Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources.

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species.

Seven strains of Legionella-like amoebal pathogens (LLAPs) were characterized on the basis of their cultural and staining characteristics, biochemical reactions, serology, cellular fatty acids (CFAs), isoprenoid quinone composition, total DNA relatedness, analysis of 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analyses. All seven strains exhibited limited growth on buffered charcoal yeast extract alpha (BCYE) agar, required cysteine for growth and contained branched-chain CFAs and quinones typical of Legionella species. The bacilli were Gram-negative and catalase-positive. There were varying degrees of serological cross-reactions between these LLAP strains and other previously described Legionella species. Results from the various tests revealed that four LLAP strains represent three unusual new species of Legionella: Legionella drozanskii sp. nov., type strain LLAP-1T; Legionella rowbothamii sp. nov., type strain LLAP-6T; and Legionella fallonii sp. nov., type strain LLAP-10T. Three other LLAP strains, designated LLAP-7FL, LLAP-7NF and LLAP-9, were shown to be members of the species Legionella lytica. The deductions made from the phenetic characteristics of these bacteria were consistent with the phylogenetic relationships inferred from 16S rRNA and mip gene sequence analyses. This study is the first to speciate LLAP strains on the basis of data including quantitative DNA hybridization.

Interaction of nitric oxide with the surface of stabilized calcium sulfate.

This work explores surface interactions between stabilized gypsum and nitric oxide. Gypsum is a common air-borne mineral particulate that has a potential two-fold relationship to the air pollution problem: as a particulate pollutant and as a catalyst or adsorbent for pollutant gases. Nitric Oxide is found in stack gases and automotive exhausts. Isotherms of nitric oxide adsorbed on stabilized gypsum were studied at 24 degrees C, 0 degree C, and -78 degrees C. Coverages were related to a monolayer based upon a surface area of 17.0 m2/g as determined from a nitrogen adsorption isotherm and the B.E.T. method. Multilayer coverages containing both reversible and irreversible adsorption were observed for nitric oxide adsorbed on stabilized hydrated calcium sulfate. An irreversible coverage of 41% at 24 degrees C and 61% at 0 degree C of the nitrogen monolayer was observed for nitric oxide adsorbed on hydration stabilized gypsum. The heat of adsorption at zero coverage was found to be 80.4 kJ/mol for nitric oxide on stabilized hydrated calcium sulfate for the irreversible adsorption and 7.5 kJ/mol for the reversible adsorption.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Outbreak of Legionnaires' disease associated with a display whirlpool spa.

BACKGROUND: Recognized outbreaks of Legionnaires' disease (LD) are rare; when they occur, they provide opportunities to understand the epidemiology of the illness and improve prevention strategies. We investigated a population-based outbreak. METHODS: After the confirmation of LD in October 1996 in five people in neighbouring towns in southwest Virginia, active surveillance for additional cases was undertaken. A case-control study was conducted to identify exposures associated with illness, followed by a cohort study among employees of the facility at which the source of the outbreak was located in order to assess unrecognized exposure and illness. Samples of likely sources of LD in the facility were cultured for LEGIONELLA: RESULTS: In all, 23 laboratory-confirmed cases of LD were eventually identified. Of the 15 cases in the case-control study, 14 (93%) reported visiting a home-improvement store, compared with 12 (27%) of 45 controls (matched odds ratio [MOR] = 23.3; 95% CI : 3-182). Among home-improvement centre patrons, 10 (77%) of 13 cases questioned recalled either visiting or walking by a display whirlpool spa, compared with 3 (25%) of 12 controls (MOR = 5.5; 95% CI : 0.7-256.0). Two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. Employees reporting more exposure to the display spas were more likely to report symptoms of LD or to have an elevated titre. CONCLUSIONS: This investigation shows that LD can be transmitted from a whirlpool spa used for display only, and highlights the need for minimizing the risk of transmission of LD from all water-filled spas. Key messages This paper describes an investigation of a population-based outbreak of Legionnaires' disease (LD). A case-control study first identified a home-improvement store as the likely source of the outbreak. An environmental investigation later confirmed that finding, as two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. The spa was intended and used for display only.

