Environmental Isolates Of Legionella Pneumophila Research Articles

Sequence-based typing of clinical and environmental Legionella pneumophila isolates in Shenyang, China

IntroductionWe performed SBT (sequence-based typing) on clinical and environmental Legionellapneumophila isolates in Shenyang (China). We analyzed and compared the results with those obtained by PFGE (pulsed field gel electrophoresis). MethodsTwenty-two L. pneumophila isolates were collected from two patients with L. pneumophila infection, two hospitals, and 13 office buildings. There were two clinical isolates, one strain isolated from domestic tap water, another from shower water and 18 strains from cooling tower water. All these isolates were analyzed by SBT and PFGE methods. ResultsThe 22 isolates were divided into 7 types by SBT. Five isolates belonged to novel sequence types (ST2345, ST2344, ST2406, ST2407, and ST2408) and one isolate belonged to ST328. The STs were not obtained for two of the isolates. The remaining 14 isolates belonged to ST1. PFGE typing divided the 22 isolates into 14 pulsotypes. The main pulsotype was SYC, which included seven isolates. ConclusionBoth typing methods showed that predominant clonal lines exist in the Shenyang region, with high levels of genetic polymorphisms. Five novel STs were identified, indicating a unique genetic composition of L. pneumophila strains in this region, which are significantly different from those found in other environmental water systems in the world.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

Sequence-based typing of clinical and environmental Legionella pneumophila isolates in Shenyang, China

IntroductionWe performed SBT (sequence-based typing) on clinical and environmental Legionellapneumophila isolates in Shenyang (China). We analyzed and compared the results with those obtained by PFGE (pulsed field gel electrophoresis). MethodsTwenty-two L. pneumophila isolates were collected from two patients with L. pneumophila infection, two hospitals, and 13 office buildings. There were two clinical isolates, one strain isolated from domestic tap water, another from shower water and 18 strains from cooling tower water. All these isolates were analyzed by SBT and PFGE methods. ResultsThe 22 isolates were divided into 7 types by SBT. Five isolates belonged to novel sequence types (ST2345, ST2344, ST2406, ST2407, and ST2408) and one isolate belonged to ST328. The STs were not obtained for two of the isolates. The remaining 14 isolates belonged to ST1. PFGE typing divided the 22 isolates into 14 pulsotypes. The main pulsotype was SYC, which included seven isolates. ConclusionBoth typing methods showed that predominant clonal lines exist in the Shenyang region, with high levels of genetic polymorphisms. Five novel STs were identified, indicating a unique genetic composition of L. pneumophila strains in this region, which are significantly different from those found in other environmental water systems in the world.

Serotyping and detection of pathogenecity loci of environmental isolates of Legionella pneumophila using MALDI-TOF MS.

The majority of Legionnaires' disease cases is attributed to Legionella pneumophila serogroup 1 (Lp1). Moreover, pathogenicity loci lvh and rtxA were associated with the ability of Lp strains to cause the disease. Consequently, except from serogroup assignment the detection of the aforementioned virulence genes during Legionella detection in water samples, could help environmental risk assessment and the implementation of targeted control measures. To establish and validate a rapid and robust MALDI-TOF MS-based method for the assignment of Lp isolates to serogroup, and identify distinct peak biomarkers for the detection of lvh and rtxA loci during environmental investigations. Fifteen reference strains and 150 Lp environmental isolates (70 Lp1 and 80 Lp2-15 strains) were used. All strains were PCR-tested for the presence of lvh and rtxA loci. Independent training and validation strain sets were constituted and all strains were protein-extracted and submitted to MALDI-TOF MS analysis. The raw spectra of the training set strains obtained, were introduced into the Mass-Up software platform for biomarker detection, for both serogroup assignment and pathogenicity loci detection. Validation of the assigned biomarkers followed using the validation set strains. For serogroup assignment, the Mass-up analysis indicated five potential discriminating peaks and correctly classified 115 out of 132 validation set strains, displaying sensitivity of 87.5%, specificity of 86.7% and 87.1% accuracy. Concerning the pathogenicity loci detection, the Mass-up analysis indicated two ion peaks for rtxA locus discrimination and one peak for lvh locus discrimination. Concerning the lvh virulence gene, the algorithm correctly classified 113 out of 137 positive and all negative strains 14 in total-showing sensitivity of 82.5%, specificity of 100.% and 84.1% accuracy. For rtxA locus, 134 out of 134 positive and 14 out of 17 negative strains were correctly classified with sensitivity of 100%, specificity of 76.5% and 97.4% accuracy. MALDI-TOF MS displayed good performance for Lp serogroup assignment and detection of the lvh and rtxA virulence genes. These findings could contribute to the rapid, inexpensive and comprehensive case investigation and risk assessment. Further studies are needed to standardize and evaluate the method using the direct target plate protein profiling instead of protein extraction in order to simplify the protocol. .

Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental Legionella pneumophila isolates.

Background & objectives:Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment.Methods:Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at pH 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR.Results:The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers.Interpretation & conclusions:The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.

Differential Proteome Between Patient-Related and Non-related Environmental Isolates of Legionella pneumophila.

Molecular epidemiologic studies of Legionella have shown different molecular types coexisting in the same environment, with only one having the ability to trigger an outbreak. We therefore studied the proteome of isolates of these different molecular types in search of the proteins responsible for infection. In this study, we performed a differential proteomic analysis between patient-related and non-patient-related environmental isolates using two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry. Sixty-three spots were observed as being different between the two groups; 31 spots were identified corresponding to 23 different proteins. Patient-related isolates overexpressed proteins associated with metabolism, with enzymes of the tricarboxylic acid cycle and the degradation pathways being the most abundant proteins identified. However, the largest group of non-patient-related proteins was associated with stress response. Furthermore, the MOMP protein was located in different spots depending on their patient-related or non-patient-related origin, suggesting different post-translational modifications. According to these results, different bacterial adaptation pathways are activated in stress conditions which influence their ability to produce infection.

New Sequence Types for Environmental Legionella pneumophila Isolated from Kuwait

New Sequence Types for Environmental Legionella pneumophila Isolated from Kuwait A total of 102 environmental isolates of Legionella pneumophila (sg 1 = 19; sg 3 = 33; sg 4= 4 sg 7= 39; sg 10=7) were genotyped by the consensus Sequenced-Based Typing (SBT) scheme developed by the ESCMID Study Group for Legionella Infections (ESGLI former EWGLI). Results showed that the Legionella isolates were discriminated into 11 distinct SBT profiles, of which six (ST1223, ST1436, ST1555, ST1604, ST1718, and ST1719) were new to the ESGLI SBT database. Results showed that ST1718 of L. pneumophila serogroup 7 (38 isolates) was the most prevalent ST. In addition to ST1718, other prevalent STs were identified included ST336 (20 isolates), ST93 (13 isolates), and ST1 (10 isolates). Furthermore, all of the ST1 L. pneumophila sergroup 1 isolates were of the Oxford/OLDA subgroup. This is the first study to describe the use of SBT to characterize environmental Legionella pneumophila isolates from domestic water systems and cooling towers in the State of Kuwait. This baseline data will form the basis for the development of a Legionella environmental surveillance program, which will be used for future epidemiological investigations.

Phylogenetic analysis of environmental Legionella pneumophila isolates from an endemic area (Alcoy, Spain)

