Legionella Isolates Research Articles

Isolation of Legionella spp. from Potting Soils, and The Growth within Acanthamoeba sp. and Antibiotics Susceptibility of Isolates

Isolation of Legionella spp. from Potting Soils, and The Growth within Acanthamoeba sp. and Antibiotics Susceptibility of Isolates

First isolation of Legionella species, including L. pneumophila serogroup 1, in Greek potting soils: possible importance for public health

A total of 21 Legionella isolates were recovered from six out of 22 samples of potting soil from the Athens area, Greece. Legionella pneumophila (serogroups 1 and 2–15) and species and serotypes included in the group of L. longbeachae serogroups 1 and 2, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei and L. anisa were isolated on BCYEα agar containing cysteine, GVPC and natamycin and on BCYEα agar containing cysteine, Wadowsky Yee supplement and natamycin. The bacterial load was 4000–120 000 CFU/g of potting soil. The isolation of L. pneumophila serogroup 1 from Greek potting soils is reported here for the first time.

Erratum: Antimicrobial Susceptibility Patterns of Legionella isolates in the Environment and in Patients

Erratum: Antimicrobial Susceptibility Patterns of Legionella isolates in the Environment and in Patients

Isolation of Legionella Spp. from Water Samples of Cooling Towers and Effects of Some Cooling Tower Biocides on the Bacterium

Isolation of Legionella Spp. from Water Samples of Cooling Towers and Effects of Some Cooling Tower Biocides on the Bacterium

Antimicrobial Susceptibility Patterns of Legionella isolates in the Environment and in Patients

Antimicrobial susceptibility of Legionella spp. has rarely been studied in Korea. Therefore, we aimed to determine the susceptibility of Legionella spp. to various antibiotics. We assessed the antimicrobial susceptibility of 66 environmental and clinical Legionella isolates collected between January 2001 and December 2008 from Korea and Japan. The minimum inhibitory concentrations (MICs) of 6 antibiotics, namely, azithromycin, ciprofloxacin, clarithromycin, clindamycin, gatifloxacin, and gemifloxacin were determined by the broth microdilution method using buffered starch yeast extract broth. The MIC ranges of the 6 antibiotics used against the Legionella isolates were as follows: 0.004-0.062 microg/mL (azithromycin), 0.002-0.5 microg/mL (ciprofloxacin), 0.004-0.5 microg/mL (clarithromycin), 0.12-4 microg/mL (clindamycin), 0.002-0.12 microg/mL (gatifloxacin), and 0.008-1 microg/mL (gemifloxacin). Legionella spp. isolates from Korea and Japan were most susceptible to gatifloxacin. Azithromycin, clarithromycin, ciprofloxacin, and gemifloxacin were also effective for treating legionellosis.

Surveillance of Legionella Contamination on Water Supply Systems of Public Utilizing Facilities in Gwangju, Korea

Legionella is a pathogenic bacteria associated to aquatic habit of natural and artificial environments. Clinical cases of legionellosis have been reported in Korea but there is a lack of information about the incidence of this bacterium on environmental sources. Thus, we investigated the contamination of Legionella on water supply systems in Gwangju, Korea, including cooling towers, public baths, hospitals and fountains. Legionella spp. were detected from 81 samples (16.9%) out of 480 samples collected and the major species of Legionella isolates was identified to L. pneumophila serogroup (sg) 1 (n=33). The result of PFGE pattern analysis for L. pneumophila sg 1 isolates was discriminated into 11 pulsotypes. In dendrogram of PFGE, the clusters of isolates from cooling tower were quite different from those derived from hot water system of public bath. Surveillance for 4 sentinel public baths showed that their water supply systems had been contaminated with Legionella spp. and L. pneumophila sg 5 or sg 6 was the major isolate. The similarity of its PFGE patterns was 92.3%, 96.6%, respectively. Thus, PFGE may be a useful tool in molecular epidemiology studies, and the monitor and control for water supply systems of public utilizing facilities will be able to provide a resolution for the reduction of Legionella infection.

