Legionnaires' Disease Bacterium Research Articles

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.

Growth of Legionella pneumophila in association with blue-green algae (cyanobacteria).

Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45 degrees C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45 degrees C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.

Identification of diaminopimelic acid in the Legionnaires disease bacterium.

Diaminopimelic acid was found to be a component of the cell wall of the Legionnaires disease bacterium, thus providing additional evidence that the organism is a bacterium. The presence of this amino acid was determined by gas-liquid chromatography and confirmed by gas chromatography-mass spectrometry.

Flagella on Legionnaires' Disease Bacteria: An Interim Report

Brief Reports1 August 1979Flagella on Legionnaires' Disease Bacteria: An Interim ReportBERENICE M. THOMASON, B.S., FRANCIS W. CHANDLER, D.V.M., Ph.D., DANNIE G. HOLLIS, M.S.BERENICE M. THOMASON, B.S.Search for more papers by this author, FRANCIS W. CHANDLER, D.V.M., Ph.D.Search for more papers by this author, DANNIE G. HOLLIS, M.S.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-91-2-224 SectionsAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail ExcerptThe Legionnaires' disease (LD) bacterium, for which the nameLegionella pneumophilahas been proposed (1), is a small gram-negative microorganism that varies considerably in length depending upon its environment (2). Early investigators did not describe motility or the presence of flagella. While examining direct fluorescent antibody-stained smears of pure cultures of LD bacterium, we have observed fluorescent polar flagella on two stock strains (Bellingham 1 and Bloomington 2). We confirmed these observations by examining smears stained with either a silver-plating stain for flagella (3) or a simplified Leifson flagella stain (4). This observation prompted us to examine other strains of...

Growth of Legionnaires disease bacterium (Legionella pneumophila) in chemically defined medium.

A chemically defined medium containing 21 amino acids and inorganic salts was developed which supported the growth of four isolates of Legionnaires disease bacterium (Legionella pneumophila). Growth in liquid defined medium at 37 degrees C with shaking approximated the generation time and growth kinetics observed for growth in complex media. After a 3-h lag, the culture grew exponentially with a generation time of 6 h and reached a maximum optical density of 230 Klett units (170 Klett units corrected for pigment). A soluble brown pigment was first observed as the culture entered late exponential to early stationary phase of growth. Morphologically, L. pneumophila grew in the liquid defined medium with extensive filamentation and numerous intracellular lipid granuoles. L-Serine, L-methionine, and L-cysteine were required for optimum growth. The latter amino acid could be replaced by L-cystine or reduced glutathione but not by D-cysteine, thiomalate, thioglycollate, or 2-mercaptoethanol. Ferric iron was needed for maximum growth, but supplemental iron was not an essential growth requirement. Carbohydrates (i.e., glucose) or organic acids did not stimulate growth. In fact, pyruvate, acetate, and citrate all gave varying degrees of inhibition (69, 37, and 0% of control growth, respectively).

Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay

An enzyme-linked immunospecific assay "sandwich" technique was developed for detecting soluble antigen from the Legionnaires disease bacterium (Legionella pneumophila). With this technique, antigen was detected in urine specimens from guinea pigs inoculated intraperitoneally with heat-killed Legionnaires disease bacteria and in urine specimens from three of four patients who attended the American Legion Convention in Philadelphia in 1976. Urine from a fifth pneumonia patient who attended the Eucharistic Congress (but who was a dubious seroconverter) was negative. Presumably, the test could also be used for detecting antigen in sputum or respiratory aspirates, but this has not been tried to date.

Further studies of the cellular fatty acid composition of Legionnaires disease bacteria.

The cellular fatty acid composition of 36 strains of the Legionnaires disease bacterium was determined by gas chromatography after growth on different media. The fatty acid profile of each strain was essentially identical on each medium and was characterized by large amounts (greater than 68%) of branched-chain acids.