Evaluation of the Binax and Biotest urinary antigen kits for detection of Legionnaires' disease due to multiple serogroups and species of Legionella.

The Binax and the Biotest urinary antigen kits for the detection of Legionnaires' disease caused by organisms other than Legionella pneumophila were compared by testing 45 urine samples from non-Legionella pneumophila serogroup 1 patients previously positive in a broad-spectrum enzyme-linked immunosorbent assay (ELISA). Eighteen were positive with the Binax kit, and 13 were positive with the Biotest. Although neither kit is as sensitive as ELISA, these results extend the number of serogroups and species of Legionella that can be diagnosed with the Binax or Biotest kit.

Strengths and limitations of molecular subtyping in a community outbreak of Legionnaires' disease.

An epidemiological and microbiological investigation of a cluster of eight cases of Legionnaires' disease in Los Angeles County in November 1997 yielded conflicting results. The epidemiological part of the investigation implicated one of several mobile cooling towers used by a film studio in the centre of the outbreak area. However, water sampled from these cooling towers contained L. pneumophila serogroup 1 of another subtype than the strain that was recovered from case-patients in the outbreak. Samples from two cooling towers located downwind from all of the case-patients contained a Legionella strain that was indistinguishable from the outbreak strain by four subtyping techniques (AP-PCR, PFGE, MAb, and MLEE). It is unlikely that these cooling towers were the source of infection for all the case-patients, and they were not associated with risk of disease in the case-control study. The outbreak strain also was not distinguishable, by three subtyping techniques (AP-PCR, PFGE, and MAb), from a L. pneumophila strain that had caused an outbreak in Providence, RI, in 1993. Laboratory cross-contamination was unlikely because the initial subtyping was done in different laboratories. In this investigation, microbiology was helpful for distinguishing the outbreak cluster from unrelated cases of Legionnaires' disease occurring elsewhere. However, multiple subtyping techniques failed to distinguish environmental sources that were probably not associated with the outbreak. Persons investigating Legionnaires' disease outbreaks should be aware that microbiological subtyping does not always identify a source with absolute certainty.

Hospital characteristics associated with colonization of water systems by Legionella and risk of nosocomial legionnaires' disease: a cohort study of 15 hospitals.

OBJECTIVE: To investigate an increase in reports of legionnaires' disease by multiple hospitals in San Antonio, Texas, and to study risk factors for nosocomial transmission of legionnaires' disease and determinants for Legionella colonization of hospital hot-water systems. SETTING: The 16 largest hospitals in the cities of San Antonio, Temple, and Austin, Texas. DESIGN: Review of laboratory databases to identify patients with legionnaires' disease in the 3 years prior to the investigation and to determine the number of diagnostic tests for Legionella performed; measurement of hot-water temperature and chlorine concentration and culture of potable water for Legionella. Exact univariate calculations, Poisson regression, and linear regression were used to determine factors associated with water-system colonization and transmission of Legionella. RESULTS: Twelve cases of nosocomial legionnaires' disease were identified; eight of these occurred in 1996. The rise in cases occurred shortly after physicians started requesting Legionella urinary antigen tests. Hospitals that frequently used Legionella urinary antigen tests tended to detect more cases of legionnaires' disease. Legionella was isolated from the water systems of 11 of 12 hospitals in San Antonio; the 12th had just experienced an outbreak of legionnaires' disease and had implemented control measures. Nosocomial legionellosis cases probably occurred in 5 hospitals. The number of nosocomial legionnaires' disease cases in each hospital correlated better with the proportion of water-system sites that tested positive for Legionella (P=.07) than with the concentration of Legionella bacteria in water samples (P=.23). Hospitals in municipalities where the water treatment plant used monochloramine as a residual disinfectant (n=4) and the hospital that had implemented control measures were Legionella-free. The hot-water systems of all other hospitals (n=11) were colonized with Legionella. These were all supplied with municipal drinking water that contained free chlorine as a residual disinfectant. In these contaminated hospitals, the proportion of sites testing positive was inversely correlated with free residual chlorine concentration (P=.01). In all hospitals, hot-water temperatures were too low to inhibit Legionella growth. CONCLUSIONS: The increase in reporting of nosocomial legionnaires' disease was attributable to increased use of urinary antigen tests; prior cases may have gone unrecognized. Risk of legionnaires' disease in hospital patients was better predicted by the proportion of water-system sites testing positive for Legionella than by the measured concentration of Legionella bacteria. Use of monochloramine by municipalities for residual drinking water disinfection may help prevent legionnaires' disease.

