Legionella-like Organisms Research Articles

Isolation of Legionella spp. from environmental samples in the Okayama area

During the period from October 1981 to March 1982, 24 water samples collected from coolingtowers and 32 environmental samples from Okayama prefecture, were cultured for isolation for Legionella spp. Environmental samples included 3 groups;(1) Seven samples of tap water, nebulizers, portable shower and baths, in Kawasaki Medical Hospital. (2) Nine samples of tap water, circulatingwater of air-conditioning systems and well water, from three private houses. (3) Seventeen variedsamples of pond soil mud and creek water, around Kawasaki Medical Hospital in autumn and winter.The isolation was carried out by guinea pig inoculation and direct plating on E-F agar after Low-p Htreatment of samples. Isolated strains were identfied as Legionella sp. by 19 ordinary biochemical tests, gaschromatography and direct fluorescent antibody techniques (DFA) useing FITC labeled antiserumwhich were prepared by injection of L. pneumophila serogroup 1, 2, 3 and 4 into rabbits. Theseserogroups were also used in biochemical tests and gaschromatography as controls.Sixteen strains (66.7%) of L. pneumophila were isolated from 24 water samples collected fromcooling towers, 4 strains of L. pneumophila and one strain of Legionella Like Organisms (L.L.O.) wereisolated from 6 samples of group 3 collected in autumn, while no Legionella sp. was isolated from othersamples in group 1, 2, and 3 collected in winter. From these result, it was very likely that amount of L.pneumophila decreased in winter season, and these organisms could not grow up in circulating water at ahigh temperature (50-75°) in air-conditioning systems set up in the private houses.Of 20 strains of L. pneumophila, only 10 stratins showed positive reaction in the Kovac oxidase test.However, several of these strains were negative when they were re-examined by the same test. Itseemed, therefore, that L. pneumophila was unstable in oxidase activity. In other biochemical tests, isolates showed identical results with those of standard strains. All isolates of L. pneumophila grew wellon B-CYE agar at 25 and 41°, indicating that B-CYE agar was more appropriate for the L. pneumophilacultivation than other media reported previously.The analysis of cellular fatty acid composition clearly demonstrated that differances existed between L. pneumophila and L.L.O., showing the usefulness of gaschromatography for classification ofthese organisms.DFA demonstrated that 18 isolates were identified as serogroup 1, one isolate serogroup 4, and theother one was seemed to be serogroup 5, 6 or nontypable.It is emphasized, that L. pneumophila is widely distributed in water in cooling towers and environmentsin Okayama prefecture.

Ecological distribution of Legionella species in Japan and pathogenicity of environmental isolates

Distribution of Legionella species over Japan was investigated.Five hundred ml of various environmental sources (343 samples of cooling tower water, 25 samples of water of puddy field. 29 samples of river, 3 samples of lake, 1 sample of fountain, 2 samples of puddle, 4 samples of well water, 6 samples of hemodialysate, 5 samples of antiseptic solution for washing hands at operating room) were cultured for isolation of Legionella spp.Isolations of the organism were applied to intraperitoneal injection to guinea pigs and direct inoculation to semiselective medium (B-CYE agar with antibiotics) after low pH treatment.One hundred sixty-six strains of Legionella spp. were isolated from 126 (36.7%) out of 343 cooling tower water samples.The origin of positive samples ranged from Hokkaido (Northern Japan) to Okinawa (Southern Japan).L. pneumophila, serogroup 1 was the most frequent isolates (66.3%); next in frequency of occurrence were Legionella-like organisms (16.3%), L. pneumophila serogroup 4 (7.2%), L. bozemanii (7.2%), L. pneumophila serogroup 6 (1.8%) and serogroup 3 (1.2%).L. pneumophila serogroup 2, serogroup 5, L. micdadei, L. dumoffii, L. gormanii, L. longbeachae and L. jordanis were not isolated from cooling tower water samples in this study.Five out of 28 soil samples were positive culture of Legionella spp. with L. pneumophila serogroup 1 (5 strains), serogroup 4 (2 strains) and serogroup 5 (1 strain).One strain of L. pneumophila serogroup 1 was isolated from one of 2 samples of puddle in construction sites. No positive culture was obtained from remaining materials.These results indicated that many types of Legionella species were widespread in all over the country.Environmental isolates possessed to have almost same endotoxin-like activity as clinical isolates by limulus test. However, there was some variations in virulence to mice among environmental isolates.

