As a type of
Humans ; Legionella/genetics* ; Legionella longbeachae/genetics* ; Legionellosis/drug therapy* ; Pneumonia/diagnosis* ; Retrospective Studies

OBJECTIVE: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. METHODS: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. RESULTS: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. CONCLUSION L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.
Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; China ; Genotyping Techniques ; Humans ; Legionella pneumophila ; genetics ; growth & development ; isolation & purification ; RNA, Ribosomal, 16S ; genetics ; Water Microbiology

OBJECTIVETo understand the mechanism of invasion by Legionella dumoffii.

METHODSThe L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR.

RESULTSThe transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain..

CONCLUSIONOur results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells.

A549 Cells ; Animals ; Genes, Bacterial ; HeLa Cells ; Humans ; Legionella ; genetics ; physiology ; Male ; Mice ; Mutation ; Operon

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged > or = 18 yr) who had an acute illness with a new cough and sputum (< or = 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and beta-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.
Anti-Bacterial Agents/therapeutic use ; Bordetella parapertussis/genetics/*isolation & purification ; Bordetella pertussis/genetics/*isolation & purification ; Bronchitis/drug therapy/*microbiology ; Chlamydophila pneumoniae/genetics/*isolation & purification ; Community-Acquired Infections/microbiology ; Female ; Humans ; Hypertension/complications ; Legionella pneumophila/genetics/*isolation & purification ; Male ; Middle Aged ; Mycoplasma pneumoniae/genetics/*isolation & purification ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Sputum/microbiology

BACKGROUND: Differentiation of atypical pathogens is important for community-acquired pneumonia (CAP). In this study, we compared sputum and nasopharyngeal swabs (NPS) for use in detection of Mycoplasma pneumoniae (MP), Chlamydophila pneumoniae (CP), and Legionella pneumophila (LP), using Seeplex PneumoBacter ACE Detection Assay (PneumoBacter; Seegene). METHODS: Sputum and NPS specimens were collected from patients in 15 hospitals. DNA was extracted from sputum using QIAamp DNA Stool Mini Kit (Qiagen) and from NPS using easyMAG (bioMerieux). Both types of specimens were evaluated by multiplex PCR using PneumoBacter. To determine the diagnostic performance of this assay, sputum samples were also tested using BD ProbeTec ET Atypical Pneumonia Assay (APA; Becton Dickinson). RESULTS: Among 217 sputum and NPS, 20 (9.2%), 2 (0.9%), and 0 sputum were positive for MP, LP, and CP, respectively, whereas 8 (3.7%) NPS were positive for MP. The sputum APA test yielded 186, 206, and 204 interpretable results for MP, LP, and CP, respectively. Of these, 21 (11.3%) were positive for MP, 2 (1.0%) were positive for LP, and 0 samples were positive for CP. Compared to APA, the sensitivity and specificity of the sputum assay for MP were 95.2% and 100.0%, respectively, whereas for the NPS assay, these were 38.1% and 93.9%. Sputum testing was more sensitive than NPS testing (P=0.002). For LP and CP diagnosis, PneumoBacter and APA tests agreed 100%. CONCLUSIONS: Specimen type is crucial and sputum is preferred over NPS for simultaneous detection of MP, LP, and CP using multiplex PCR in CAP.
Chlamydophila Infections/diagnosis ; Chlamydophila pneumoniae/*genetics/isolation & purification ; Community-Acquired Infections/*diagnosis ; DNA, Bacterial/analysis/isolation & purification ; Humans ; Legionella pneumophila/*genetics/isolation & purification ; Legionnaires' Disease/diagnosis ; Multiplex Polymerase Chain Reaction ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Nasopharynx/*microbiology ; Pneumonia, Mycoplasma/diagnosis ; Reagent Kits, Diagnostic ; Sputum/*microbiology

OBJECTIVETo investigate the contamination state of Legionella in cooling water samples from different places in Wuxi city and reveal the molecular biological characteristics of Legionella strains.

METHODS112 parallel water samples (500 ml each) were collected from 56 sites in Wuxi city during year 2009 - 2010. The samples were used for Legionella test and quantitative culture. The isolated Legionella strains were used for serotyping, pulsed-field gel electrophoresis (PFGE), sequence-based typing (SBT), and intracellular growth were tested.

RESULTSThe positive proportion of Legionella was 39. 3% (22/56) among all sampling sites. A total of 29 Legionella strains were isolated, and the serotypes include LP1, LP3, LP5 and LP6. LP1 serotype was the major one with a proportion of 65.5% (19/29). 29 Legionella strains got 17 PFGE types. There were 10 SBT types among 10 Legionella strains with different PFGE types. Comparing to LP1 strain (ATCC 33152), WX2011062 (LP6) and WX2011067 (LP5) had strong intracellular growth ability in mouse peritoneal macrophages J774 cell line (the amount of intracellular bacteria on day 0 after infection were (5.5 +/- 1.32) x 10(5), (3.9 +/- 0.60) x 10(5), (7.8 +/- 0.76) x 10(5) CFU/ml, respectively; the amount of intracellular bacteria on day 3 after infection were (58.3 +/- 1.61) x 10(5), (2700.0 +/- 655.74) x 10(5), (3066.7 +/- 208.17) x 10(5) CFU/ml, respectively).