Classification of the genus Legionella.

There are currently 42 described species of legionellae representing 64 serogroups in the family Legionellaceae and the genus Legionella. The phenotypic characteristics of legionellae are described, including growth requirements, and biochemical characteristics. Identification of legionellae by biochemical tests, fatty acid analysis, ubiquinones, protein profiles, carbohydrate analysis, serology, monoclonal antibodies, and molecular techniques is described. The occurrence and description of Legionella-like amebic pathogens are discussed. The problems of identification to the species level are discussed, along with the need for further evaluations of additional strains from all known species using biochemical and molecular techniques.

Isolation of an amoeba naturally harboring a distinctive Legionella species.

There are numerous in vitro studies documenting the multiplication of Legionella species in free-living amoebae and other protozoa. It is believed that protozoa serve as host cells for the intracellular replication of certain Legionella species in a variety of environmental settings. This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample. In the laboratory, the bacterium multiplied within and was highly pathogenic for Acanthamoeba polyphaga. Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media. A 16S rRNA gene analysis placed the bacterium within the genus Legionella. Serological studies indicate that it is distinct from previously described species of the genus. This report also describes methods that should prove useful for the isolation and characterization of additional Legionella-like bacteria from free-living amoebae. In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens.

Epidemic Legionnaires' disease two decades later: old sources, new diagnostic methods.

In July 1995 we investigated a pneumonia outbreak in a Pennsylvania town. We conducted epidemiological and molecular microbiological studies to determine the outbreak source and interrupt transmission of disease. Legionnaires' disease (LD) was quickly identified by urine antigen testing, and a newly developed immunohistochemical stain confirmed nosocomial transmission to a hospital inpatient. LD was confirmed in 22 patients. Case-patients were more likely than controls to have been within 1,000 feet of the hospital (matched odds ratio, 21.0; 95% confidence interval, 2.9-368) during the 2 weeks prior to illness. Legionella pneumophila serogroup 1 (Lp-1) was isolated from hospital cooling towers (CTs) and rooftop air samples but not from hospital potable water or community CTs. Hospital CT and air Lp-1 isolates matched all five patient isolates by monoclonal antibody, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis subtyping. Strategies to prevent LD must include minimizing transmission from CTs.

More than 10 years of unrecognized nosocomial transmission of legionnaires' disease among transplant patients.

OBJECTIVE: To investigate a cluster of cases of legionnaires' disease among patients at a hospital. SETTING: A university hospital that is a regional transplant center. DESIGN: Retrospective review of microbiology and serology data from the hospital laboratories and prospective surveillance via the radiology department; a case-control study and environmental sampling within the hospital and from nearby cooling towers. RESULTS: Diagnosis of seven cases of legionnaires' disease in the first 9 months of 1996 led to recognition of a nosocomial outbreak that may have begun as early as 1979. Review of charts from 1987 through September 1996 identified 25 culture-confirmed cases of nosocomial or possibly nosocomial legionnaires' disease, including 18 in bone marrow and heart transplant patients. Twelve patients (48%) died. During the first 9 months of 1996, the attack rate was 6% among cardiac and bone marrow transplant patients. For cases that occurred before 1996, intubation was associated with increased risk for disease. High-dose corticosteroid medication was strongly associated with the risk for disease, but other immunosuppressive therapy or cancer chemotherapy was not. Several species and serogroups of Legionella were isolated from numerous sites in the hospital's potable water system. Six of seven available clinical isolates were identical and were indistinguishable from environmental isolates by pulsed-field gel electrophoresis. Initial infection control measures failed to interrupt nosocomial acquisition of infection. After extensive modifications to the water system, closely monitored repeated hyperchlorinations, and reduction of patient exposures to aerosols, transmission was interrupted. No cases have been identified since September 1996. CONCLUSIONS: Legionella can colonize hospital potable water systems for long periods of time, resulting in an ongoing risk for patients, especially those who are immunocompromised. In this hospital, nosocomial transmission possibly occurred for more than 17 years and was interrupted in 1996, after a sudden increase in incidence led to its recognition. Hospitals specializing in the care of immunocompromised patients (eg, transplant centers) should prioritize surveillance for cases of legionnaires' disease. Aggressive control measures can interrupt transmission of this disease successfully.