Reactivity of serum from patients with suspected legionellosis against 29 antigens of legionellaceae and Legionella-like organisms by indirect immunofluorescence assay.

Sets of sera (444) submitted for diagnostic testing for legionellosis were tested against 29 indirect immunofluorescence assay (IFA) antigens prepared from the characterized Legionella species and Legionella-like organisms to determine the prevalence of antibodies to Legionella organisms. Reciprocal titers of 15% of the serum sets rose fourfold or more to greater than or equal to 128 (indicating seroconversion) against one or more Legionella antigens. The specificity of the test was 96% when evaluated in patients with pneumonia due to non-Legionella organisms. Antibodies were of the IgG, IgM, and (infrequently) IgA classes and were either specific for a single species (as defined by a difference in titer of fourfold or more) or reacted with common Legionella antigens (30 [45%] vs. 36 [55%] of 66 seroconversions, respectively). No single antigen detected half of the positive sera. Elevated IFA titers (of greater than or equal to 256) against single or multiple Legionella antigens occurred in 12% of 184 normal control sera. Therefore, only seroconversions to titers of greater than or equal to 128 should be considered indicative of recent Legionella infection.

Enzymatic activities of Legionella pneumophila and Legionella-like organisms.

The API ZYM system was used to investigate enzymatic activities of Legionella pneumophila and other Legionella-like organisms. Leucine aminopeptidase, alkaline and acid phosphatase, butyrate and caprylate esterase, and phosphoamidase activities were consistently detected in all strains tested. No evidence of myristate lipase, trypsin, chymotrypsin, or glycosidase activity was found.

Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium.

A selective medium was developed and used successfully to isolate Legionella pneumophila and Legionella-like organisms from environmental specimens previously positive by animal inoculation methods. This medium consists of charcoal-yeast extract agar to which have been added cephalothin (4 micrograms/ml), colistin (16 micrograms/ml), vancomycin (0.5 microgram/ml), and cycloheximide (80 micrograms/ml). Pretreating of the environmental water samples with an acid buffer (pH 2.2), followed by plating on the selective medium, improved the rate of recovery of both Legionella and Legionella-like organisms relative to that with direct plating on selective media.

Fluoribacter dumoffii (Brenner et al.) comb. nov. and Fluoribacter gormanii (Morris et al.) comb. nov.

Strains of “atypical Legionella-like organisms”(ALLO) were studied by deoxyribonucleic acid homology. Strains WIGA (ALLO1) and MI-15 (ALLO2) were previously shown by Garrity et al. (G. M. Garrity, A. Brown, and R. M. Vickers, Int. J. Syst. Bacteriol. 30:609-614, 1980) to be related, and the name Fluoribacter bozemanae was proposed for them. We now show that strains NY-23 (ALLO4) and TEX-KL are also closely related genetically. Studies with strain LS-13 demonstrate a lower, but significant, degree of homology of LS-13 with strains WIGA, MI-15, and NY-23, justifying inclusion of all of these strains in a single genus, distinct from Legionella. It is proposed, therefore, that because of phenotypic similarities and genetic relatedness, strains NY-23, TEX-KL, and LS-13 be classified as members of the genus Fluoribacter, which also includes strains WIGA and MI-15. Because NY-23 and LS-13 are the type strains of Legionella dumoffii and L. gormanii, respectively, we propose the transfer of these species to the genus Fluoribacter as F. dumoffii (Brenner et al.) comb. nov. and F. gormanii (Morris et al.) comb. nov.

Isolation of plasmids in Legionella pneumophila and Legionella-like organisms.

Agarose gel electrophoresis was employed to screen nine strains of Legionella-like bacteria and one strain of Legionella pneumophila for the presence of extrachromosomal deoxyribonucleic acid. Cryptic plasmids with molecular weights ranging from 46.6 x 10(6) to 59.8 x 10(6) were found in three of the isolates examined.