Classification of the Legionnaires' Disease Bacterium: Legionella pneumophila, genus novum, species nova, of the Family Legionellaceae, familia nova

Deoxyribonucleic acid (DNA) relatedness was used to classify strains of the Legionnaires' disease (LD) bacterium. These DNA comparisons showed that all strains of the LD bacterium were members of the same species. Included were strains isolated from the environment and strains with three different O-antigens. The DNA from the LD bacterium was not significantly related to DNA from any other group of bacteria that was tested. Biochemical data, growth characteristics, and guanine-plus-cytosine ratios were used to rule out the possibility that the LD bacterium was significantly related to members of genera whose DNA was not tested. In view of these data we propose that the LD bacterium be named Legionella pneumophila species nova, the type species of Legionella, genus novum. The type strain of L. pneumophila is Philadelphia 1.

Legionnaires' Disease Bacterium Isolated in 1947

The results of serologic, cultural, and DNA relatedness studies have shown that the Legionnaires' disease (LD) bacterium and an unclassified agent isolated in 1947 are the same species. Both organisms grew on charcoal-yeast extract agar, enriched Mueller-Hinton agar, and F-G agar, but neither grew on blood agar, trypticase soy agar, or in thioglycollate broth. Both agents reacted with convalescent sera from patients with Legionnaires' disease and convalescent sera from guinea pigs infected experimentally with the LD bacterium. The percentage of guanine plus cytosine in DNA preparations from each organism was ascertained by thermal denaturation to be 39%. In DNA hybridization reactions the 1947 isolate showed the same degree of relatedness to Philadelphia 1 strain of the LD bacterium as did three recent isolates of the bacterium. The LD bacterium was also shown to be antigenically related to another unclassified organism isolated in 1959.

Ultrastructure of the Legionnaires' disease bacterium. A study using transmission electron microscopy.

The Legionnaires' disease (LD) bacterium appeared ultrastructurally identical in human lung, egg yolk membrane, and artificial media, seen as a blunt or tapering rod measuring 0.3 to 0.9 micron in diameter and greater than or equal to 2.0 micron long. Greatly elongated forms were commonly found in cultures and yold sac membranes after 5 to 7 days of growth but were only rarely seen in human lung. The LD bacterium was clearly prokaryotic. Prominent features included electron-lucent nucleoids interspersed among areas of well-defined ribosomes; cleanly circumscribed cytoplasmic vacuoles or granular inclusions; and a double envelope enclosure, each portion consisting of a triple-layered "unit" membrane, approximately 75 A wide. Division always occurred as a pinching, nonseptate process typical of bacteria with a double, gram-negative type of envelope. No definite structure was seen in the periplasmic space that might represent the peptidoglycan layer. These features of the LD bacterium confirm earlier reports of the gram-negative staining reaction of organisms obtained from cultures and preliminary evidence of their gram-negative ultrastructure. We found no unique features that would aid in the ultrastructural differentiation of the LD bacterium from other small gram-negative bacilli.

Isolation of the Legionnaires' Disease Bacterium from Environmental Samples

We analyzed 24 environmental samples collected in or near the Indiana Memorial Union, where an epidemic of Legionnaires' disease occurred in early 1978. We conducted fluorescent antibody analyses and culture on F-G and charcoal yeast extract agars of each sample directly; splenic tissue of guinea pigs inoculated with the sample; and yolk sacs from embryonated eggs inoculated with splenic tissue of guinea pigs injected with the sample. Legionnaires' disease (LD) bacterium was isolated from seven of the 24 samples: one water sample from the air-conditioner cooling tower of the Union; three water samples from a stream near the Union; and three mud samples from the same stream. The LD bacterium strains were of three different serotypes. These findings indicate that LD bacteria may be widespread in nature.

A High Molecular Weight Antigen in Legionnaires' Disease Bacterium: Isolation and Partial Characterization

We isolated a high molecular weight antigen of the Legionnaires' disease (LD) bacterium by column chromatography. The antigen was composed of 35% carbohydrate, 2.6% protein, 1.8% phospholipid, and 1% 2-keto-3-deoxyoctonate and was important in the host's antibody response because it inhibited the indirect immunofluorescent and microagglutination titers of convalescent sera from patients with Legionnaires' disease. The antigen also formed precipitin bands with seven of 10 convalescent sera from patients with Legionnaires' disease. We found chemical and biological evidence of endotoxinlike activity associated with the antigen. Cell sonicates and acid extracts of the LD bacterium gave multiple bands in immunodiffusion with human convalescent serum and rabbit antisera prepared against heat-killed LD bacteria. The antigenic structure of the LD bacterium therefore appears complex.

