions. The suspensions which contained 10(3), 10(5), or 10(7) CFU of the non-Legionellaceae bacteria per ml appeared to enhance survival or cryptic growth of agar-grown L. pneumophila. A decline of 1.3 log CFU of L. pneumophila per ml occurred within the first week of incubation in the sample which contained 10(8) CFU of the non-Legionellaceae bacteria per ml. In contrast to these results, naturally occurring L. pneumophila multiplied in the presence of associated microbiota. The necessity to subculture L. pneumophila and the non-Legionellaceae bacteria on artificial medium to obtain pure cultures may have affected the multiplication of L. pneumophila in tap water. Alternatively, other microorganisms may be present in the naturally occurring suspension which support the growth of this bacterium.

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A water culture containing naturally occurring Legionella pneumophila and associated microbiota was maintained in the laboratory by serially transferring the culture in tap water which had been sterilized by membrane filtration. Successful maintenance of the water culture depended upon transferring the culture when the growth of L. pneumophila was in the late-exponential to early-stationary phase. The water culture was used as a source of naturally occurring bacteria to determine some of the parameters which affect the multiplication of L. pneumophila in tap water. Naturally occurring L. pneumophila multiplied at a temperature between 25 and 37 degrees C, at pH levels of 5.5 to 9.2, and at concentrations of dissolved oxygen of 6.0 to 6.7 mg/liter. Multiplication did not occur in tap water which contained less than 2.2 mg of dissolved oxygen per liter. An association was observed between the multiplication of L. pneumophila and the non-Legionellaceae bacteria which were also present in the water culture. The method of preserving naturally occurring L. pneumophila and associated microbiota may facilitate studies on the symbiosis of L. pneumophila with other microorganisms.

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During a 3-month period, 1,062 mother-infant pairs were studied for infections following internal fetal monitoring during labor. Six infants (0.56%) developed septic scalp dermatitis at the site of the spiral electrode application. Factors associated with septic scalp dermatitis included the number of vaginal examinations, the use of an intrauterine pressure catheter or of more than one spiral electrode, and fetal scalp blood sampling. Maternal diabetes and endomyometritis were also associated with an increased risk of scalp infection. The duration of spiral electrode use and duration of ruptured membranes were not significant risk factors. Endomyometritis was documented in 41 mothers, an overall incidence of 3.9%. In women whose babies were delivered by cesarean section, the incidence of endomyometritis was 28/117 (23.9%). Using multivariate analysis by logistic regression, endomyometritis was associated with the number of vaginal examinations during labor but not with the duration of internal monitoring, duration of labor, or duration of ruptured membranes.

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Two children with legionellosis complicating a relapse of acute lymphoblastic leukemia are reported. A 5-year-old boy with pneumonia had Legionella pneumophila cultured from a tracheal aspirate following a rapid deterioration in his respiratory status and intubation. This child had severe and irreversible granulocytopenia and died in spite of therapy with erythromycin and rifampin added five days later. Combination antimicrobial therapy is suggested for immunosuppressed children with legionellosis if resolution of neutropenia is not readily anticipated. Culture of Legionella sp from respiratory tract secretions or sputum, as reported for the first time in the pediatric literature, should be attempted in all children in whom this infection is suspected. A 13-year-old boy with pneumonia recovered in spite of therapy with antimicrobial agents not proven to be effective against the legionellae. Clinical improvement coincided with increase in absolute granulocyte count. A retrospective diagnosis was made when seroconversion to Legionella micdadei (less than 1:16 to 1:1,024) was determined during a survey of unselected sera from 255 hospitalized children. This is the first documented case of Pittsburgh pneumonia described in a child.

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In an analysis of legionellosis at Presbyterian-University Hospital in Pittsburgh during the 28 months after the original outbreak of pneumonia caused by Legionella micdadei , 101 consecutive renal-allograft recipients were studied. The prevalence of elevated titers of antibody to Legionella pneumophila or L. micdadei among the patients at the time of transplantation (21.6%) was not significantly different from that among kidney donors (13.4%; P greater than .25). Seroconversion occurred in 21 (23.6%) of 89 recipients during the six months after transplantation. Seven recipients who seroconverted were diagnosed as having legionella pneumonia, eight had fever and pneumonitis attributed to other causes but compatible (on clinical grounds) with legionella infection, and six had no clinical evidence of infectious pneumonia. The interval from transplantation to onset of pneumonia was shorter for recipients with evidence of L. micdadei pneumonia than for patients with L. pneumophila pneumonia (P = .006); this finding suggested that the former disease occurred when the recipients were most immunocompromised. The original outbreak of L. micdadei pneumonia was found to have subsided nine months after the last formally recognized case, but infections with L. pneumophila continued to occur thereafter.

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Legionella pneumophila serogroup 1 was observed to satellite around colonies of Flavobacterium breve on an L-cysteine-deficient medium which did not support growth of legionellae. Both isolates were recovered from the hot water tanks of hospitals. Ferric PPi stimulated satellite growth between 0.01 and 0.1%.

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A study was conducted to compare the susceptibility of legionellae and coliforms to disinfection by chlorine. The chlorine residuals used were similar to concentrations that might be found in the distribution systems of large public potable water supplies. The effects of various chlorine concentrations, temperatures, and pH levels were considered. A number of different Legionella strains, both environmental and clinical, were tested. The results indicate that legionellae are much more resistant to chlorine than are coliform bacteria. At 21 degrees C, pH 7.6, and 0.1 mg of free chlorine residual per liter, a 99% kill of L. pneumophila was achieved within 40 min, compared with less than 1 min for Escherichia coli. The observed resistance is enhanced as conditions for disinfection become less optimal. The required contact time for the removal of L. pneumophilia was twice as long at 4 degrees C than it was at 21 degrees C. These data suggest that legionellae can survive low levels of chlorine for relatively long periods of time.