Prevalence study of Legionella spp. contamination in Greek hospitals

Water and swab samples were collected from 13 hospitals and analyzed for Legionella counts. Legionella was detected in eight out of 13 hospitals and in 22 of 130 water and swab-collected samples. A total of 72.7% of the strains were L. pneumophila ser. 1, 22.7% were L. pneumophila ser. 2-14, and 4.5% did not belong to any of these groups. AFLP typing of the L. pneumophila ser. 1 strains generated two distinguishable AFLP types. There was no significant correlation to the sample type with Legionella recovery. Legionella isolation was more likely to occur in the cooling towers than the water system. Water temperatures of 30–40°C seem to favor Legionella growth. Of the 265 serum samples taken from the medical and technical staff for the control of IgG titre, 89.4% were negative, 7.2% were positive, and for 3.4% the result was doubtful. No association between IgG titre and maximum observed level of Legionella occurrence was detected.

A Microbiological Study of Nocardia, Legionella, and Mycoplasma Isolated from Lower Respiratory Tract Infections in Iraqi Patients

DOI: http://dx.doi.org/10.5915/40-3-5487 A total of 295 patients suffering from lower respiratory tract infections, including bronchitis (99), pneumonia (156), and bronchiectasis (40), were enrolled in this prospective microbiological study. The patients were of two categories: 173 were immunocompromised (with underlying diseases), and 122 were immunocompetent. Two types of clinical specimens, including sputum (216) and bronchial wash (79), were collected from these patients. Direct examination, culture, and biochemical tests were performed to test for the presence of bacteria. A total of 311 bacterial isolates and 32 fungal growths were detected. The bacterial isolates were encountered from 62.8% of the immunocompromised patients and from 37.2% of the immunocompetent patients. Out of these, 311 isolates, 17 (4.8%) were relatively uncommon pathogens of the respiratory tract, namely 11 (3.5%) were Nocardia, 4 (1.3%) were Mycoplasma, and 2 (0.6%) were Legionella. The Nocardia isolates were fully sensitive to amikacin and cotrimoxazole, and Legionella isolates were fully sensitive to erythromycin, azithromycin, ciprofloxacin, and levofloxacin. Also, 294 common pathogens were isolated, namely Gram-positive (49.2%), Gram-negative (33.5%), and anaerobic (11.9%) bacteria.

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002–2006

This study investigated the hypothesis that the genotype distribution of Legionella isolates from sporadic patients with Legionnaires' disease differs from that of Legionella strains in the environment. An amplified fragment-length polymorphism (AFLP) assay was used to genotype patient-derived and environmental Legionella isolates. The three Legionella pneumophila genotypes isolated most frequently from human respiratory secretions were AFLP types 004 Lyon, 010 London and 006 Copenhagen. These genotypes were cultured significantly less frequently from environmental samples (50% vs. 4%; p <0.001). The most frequently observed L. pneumophila serogroup 1 genotype among patient-derived isolates was 004 Lyon (32%). This genotype was cultured from only one of 6458 environmental samples. The positive sample contained 1.26 x 10(6) CFU/mL and originated from a whirlpool spa that had not been disinfected and had been maintained at 36 degrees C for several months. Overall, the distribution of genotypes differed significantly among patient and environmental isolates. A possible explanation is that virulent strains may exist in potential environmental sources at undetectable concentrations.

Lorraine Strain ofLegionellapneumophilaSerogroup 1, France

Lorraine Strain of<i>Legionella</i><i>pneumophila</i>Serogroup 1, France

Profiling of environmental Legionella pneumophila strains by randomly amplified polymorphic DNA method isolated from geographically nearby buildings

Legionella pneumophila (L. pneumophila) which is also known as etiologic agent Legionnaires Disease lives in natural water and man made water systems. These bacteria belonging to Legionellaceae family are divided 15 serogroups. Phenotypical methods used for the identification of Legionella isolates are not very discriminatory. In this study we investigated genotypic features of eight L. pneumophila serogroup 1 and 18 L. pneumophila serogroup 2-14 strains isolated from different buildings in Istanbul by randomly amplified polymorphic DNA (RAPD) method. Eight L. pneumophila serogroup 1 strains (37.5%) were similar RAPD profile and they were isolated from buildings located in a short distance (about 500 m). Four L. pneumophila serogroup 2-14 strains (22%) were identical genotypically. Three of these strains were isolated from buildings located in a short distance.