Legionnaires' Disease in Pneumonia Patients in Iowa

The frequency of Legionnaires' disease among 586 cases of pneumonia that occurred in Iowa between fiscal years 1972 and 1977 was studied retrospectively on the basis of paired sera. The frequency of confirmed Legionnaires' disease was 4.1% and of presumptive Legionnaires' disease was 11.4%. Infections with the Legionnaires' disease (LD) bacterium were most frequent in the summer. Of the 22% of pneumonias for which a cause could be defined, Legionnaires' disease was third in frequency behind Mycoplasma pneumoniae and influenza A virus infections. Infections with the LD bacterium occurred in association with pneumonias in most age groups. The youngest patient with LD infection was a 5-year-old boy with pneumonia. The disease occurred 3.2 times more often in males than in females. In males, the frequency of confirmed and presumptive Legionnaires' disease increased steadily to plateau after the fourth decade at about 12% and 28%, respectively. In females the frequency of presumptive Legionnaires' disease was 7% to 16%, relatively evenly distributed over all age groups. Pneumonias associated with LD bacterium infection should be considered in the differential diagnosis of community-acquired pneumonias in most age groups.

Nosocomial Legionnaires' Disease: A Continuing Common-Source Epidemic at Wadsworth Medical Center

Forty-nine cases of Legionnaires' disease were identified from May 1977 through July 1978 in patients and employees at Wadsworth Medical Center. Cases clustered in October and November 1977. Fifteen patients died. All Legionnaires' disease (LD) patients were in the hospital before onset of illness (median time from admission to onset, 17 days; range, 3 to 276 days). Twenty patients were immunosuppressed or compromised by malignancy. In 1977, six of 12 renal-homograft recipients acquired LD pneumonia in contrast to three of 22 during the preceding 3 years (P = 0.031, Fisher's exact test). In a prospective survey of 1658 consecutive hospital admissions, seven cases of Legionnaires' diseases occurred (0.4%), including six among 14 patients who seroconverted to the LD bacterium. Prevalence of a reciprocal titer of 128 or above in Wadsworth employees was significantly greater than in a nearby control population (P = 0.044, Fisher's exact test). Exposure to the external hospital environment may be an important factor, and soil may be a reservoir for the LD bacterium. Legionnaires' disease at Wadsworth may be a nosocomial pneumonia affecting a small group of patients with particular risk factors.

Relation ofMycoplasma pneumoniaeSeroreactivity, Immunosuppression, and Chronic Disease to Legionnaires' Disease

From April 1977 through March 1978, 28 presumptive cases of Legionnaires' disease were identified among 432 consecutive candidates having paired sera or tissue samples submitted to the Massachusetts Public Health Laboratories. Among the subgroup of 209 candidates with documented diffuse pneumonia and temperature of 39 degrees C or above, 24 (11.5%) had Legionnaires' disease whereas the diagnostic yield was only four of 223 (1.8%) among the remainder. The case-fatality rate was two of 28 (7%). Patients with Legionnaires' disease when compared to the entire group of candidates were similar in mean age (49 versus 48 years) and frequency of immunosuppressant therapy (15% versus 12%) but were more often male (64% versus 47%) with underlying chronic illness (46% versus 22%). Complement fixation tests against Mycoplasma pneumoniae (whole organisms) showed seroreactivity in 81% of Legionnaires' disease (LD) cases (22 to 27) compared to 13% of non-LD cases; conversely, 29% of all cases seropositive for M. pneumoniae (22 of 75) were seropositive for the LD bacterium compared to only 1% (five of 357) of the remainder. The coincidence of seroreactivity for M. pneumoniae and the LD bacterium is unexplained but suggests that M. pneumoniae seropositive cases should be evaluated for the possibility of Legionnaires' disease.