CONCLUSIONThe Legionella contamination existed in cooling water samples from different places in Wuxi city. Legionella strains isolated showed high genetic variation. Some Legionella strains had vigorous intracellular growth ability.

Air Conditioning ; Animals ; Cells, Cultured ; Environmental Microbiology ; Legionella ; genetics ; growth & development ; isolation & purification ; Legionella pneumophila ; growth & development ; isolation & purification ; Macrophages ; microbiology ; Mice ; Serotyping ; Water Microbiology

OBJECTIVETo analyze the characteristics of Sequence-based Typing (SBT) of the Serotype 1 Legionella pneumophila (Lp1) isolated from environmental water in China, and then create a preliminary database.

METHODSA total of 82 strains of Lp1 isolated from environmental water in 9 provinces of China between 2005 and 2008 were genotyped by SBT method and Pulsed-field Gel Electrophoresis (PFGE) method. The results of the two different typing methods were then compared by cluster analysis, adopting BioNumerics version 5.1 software.

RESULTSBy SBT method, the 82 strains of Lp1 were divided into 22 ST types, of which 17 new types and one new allele was discovered. The dominant type was ST-1 type, found in 8 provinces, accounting for 46.3% (38/82). ST-1, ST-150, ST-154, ST-159, ST-160 and ST-630 types were found in more than 2 isolated-sites; while more than 2 different ST types were found in 5 isolated-sites, as site B4, B5, B6, S3 and S8. In cluster analysis, 15 ST types were grouped into three complexes (ST-1 complex, ST-154 complex and ST-149 complex); and the other 7 ST types were not assigned complex. By PFGE method, 46 banding patterns were observed. As a result of the combination of the two methods, the 82 isolates strains could be divided into 54 molecular types, which showed a reliable accordance in the cluster analysis between the two methods.

CONCLUSIONThe SBT of the Lp1 in environmental water in China was unique. From the study, a preliminary SBT database was set up.

China ; Cluster Analysis ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Legionella pneumophila ; classification ; genetics ; isolation & purification ; Serotyping ; methods ; Water Pollution

OBJECTIVETo establish a simple and rapid molecular detection for Legionella pneumophila.

METHODSThe loop-mediated isothermal amplification (LAMP) was applied for detection of Legionella pneumophila. A set of primers were designed to identify six special areas in mip gene of Legionella pneumophila. Genomic DNAs from 13 bacterial strains,including 8 Legionella pneumophila strains and 5 other bacterial strains were amplified by LAMP and general PCR method to evaluate the specificity and sensibility of LAMP.

RESULTAll positive tubes produced visible white precipitation, and no precipitation was observed in others. By adding smart green fluorescent dye, all Legionella pneumophila positive tubes presented a strong green fluorescence, while others showed weak fluorescence. The detection rate of LAMP was higher than that of general PCR. The detection limits were 576fg with genomic DNA of Legionella pneumophila,and 8 cfu/mL with positive water samples.

CONCLUSIONLAMP detection of Legionella pneumophila is an effective and low-cost method with high specificity and sensitivity requiring no special equipment.

DNA Primers ; Legionella pneumophila ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; Sensitivity and Specificity

OBJECTIVETo construct the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NIH3T3 cells.

METHODSlvgA gene flanked with CpG motifs of Legionella pneumophila was amplified by PCR. The PCR products was inserted into the eukaryotic expression plasmid pcDNA3.1/myc-his(+) to construct the recombinant plasmid pclvgA/CpG, which was subsequently transfected into NIH3T3 cells via lipofection. Immunofluorescence analysis was carried out to detect the transient expression of the plasmid in the cells.

RESULTSSequence analysis showed that the recombinant plasmid pclvgA/CpG contained the lvgA/CpG fragment with a length of 657 bp, encoding a protein of 27.7 Ku. Immunofluorescence analysis identified the transient expression of the recombinant plasmid pclvgA/CpG in NIH3T3 cells.

CONCLUSIONThe lvgA gene flanked with CpG motifs of Legionella pneumophila has been constructed successfully, and the transient expression of the recombinant plasmid pclvgA/CpG can be detected in NIH3T3 cells.

Animals ; Bacterial Vaccines ; genetics ; CpG Islands ; genetics ; Legionella pneumophila ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; Virulence Factors ; biosynthesis ; genetics

OBJECTIVETo establish a set of procedure for recovery and species identification of Legionella from the surface environmental water.

METHODSForty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEalpha plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism (AFLP) method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing.

RESULTSLegionella was recovered among 27 (61.36%) samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of L. feeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition . However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis.

CONCLUSIONThe taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.

Bacteriological Techniques ; DNA, Bacterial ; genetics ; Environmental Monitoring ; methods ; Legionella ; genetics ; isolation & purification ; RNA, Bacterial ; RNA, Ribosomal, 16S ; Water Microbiology