A Legionnaires’ disease outbreak: A water blaster and roof-collected rainwater systems

In February 2006, an outbreak of Legionnaires’ disease (LD) was identified in Beachlands, a small, isolated east Auckland suburb. It was investigated through case finding, a case–control study, sampling potential sources of infection and by molecular typing (using sequence-based typing (SBT) of all Legionella pneumophila serogroup 1 (Lp1) isolates). Lp1 was isolated from the respiratory tract of one case, the roof-collected rainwater systems of five households (three associated with cases) and from a water blaster at a nearby marina. All isolates were indistinguishable, exhibiting the same SBT allele pattern. Three LD cases lived within 500 m of the water blaster (the fourth case within 1250 m) and downwind in prevailing conditions. Another domestic roof-collected rainwater supply contaminated by Lp1 (identical SBT pattern) was incidentally identified in another suburb 4 km east of Beachlands. This is the first outbreak of LD linked to roof-collected rainwater supplies and the first isolation of Legionella from these systems in New Zealand. Aerosols containing Legionella discharged to air by the marina water blaster may have infected some cases directly or may have seeded roof-collected rainwater systems. Some cases may have been exposed by contaminated bathroom showers. Roof-collected rainwater systems need appropriate design, careful cleaning and the maintenance of hot water temperatures at a minimum of 60 °C to reduce the chances of Legionella multiplying. Further research into the ecology of Legionella in roof-collected rain water systems is indicated.

Isolation of Legionella species/serogroups from water cooling systems compared with potable water systems in Spanish healthcare facilities

Surveillance of Legionella spp. in hospital water systems was performed in forty-four inpatient healthcare facilities in Spain during 2005-2006. A total of 2,341 samples were collected: 470 from cooling systems (cooling towers) and 1,871 from potable water systems. The latter included 211 from cold-water tanks and 260 from hot-water tanks, totalling 471 from central water reservoirs 136 from showers, 1,172 from unfiltered taps and 92 from filtered taps, totalling 1,400 from peripheral points. Temperature, chlorine levels and the presence of Legionella spp. were determined. In all, 373 (15.9%) samples yielded Legionella spp. Significantly higher isolation rates were obtained from cooling towers (23.8%) versus cold- and hot-water tanks (approximately 4.7%), due to the significantly higher number of samples positive for serogroup 1 (19.4 vs 0.9-3.5%). In potable water systems, no differences were found between central water tanks and showers, but significant differences in isolation rates between central water tanks and unfiltered taps were observed (4.7 vs 19.6%) due to differences in non-serogroup 1 L. pneumophila. Filters significantly decreased isolation rates of these serotypes (11 vs 0%). Some seasonal differences were noted, with higher isolation rates in summer for legionella serogroup 1 in cooling systems and for L. pneumophila serogroups 2-14 in potable water systems. In regression models, higher temperatures were associated with colonisation in cooling systems, while lower chlorine levels were associated with colonisation in potable water systems.

Outbreak of Legionnaires’ disease in West Fife: Review of environmental guidelines needed

The objectives of this study were to investigate the source of and outbreak of Legionnaires' disease in West Fife in 2004; to control the spread of legionella from this source; and to make recommendations for future practice based on the findings from the investigation. Outbreak investigation. A multi-agency team investigated links between the cases of legionellosis using detailed interviews, movement mapping, a timeline and extensive environmental sampling. The investigation found evidence that individuals affected by legionella during this outbreak had been in, or near (within 800 m), sullage tanks in Rosyth Dockyard within a period of 4 months. In the absence of laboratory isolation of Legionella spp. from the human cases, it was impossible to state definitely that a previously unrecognized source of environmental legionella aerosolization was responsible for the outbreak. However, strong epidemiological and environmental evidence would support this hypothesis.