A recurrent outbreak of nosocomial legionnaires' disease detected by urinary antigen testing: evidence for long-term colonization of a hospital plumbing system.

BACKGROUND: In 1994, a hospital reported an increase in nosocomial legionnaires' disease after implementing use of a rapid urinary antigen test for Legionella pneumophila serogroup 1 (Lp-1). This hospital was the site of a previous nosocomial legionnaires' disease outbreak during 1980 to 1982. METHODS: Infection control records were reviewed to compare rates of nosocomial pneumonia and the proportion of cases attributable to legionnaires' disease during the 1994 outbreak period with those during the same period in 1993. Water samples were collected for Legionella culture from the hospital's potable water system and cooling towers, and isolates were subtyped by monoclonal antibody (MAb) testing and arbitrarily primed polymerase chain reaction (AP-PCR). RESULTS: Nosocomial pneumonia rates were similar from April through October 1993 and April through October 1994: 5.9 and 6.6 per 1,000 admissions, respectively (rate ratio [RR], 1.1; P=.56); however, 3.2% of nosocomial pneumonias were diagnosed as legionnaires' disease in 1993, compared with 23.9% in 1994 (RR, 9.4; P<.001). In 1994, most legionnaires' disease cases were detected by the urinary antigen testing alone. MAb testing and AP-PCR demonstrated identical patterns among Lp-1 isolates recovered from a patient's respiratory secretions, the hospital potable water system, and stored potable water isolates from the 1980 to 1982 outbreak. CONCLUSIONS: There may have been persistent transmission of nosocomial legionnaires' disease at this hospital that went undiscovered for many years because there was no active surveillance for legionnaires' disease. Introduction of a rapid urinary antigen test improved case ascertainment. Legionella species can be established in colonized plumbing systems and may pose a risk for infection over prolonged periods.

Flagella are a positive predictor for virulence in Legionella.

Pathogenesis of Legionnaires>> disease is strictly related to the ability of the legionellae to infect phagocytic cells, yet surface markers of virulence in Legionella isolates are currently unknown. Rabbit antibodies raised against purified flagella of Legionella pneumophila serogroup 1 recognized a total of 24 of 30 laboratory-maintained isolates of L. pneumophila serogroups 1-15 and 16 of 24 other Legionella species tested by rapid immunoblot and indirect immunofluorescence assay. All isolates possessing flagella detectable with these anti-flagella antibodies, regardless of species, were capable of infecting Hartmannella vermiformis. Isolates lacking immunologic cross-reactivity were shown to lack purifiable flagella. The majority of aflagellate isolates were not motile and failed to multiply intracellularly in co-culture with Hartmannella vermiformis. Some isolates characterized as aflagellate when harvested from BCYE agar were able to multiply in amoebae, and flagella were subsequently detectable by immunologic methods. These data suggest that lack of immunologic recognition of flagella in laboratory-maintained isolates of Legionella is due to their attenuation and a corresponding loss of expression of flagella. More importantly, the presence of flagella can serve as a positive predictive marker for strain virulence and is useful in determining the virulence status of Legionella isolates.

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.

Stability of Legionella urinary antigens over time.

Twenty-two urine samples positive for Legionella pneumophila serogroup 1 antigen by EQUATE radioimmunoassay (RIA) (Binax, Portland, ME, USA) were stored at various temperatures and the RIA repeated at 1, 7, 30, 90, and 120 days to evaluate stability of the urinary antigens. The mean ratios of patient/negative control remained stable. Although there was a 10% decrease in the mean ratios after 1 month, changes were not significant. However, individual samples with ratios close to 3 may fall to < 3.