Environmental surveillance of Legionella pneumophila is a key component of the control measures established in urban settlements to ensure water safety and quality, with the aim of minimizing and limiting opportunistic infections in humans. In this work, we present results on the detection and genetic characterization of these bacteria in the outbreak-recurrent region of Alcoy (Comunidad Valenciana, Spain) using water and biofilm samples. We were particularly interested in studying the presence and distribution of L. pneumophila in the absence of outbreak or sporadic cases of legionellosis and in comparing the efficacy of culturing from water samples with a biofilm-based detection procedure using molecular amplification. To this end, water samples were taken from 120 sites distributed all around the city and its surroundings, as well as 60 biofilm swabs from half of the sampling sites. L. pneumophila could be isolated from water in just 4 of the locations. Touchdown PCR was applied to DNA extracted from water and also biofilm swabs, as a rapid method for both routine and outbreak investigations. L. pneumophila was detected by this method in 14 of the sites in which both water and biofilms were taken, although 13 of them tested positive using only the biofilm samples. These results show a ten-fold increase in the success rate of Legionella detection over water samples. The application of this method to study the presence of L. pneumophila in the water-supply system and risk facilities of Alcoy revealed different strains distributed in different areas of the city. Sequence Type ST578, endemic in the area and responsible for most clinical cases, was detected in one of the sampling sites. The number of positive samples correlated with water temperature but not with chlorine levels. The direct analysis of biofilm swabs improves the detection rate and genetic characterization of L. pneumophila and can complement analyses based on bacterial culture.

Molecular characterization of clinical and environmental isolates of Legionella pneumophila in Norway, 2001–2008

The aims of the study were to determine the molecular characteristics of a collection of Legionella pneumophila isolates from 45 cases with Legionnaires' disease and from 96 environmental samples, received by the national reference laboratory in Norway between 2001 and 2008, to use these characteristics to identify links between cases and suspected sources of infection, and to compare the isolate characteristics with those in other European countries. The isolates were characterized by 7-gene locus sequence-based typing and dot-blotting with monoclonal antibodies to various serogroups and subgroups. The clinical isolates represented 12.6% of the 357 cases notified in Norway between 2001 and 2008, during which 3 outbreaks of L. pneumophila serogroup 1 occurred. Outbreak cases constituted 62.2% of the cases, followed by travel-associated (24.4%) and sporadic cases (11.1%). Forty-two (93.3%) of the clinical and 69 (71.9%) of the environmental isolates were serogroup 1, and 39 (86.7%) and 50 (52.1%) isolates, respectively, carried the monoclonal antibody (Mab) 3/1 virulence-associated epitope. The clinical isolates belonged to 17 sequence types and the environmental isolates to 19 sequence types. neuA was not detected in 23 environmental isolates. Matching characteristics of sequence types and monoclonal subgroups for case and environmental isolates were obtained for all 3 outbreaks and for 2 of 5 cases of sporadic disease. Sampling during the outbreaks accounted for the higher proportion of serogroup 1 and Mab 3/1-positive environmental isolates in comparison with other European strain collections.

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.

Serotyping, ribotyping, PCR-mediated ribosomal 16S–23S spacer analysis and arbitrarily primed PCR for epidemiological studies on Legionella pneumophila

Fifty clinical and environmental isolates of Legionella pneumophila were typed serologically and by DNA fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR). Furthermore, variability in and around ribosomal operons was assessed by conventional ribotyping and PCR-mediated amplification of the spacer region separating the 16S and 23S genes. It appears that serotyping suffers from low resolution capabilities, and ribotyping and spacer PCR display intermediate resolving capabilities, whereas AP-PCR is more discriminating. Results from AP-PCR and both forms of ribotyping analysis correlate with epidemiological and environmental data. It is suggested that AP-PCR typing may be the method of choice for rapidly determining clonality among L. pneumophila isolates.

DNA polymorphisms in strains of Legionella pneumophila serogroups 3 and 4 detected by macrorestriction analysis and their use for epidemiological investigation of nosocomial legionellosis.

Genomic DNAs of clinical and environmental isolates of Legionella pneumophila belonging to serogroups 3 and 4 were analyzed by macrorestriction analysis by pulsed-field gel electrophoresis. The restriction enzymes SfiI and NotI allowed easy visual separation of epidemiologically unrelated serogroup 3 strains. Three unrelated serogroup 3 strains that were isolated from different locations were identical by this genome mapping technique. Five unrelated serogroup 4 strains were separable by this technique. The electrophoretic patterns obtained after SfiI or NotI cleavage of the DNA of strains isolated from four patients with hospital-acquired legionellosis were identical to the patterns of strains isolated from the hot water supply systems of the buildings in which the patients were hospitalized. In conclusion, macrorestriction analysis is a valuable tool for epidemiological studies of infections caused by L. pneumophila serogroups 3 and 4.