Dye-containing buffered charcoal-yeast extract medium for differentiation of members of the family Legionellaceae.

The addition of 0.001% bromocresol purple and 0.001% bromothymol blue to buffered charcoal-yeast extract agar allowed differentiation between members of the family Legionellaceae. On this medium, Legionella pneumophila grew as relatively flat, pale green colonies, whereas Tatlockia micdadei (gen. nov., comb. nov., Pittsburgh pneumonia agent) produced blue-gray colonies. Fluoribacter spp. (gen. nov., atypical Legionella-like organisms) developed glistening colonies which were brighter green than those of L. pneumophila.

Laboratory diagnosis of legionnaires’ disease

Legionnaires’ Disease is a pulmonary infection caused by Legionella pneumophila . Most sporadic and epidemic-forms of the disease are due to serogroup 1 strains. Legionella of the other serogroups and other legionella-like organisms can cause opportunistic infections in some individuals. Laboratory diagnosis is most simply achieved by demonstrating legionella in pulmonary tissue or respiratory secretions. Special stains are necessary since legionella stain poorly or not at all in the Gram stain. Direct fluorescent antibody staining is the most sensitive and specific test for delecting organisms, but the range of sera and their availability are limited. Tissue and or secretions should be injected into guinea pigs and cultured on to charcoal yeast extract (CYF.) agar or a suitable alternative. Organisms infecting the guinea pigs can be isolated on CYE agar by plating splenic tissue. Serological testing of acute and convalescent sera to demonstrate a significant rise in antibody by the indirect fluorescent test (IFA) can be used to make a retrospective diagnosis. Most patients show increasing litres of specific IgM antibody in the second week of their illness. No single level of antibody can be considered diagnostic. At present only a limited range of strains (serogroups 1 -4) are available as antigen. A negative serological test does not exclude infection with one of the other serogroups or with other /egione/la-Yikc organisms which are antigenically distinct from those used in this test.

Pittsburgh pneumonia agent: direct isolation from human lung tissue.

Pittsburgh pneumonia agent (PPA) was recently cultivated from infected egg yold on charcoal yeast extract agar. PPA has now been isolated both from infected egg yolk and human lung tissue on charcoal yeast extract agar and on a new medium, buffered charcoal yeast extract agar. PPA resembles Legionella pneumophila and other Legionella-like organisms in requirements for growth and composition of fatty acids. It differs in genetic relatedness, antigenic composition, and colonial morphology and has distinctive characteristics that allow it to be identified. The name Legionella pittsburgensis species nova is proposed for this organism.

Slide agglutination test for serogrouping Legionella pneumophila and atypical Legionella-like organisms.

A simple, inexpensive slide agglutination test correctly identified the serogroup to which 38 Legionella pneumophilia strains belonged, and distinguished them from two atypical, Legionella-like organisms.

ATYPICAL LEGIONELLA-LIKE ORGANISMS: FASTIDIOUS WATER-ASSOCIATED BACTERIA PATHOGENIC FOR MAN

A group of related bacteria designated atypical Legionella-like organisms (ALLO) has been identified. ALLO, like L. pneumophila, are fastidious gram-negative rods that grow well on charcoal yeast extract (CYE) agar and produce ground glass colonies and browning of modified yeast extract agar. Unlike L. pneumophila, ALLO do not grow well on Feeley-Gorman (FG) agar, and on CYE agar they fluoresce under longwave ultraviolet light. ALLO and L. pneumophila have a similar predominance of branched-chain forms among total cellular fatty acids but have distinctive fatty-acid profiles. 2 patients with culture-verified ALLO pneumonia and 10 with pneumonia of uncertain ætiology who seroconverted to ALLO offer evidence that ALLO may be a cause of community-acquired pneumonia. Like L. pneumophila,ALLO appear to be water-associated; both persons with culture-verified ALLO infection were exposed to fresh water or its contents before becoming ill, and two strains of ALLO were isolated from water or wet environments.