Legionella impletisoli sp. nov. and Legionella yabuuchiae sp. nov., isolated from soils contaminated with industrial wastes in Japan

In this study, we tried to isolate legionellae from nine Legionella DNA-positive soil samples collected from four different sites contaminated with industrial wastes in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 22 isolates of legionellae were obtained from five of the nine samples. Identification of species and/or serogroups (SGs), performed by DNA–DNA hybridization and agglutination tests, revealed that the 22 isolates consisted of ten isolates of Legionella pneumophila including five SGs, five Legionella feeleii, and one each of Legionella dumoffii, Legionella longbeachae, and Legionella jamestownensis. The species of the remaining four isolates (strains OA1-1, -2, -3, and -4) could not be determined, suggesting that these isolates may belong to new species. The 16S rDNA sequences (1476–1488 bp) of the isolates had similarities of less than 95.0% compared to other Legionella species. A phylogenetic tree created by analysis of the 16S rRNA (1270 bp) genes demonstrated that the isolates formed distinct clusters within the genus Legionella. Quantitative DNA–DNA hybridization tests on the OA1 strains indicated that OA1-1 should be categorized as a new taxon, whereas OA1-2, -3, and -4 were also genetically independent in another taxon. Based on the evaluated phenotypic and phylogenetic characteristics, it is proposed that one of these isolates from the soils, OA1-1, be classified as a novel species, Legionella impletisoli sp. nov.; the type strain is strain OA1-1 T (=JCM 13919 T=DSMZ 18493 T). The remaining three isolates belong to another novel Legionella species, Legionella yabuuchiae sp. nov.; the type strain is strain OA1-2 T (=JCM 14148 T=DSMZ 18492 T). This is the first report on the isolation of legionellae from soils contaminated with industrial wastes.

Comparison of clinical and environmental isolates of Legionella pneumophila obtained in the UK over 19 years

Between January 1980 and December 1998, 3458 cases of Legionnaires' disease were reported to the national surveillance scheme in England and Wales. Of these, 463 (13.4%) were reported as proven by culture and isolation of Legionella spp., with 96.3% being Legionella pneumophila. Serogroup (Sgp), monoclonal antibody (mAb) subgrouping and restriction fragment length polymorphism (RFLP) analysis data were obtained for 321 (69.3%) of these, of which 284 were classified as being unrelated to any other isolate in the study. Typing data were also available for 117 unrelated environmental isolates of L. pneumophila obtained from England and Wales, giving a total of 401 unrelated isolates in the study. Of the clinical isolates, 88.0% were Sgp1, compared with only 42.7% of environmental isolates (p <0.001); 79.6% of clinical isolates were subgrouped as mAb2+, compared with only 12.8% of environmental isolates (p <0.001). RFLP typing identified 67 types among the 401 isolates, with clinical isolates showing significantly less diversity than environmental isolates (index of diversity (IOD) 0.944 vs. 0.958; p <0.05), with three RFLP types (1, 5 and 14) accounting for 40.0% of all clinical isolates. Combining the phenotypic and genotypic data resulted in 173 distinct phenons, with clinical isolates showing significantly less diversity than environmental isolates (IOD 0.964 vs. 0.996; p <0.01). Three phenons accounted for 30% of all clinical isolates. These data strongly suggest that some strains of L. pneumophila are more likely to cause human infection than would be expected from their distribution in the environment.