Risk factors for domestic acquisition of legionnaires disease. Ohio legionnaires Disease Group.

BACKGROUND: Legionnaires disease is a common cause of adult pneumonia. Outbreaks of legionnaires disease have been well described, but little is known about sporadically occurring legionnaires disease, which accounts for most infections. Exposure to contaminated residential water sources is I plausible means of disease acquisition. METHODS: Employing a matched case-control study design in 15 hospitals in 2 Ohio counties, we prospectively enrolled 146 adults diagnosed as having nonepidemic, community-acquired legionnaires disease and compared each with 2 hospital-based control patients, matched for age, sex, and underlying illness category. An interview regarding potential exposures was followed by a home survey that included sampling residential sources for Legionella. Interview and home survey data were analyzed to estimate the risk of acquiring legionnaires disease associated with various exposures. RESULTS: Multivariate analysis showed that a nonmunicipal water supply (odds ratio [OR], 2.26; 95% confidence interval [CI], 1.17-4.37), recent residential plumbing repair (OR, 2.39; 95% CI, 1.10-5.18), and smoking (OR, 3.48; 95% CI, 2.09-5.79) were independent risk factors for legionnaires disease. Univariate analysis suggested that electric (vs gas) water heaters (OR, 1.97; 95% CI, 1.10-3.52), working more than 40 hours weekly (OR, 2.13; 95% CI, 1.12-4.07), and spending nights away from home before illness (OR, 1.68; 95% CI, 1.03-2.74) were additional possible risk factors. Lower chlorine concentrations in potable water and lower water heater temperatures were associated with residential Legionella colonization. CONCLUSIONS: A proportion of sporadic cases of legionnaires disease may be residentially acquired and are associated with domestic potable water and disruptions in residential plumbing systems. Potential strategies to reduce legionnaires disease risk include consistent chlorination of potable water, increasing water heater temperatures, and limiting exposure to aerosols after domestic plumbing repairs.

Legionella waltersii sp. nov. and an unnamed Legionella genomospecies isolated from water in Australia.

Two Legionella-like organisms were isolated from water samples obtained in Adelaide, Australia. One organisms was isolated from a drinking water distribution system, and the other was isolated from a cooling tower at a sewage treatment plant. Both strains required L-cysteine for growth and contained cellular branched-chain fatty acids and ubiquinones typical of the genus Legionella. These strains were serologically distinct from each other as determined by a slide agglutination test. STrain 2074-AUS-ET (T = type strain) was serologically distinct from all previously described Legionella species and serotypes. Strain 2055-AUS-E could not be differentiated biochemically or serologically from Legionella quinlivanii. Both strains were shown by DNA hybridization studies (Hydroxyapatite method) to be members of new Legionella species. Legionella waltersii sp. nov. is the name proposed for strain 2074-AUS-ET (= ATCC 51914T). L. waltersii was less than 10% related to other Legionella species. Strain 2055-AUS-E (= ATCC 51913) was informally named Legionella genomospecies 1, since it could not be phenotypically distinguished from L. quinlivanii. Legionella genomospecies 1 was closely related to L. quinlivanii strains (53 to 69% related with 4.5 to 6.5% divergence at 60 degrees C and 31 to 52% related at 75 degrees C).

Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen.

The Legionella Urinary Antigen EIA kit (Binax, Portland, Maine) was compared with the EQUATE RIA Legionella Urinary Antigen kit (Binax) for its ability to detect the presence of urinary antigens to Legionella pneumophila serogroup 1. Urine specimens from patients without Legionnaires' disease (n = 33) were negative by both methods (specificity, 100%). Twenty (77%) of 26 urine specimens from patients with Legionnaires' disease positive by the radioimmunoassay kit were also positive by the enzyme immunoassay (EIA) kit. If the cutoff for a positive EIA result were lowered to a ration of > or = 2.5, 23 of 26 (88%) urine specimens would have been positive by EIA and the specificity would remain 100%. Use of the EIA kit is an acceptable method for detecting L. pneumophila serogroup 1 urinary antigens by laboratories that do not want to handle radioactive materials.