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MICs of ticarcillin, tobramycin, and rifampin alone and in combination were determined by a three-dimensional checkerboard method for 33 isolates of Pseudomonas aeruginosa. Twenty-nine of 33 isolates were affected synergistically (FIC index less than 1.0) by the combination of ticarcillin-tobramycin, including 13 of the 16 isolates resistant to ticarcillin and/or tobramycin alone. However, despite fulfilling the criteria for synergy, the MIC of the ticarcillin and/or tobramycin when in combination still exceeded attainable serum concentrations for 12 of the 13 resistant isolates. When rifampin was added to ticarcillin-tobramycin, a synergistic interaction was observed for all 33 isolates. Furthermore, of the 16 isolates resistant to ticarcillin and/or tobramycin, eight were inhibited by attainable concentrations of all three antibiotics in combination. If these results can be confirmed in the clinical situation, application of the three-drug combination of ticarcillin-tobramycin-rifampin might improve survival in selected patients with serious P. aeruginosa infections.

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An immunocompromised patient developed pneumonia in which both Legionella micdadei and L. pneumophila, serogroup 6, were isolated from transtracheal aspirates in the absence of any other bacteria. Unusual features included the development of disseminated intravascular coagulation and multiple pulmonary abscesses during treatment with erythromycin, and relapse of the legionellosis after extended therapy with both erythromycin and rifampin, which was heralded by septic shock. Because simultaneous infection with two distinct species of Legionella occurs, concurrent increases in antibody titer to more than one Legionella may, in some instances, reflect multiple infection rather than cross-reacting antibody.

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Correlation between in vitro and in vivo test results for synergy between carboxypenicillins and aminoglycosides against Pseudomonas aeruginosa is poor. Although the divalent cation content of culture media is known to affect aminoglycoside susceptibility testing for P. aeruginosa, this effect of divalent cations has not been examined for synergy testing of carboxypenicillin-aminoglycoside interaction against P. aeruginosa. The minimal inhibitory concentrations (MICs) of tobramycin and ticarcillin and the interaction of these drugs in combination were studied by a microtitration method for 36 strains of P. aeruginosa in Mueller-Hinton broth with varying supplements of calcium, magnesium, and zinc. The supplementation of Mueller-Hinton broth to 50 or 100 mg of calcium per liter had a significant effect in increasing the tobramycin MIC (P less than 0.01), as well as decreasing the degree of synergy between ticarcillin and tobramycin (P less than 0.01). Supplementation to 20 mg of magnesium per liter, 1.0 mg of zinc per liter, or both did not significantly affect tobramycin MIC or the interaction of tobramycin and ticarcillin. Supplementation to 50 or 100 mg of calcium per liter rendered any additional effect of magnesium and zinc on aminoglycoside MIC and aminoglycoside-carboxypenicillin interaction negligible. If these results for ticarcillin and tobramycin are confirmed for other carboxypenicillins and aminoglycosides, then the Mueller-Hinton broth used for P. aeruginosa aminoglycoside susceptibility and synergy testing may need to be supplemented only with calcium at a concentration of 50 mg/liter.

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Naturally occurring Legionella pneumophila, an environmental isolate which had not been grown on artificial medium, was tested for the ability to multiply in tap water. A showerhead containing L. pneumophila and non-Legionellaceae bacteria was immersed in nonsterile tap water supplying this fixture. Also L. pneumophila and non-Legionellaceae bacteria were sedimented from tap water from a surgical intensive care unit. This bacterial suspension was inoculated into tap water from our laboratory. The legionellae in both suspensions multiplied in the tap water at 32, 37, and 42 degrees C. The non-Legionellaceae bacteria multiplied at 25, 32, and 37 degrees C. A water sample which was collected from the bottom of a hot water tank was found to contain L. pneumophila and non-Legionellaceae bacteria. These legionellae also multiplied when the water sample was incubated at 37 degrees C. These results indicate that L. pneumophila may multiply in warm water environments such as hot water plumbing fixtures, hot water tanks, and cooling towers.

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Samples obtained from plumbing systems of hospitals, nonhospital institutions and homes were cultured for Legionella spp. by plating the samples directly on a selective medium. Swab samples were taken from the inner surfaces of faucet assemblies (aerators, spouts, and valve seats), showerheads, and shower pipes. Water and sediment were collected from the bottom of hot-water tanks. Legionella pneumophila serogroups 1, 5, and 6 were recovered from plumbing fixtures of the hospitals and nonhospital institutions and one of five homes. The legionellae (7 to 13,850 colony-forming units per ml) were also present in water and sediment from hot-water tanks maintained at 30 to 54 degrees C, but not in those maintained at 71 and 77 degrees C. Legionella micdadei was isolated from one tank. Thus legionellae are present in hot-water tanks which are maintained at warm temperatures or whose design results in warm temperatures at the bottom of the tanks. We hypothesize that hot-water tanks are a breeding site and a major source of L. pneumophila for the contamination of plumbing systems. The existence of these bacteria in the plumbing systems and tanks was not necessarily associated with disease. The extent of the hazard of this contamination needs to be delineated.

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