Pathologic Findings in Guinea Pigs Inoculated lntraperitoneally with the Legionnaires' Disease Bacterium

Tissues from guinea pigs inoculated intraperitoneally with the Legionnaires' disease (LD) bacterium were studied with light, immunofluorescent, and electron microscopy. The principal gross lesion was diffuse peritonitis of varying severity. Microscopically, the peritonitis of covered by a mixed inflammatory infiltrate of macrophages, neutrophils, fibrin, and cellular debris. Foci of inflammation and necrosis were consistently observed in the splenic parenchyma, and similar lesions were often found in the lungs, liver, lymph nodes, pancreas, heart, and other organs. Numerous LD bacteria were seen in the peritoneal exudate; fewer were found in disseminated lesions. In electron micrographs, the highest concentrations were seen in macrophages, with fewer organisms present in neutrophils or extracellular spaces. Although the lung is the primary organ konwn to be affected by Legionnaires' disease in humans, our findings indicate that the LD bacterium is capable of dissemination.

Aromatic substrate specificity of browning by cultures of the Legionnaires' disease bacterium.

When the Legionnaires' disease (LD) bacterium is grown on supplemented Mueller-Hinton agar, brown pigmentation of the medium occurs. Since this browning may result from tyrosinase-mediated formation of melanin, we supplemented yeast-extract agar with various aromatic precursors of melanin and inoculated it with eight strains of the LD bacterium. Browning occurred with growth of each LD strain of the bacterium on yeast-extract agar enriched with 2.5 mmol/L of L-phenylalanine or L-tyrosine but not without such enrichment. Equimolar D-phenylalanine or D-tryosine in yeast-extract agar did not enhance browning. The LD bacterium may possess L-phenylalanine hydroxylase activity, but it does not use D-aromatic amino acids effectively in pigment production.

Nosocomial Legionnaires' Disease in Columbus, Ohio

Three patients with severe pneumonia at a community hospital in Columbus, Ohio, were found to have Legionnaires' disease in late August 1977. A subsequent serologic survey of patients with pneumonia at this hospital identified three additional cases. Among patients with pneumonia, hospital exposure in the 2 weeks before onset of illness was significantly associated with Legionnaires' disease (P = 0.003). Serosurveys of hospital employees with a recent history of upper respiratory illness, healthy employees, and workers at the hospital construction site showed that one of 101, one of 107, and none of 114, respectively, has a single reciprocal titer of greater than or equal to 256 to the Legionnaires' disease (LD) bacterium. Serosurveys of patients with pneumonia at three control hospitals identified five additional patients with Legionnaires' disease, three of them with pneumonia that was apparently hospital acquired in a single renal transplant unit. A fourth patient from that unit without clinical illness had a fourfold rise in titer to LD bacterium.

Growth, survival, and resistance of the Legionnaires' disease bacterium.

The Legionnaires' disease (LD) bacterium can now be readily cultured on artificial media. Studies were done to define the growth and survival of the LD bacterium in these media and ascertain its susceptibility to disinfecting agents. Growth-curve studies of the Philadelphia 1 strain using Mueller-Hinton broth with ferric pyrophosphate and L-cysteine (Feeley-Gorman broth) showed a lag phase of less than 24 h, a generation time of 3.8 h during the logarithmic phase, a plateau of 2 x 10(7) organisms per millilitre, and continued viability for as long as 110 d. Viability on chocolate agar with 1% hemoglobin and 2% IsoVitaleX added reached 150 d. This strain was susceptible to a variety of commonly recommended hospital and laboratory disinfectants, often in low concentrations. These investigations suggest that prolonged survival may occur in natural as well as artificial milieus and that low concentrations of phenolics, quaternary ammonium compounds, glutaraldehyde, formaldehyde, and hypochlorite could eradicate potential reservoirs for human infection.

Legionnaires' Disease in Patients with Associated Serious Disease

Of nine patients with Legionnaires' disease, seven were receiving corticosteroids, and all nine had serious underlying diseases. Direct immunofluorescent examination of respiratory secretions, including sputum and transtracheal aspirates, showed the Legionnaires' disease (LD) bacterium in five of seven patients who seroconverted and in a sixth patient with a single elevated titer to the LD bacterium. All nine patients received erythromycin therapy, and five survived. Two patients showed persistence of their infection after receiving 2 weeks of erythromycin therapy, and two patients developed pulmonary abscesses. These cases of Legionnaires' disease show the occurrence of pulmonary abscesses, the possibility of relapse after giving only 2 weeks of erythromycin therapy, and the utility of direct immunofluorescence for early diagnosis.