Identification of clinical and environmental isolates of Legionella pneumophila by analysis of outer-membrane proteins, ubiquinones and fatty acids

47 strains of L. pneumophila, 9 reference strains of eight serogroups, 15 clinical isolates and 23 water-derived strains from different cities were analysed with respect to three chemical constituents of the cell envelope: outer- membrane proteins (OMPs), ubiquinones and fatty acids. The OMPs were obtained by Sarkosyl-extraction of a total membrane preparation and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA-GE). The ubiquinone content was quantitated by high-performance liquid chromatography (HPLC) and the fatty acid composition was established by gas-liquid chromatography coupled with mass spectrometry (GLC-MS). In all strains the OMP yielded the known characteristic pattern with the major component of 29 kilodalton. The ubiquinone content was very consistent within different strains revealing Q-12 with 65% as the predominant feature. Q-11 and Q-13 were present in amounts of 11% and 23%, respectively. Q-14 was found in trace amounts of about 0.65%. Only a very few ubiquinones with side chains shorter than 11 residues were present. GLC-MS allowed an unequivocal identification of each fatty acid. The branched chain fatty acid i-16:0 was the predominant feature with 33% followed by the unsaturated straight-chain acid 16:1. In this respect earlier reports by other authors were confirmed but minor compounds not detected so far could be revealed as 15:1, the doublet 18:1 and i-18:0, 19:1 and 20:1. OMP profiles are easy to establish and to interpret. They are well suited for identification of the species L. pneumophila. Ubiquinone analysis permits rapid but preliminary species identification, whereas fatty acid analysis by GLC-MS is an intricate technique providing a valuable chemical marker for L. pneumophila. The biochemical analyses presented here can supplement immunological studies, clarify immunological findings and help to identify new serogroups of L. pneumophila.

Difference in virulence of environmental isolates of Legionella pneumophila.

Endemic nosocomial Legionnaires disease has occurred at our medical center for several years. Two subtypes of Legionella pneumophila serogroup 1 (UH-1 and RH-1) have been isolated in approximately equal numbers from hospital potable water. However, almost all clinical isolates have been UH-1. To assess potential differences in virulence, 50% lethal doses (LD50) and 50% infective doses (ID50) of UH-1 and RH-1 were determined by intraperitoneal infection in guinea pigs. The UH-1 LD50 was 7.41 X 10(6) CFU, which was significantly lower than the RH-1 LD50 of 9.12 X 10(7) CFU (P = 0.0001). The mean time to death in UH-1-infected guinea pigs was also significantly shorter than in RH-1-infected animals (P = 0.0008). The UH-1 ID50 was 5.8 X 10(3) CFU, and although it was lower than the RH-1 ID50 of 1.4 X 10(4) CFU, this difference was not statistically significant (P = 0.21). This study demonstrates a difference in virulence between UH-1 and RH-1 in guinea pigs. Differences in strain virulence, as demonstrated between these two subtypes, may help to explain the widespread isolation of L. pneumophila from the environment contrasted with the limited occurrence of human Legionnaires disease.

Characteristics of environmental isolates of Legionella pneumophila.

Thirty-eight cultures of Legionella pneumophila isolated from surface waters were characterized by their morphological, tinctorial, biochemical, and serological properties and by their ability to produce disease in guinea pigs. Their susceptibility to antimicrobial agents also was tested. When they were compared with clinical isolates, no important differences were found between cultures from the two sources. Sodium hippurate hydrolysis, gelatin liquefaction, pigment formation, and beta-lactamase and alkaline phosphatase activity were useful in differentiating the four described species of Legionella. Hydrolysis of diacetylfluorescein and the inability to reduce nitrate help to distinguish Legionella species from other gram-negative bacterial rods.