Nosocomial Legionnaires' disease caused by Legionella pneumophila serogroup 6: Implication of the sequence-based typing method (SBT)

Sequence-based typing (SBT) was used to determine the allelic profiles of 3 sporadic clinical isolates as well as 7 environmental isolates of Legionella pneumophila serogroup 6, isolated at the Karolinska Hospital during 2004. The clinical isolates were cultured from patients with nosocomial Legionnaires' disease (LD), while the environmental isolates were cultured from potable water sources of the hospital wards in the close vicinity of the 3 patients being investigated. The genes sequenced for the construction of the SBT profile included flaA, pilE, asd, mip, mompS and proA, in this pre-determined order and the allelic profile of the 10 isolates was identical (3, 13, 1, 28, 14, 9). Furthermore, 2 of the isolates, 1 clinical and 1 environmental, were analysed using the amplified fragment length polymorphism analysis (AFLP). The AFLP genotype of both isolates was congruent. Eight of 9 control L. pneumophila serogroup 6 isolates had the same SBT profile as the study isolates. We conclude that the environmental strain isolated from our hospital's drinking water is indistinguishable genotypically from the 3 clinical isolates of Legionella. However, this genotype of L. pneumophila is geographically widespread. Thus, results of genotyping must be evaluated in conjunction with the clinical and epidemiological data.

Legionnaires’ disease and gardening

Legionella longbeachae was cultured from the sputum of a patient suffering from Legionnaires' disease. Source identification efforts included analysis of samples of potting soil from the patient's garden, and a genotypically indistinguishable strain of L. longbeachae was cultured from this material. Following examination of a national collection of Legionella isolates, two more patients with indistinguishable genotypes were identified. One of these patients had visited a garden centre in the same municipality in which the index patient had acquired his potting soil. The study demonstrated the value of systematic collection of identification data and patient isolates over a prolonged period.

A community outbreak of Legionnaires' disease: evidence of a cooling tower as the source

A community outbreak of Legionella pneumonia in the district of Cerdanyola, Mataró (Catalonia, Spain) was investigated in an epidemiological, environmental and molecular study. Each patient was interviewed to ascertain personal risk-factors and the clinical and epidemiological data. Isolates of Legionella from patients and water samples were subtyped by pulsed-field gel electrophoresis. Between 7 August and 25 August 2002, 113 cases of Legionella pneumonia fulfilling the outbreak case definition criteria were reported, with 84 (74%) cases being located within a 500-m radius of the suspected cooling tower source. In this area, the relative risk of being infected was 54.6 (95% CI 25.3-118.1) compared with individuals living far from the cooling tower. Considering the population residing in the Cerdanyola district (28,256 inhabitants) as a reference population, the attack rate for the outbreak was 399.9 cases/100,000 inhabitants, and the case fatality rate was 1.8%. A single DNA subtype was observed among the ten clinical isolates, and one of the subtypes from the cooling tower matched exactly with the clinical subtype. Nine days after closing the cooling tower, new cases of pneumonia caused by Legionella ceased to appear. The epidemiological features of the outbreak, and the microbiological and molecular investigations, implicated the cooling tower as the source of infection.

Isolation and characterization of Legionella spp. and Pseudomonas spp. from greenhouse misting systems

Greenhouse misting systems used for watering plants produce fine aerosols. They are a possible cause for bacterial infections. This study investigates the colonization of greenhouse misting systems with Legionella spp. and Pseudomonas spp. and evaluates a possible health hazard. Between June and September 2003, a total of 80 water samples were collected in 20 different greenhouse systems in Germany, each tested on two different occasions. Each time, water was drawn at a central tap and at the outlet of spray nozzles. Sampled greenhouses were used to cultivate various plants and trees for commercial, recreational or scientific reasons, some of them in tropical conditions. Legionella spp. were detected in 10% of the systems (two systems), but only in low numbers. On the contrary, Pseudomonas spp. were recovered from 70% of the greenhouse watering systems (14 systems), occasionally at counts greater than 10,000 CFU per 100 ml. A random amplified polymorphic DNA polymerase chain reaction typing method was used to demonstrate that each colonized greenhouse had one or several individual strains of Legionella and Pseudomonas that could not be detected in any other system. This study demonstrates that aerosolizing greenhouse watering systems may be contaminated with Legionella or Pseudomonas which under certain circumstances could become a potential source of infection for workers and visitors. The study results indicate that greenhouse misting systems should be included in Legionella and Pseudomonas monitoring and control programs.