Journal of Microbiology

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Application of Multilocus Sequence Analysis (MLSA) for Accurate Identification of Legionella spp. Isolated from Municipal Fountains in Chengdu, China, Based on 16S rRNA, mip, and rpoB Genes: Wang Guan , Ying Xu , Da-li Chen , Jia-nan Xu , Yu Tian , Jian-ping Chen; J. Microbiol. 2012;50(1):127-136. Published online February 27, 2012; DOI: https://doi.org/10.1007/s12275-012-1243-1

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Abstract: Legionellosis (Legionnaires’ disease; LD) is a form of severe pneumonia caused by species of Legionella bacteria. Because inhalation of Legionella-contaminated aerosol is considered the major infection route, routine assessments of potential infection sources such as hot water systems, air-conditioner cooling water, and municipal fountains are of great importance. In this study, we utilized in vitro culture and multilocus sequence analysis (MLSA) targeting 16S rRNA, mip, rpoB, and mip-rpoB concatenation to isolate and identify Legionella spp. from 5 municipal fountains in Chengdu City, Sichuan Province, China. Our results demonstrated that 16S rRNA was useful for initial identification, as it could recognize isolates robustly at the genus level, while the genes mip, rpoB, and mip-rpoB concatenation could confidently discriminate Legionella species. Notably, the three subspecies of L. pneumophila could be distinguished by the analysis based on rpoB. The serotyping result of strain CD-1 was consistent with genetic analysis based on the concatenation of mip and rpoB. Despite regular maintenance and sanitizing methods, 4 of the 5 municipal fountains investigated in this study were positive for Legionella contamination. Thus, regularly scheduled monitoring of municipal fountains is urgently needed as well as vigilant disinfection. Although the application of MLSA for inspection of potential sites of infection in public areas is not standard procedure, further investigations may prove its usefulness.

Journal Article

Screening-Level Assays for Potentially Human-Infectious Environmental Legionella spp.: Helen Y. Buse , Abby Brehm , Jorge W. Santo Domingo , Nicholas J. Ashbolt; J. Microbiol. 2011;49(2):200-207. Published online May 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0233-z

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Abstract: In spite of the fact that various Legionella species are isolated from nonclinical water settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. Here we provide a screening-level approach based on an in vivo murine (A/J mouse) model and three in vitro proliferation assays using Acanthamoeba polyphaga, and THP-1 human and J774 murine macrophage cell lines to identify potentially human-infectious legionellae. As an initial demonstration the infectivity potential of three clinical (Legionella pneumophila, L. longbeacheae, and L. micdadei) and three environmental (L. dumoffii, L. maceachernii, and L. sainthelensi) legionellae were evaluated. A/J mice were intranasally infected and by 6 h post infection (p.i.), there were significant bacterial titers in the lungs. L. pneumophila, L. dumoffii, and L. micdadei densities were higher than L. longbeacheae, L. maceacherni, and L. sainthelensi at 24 h p.i. However, only L. pneumophila and L. micdadei persisted in the lungs after 48 h, indicating that the other isolates were rapidly cleared. Results from the in vitro assays showed that only L. pneumophila significantly multiplied within A. polyphaga, THP-1 and J774 cells after 72 h, but lysis of any of the in vitro hosts also flagged the strains for potential concern (e.g. L. dumoffii and L. micdadei). The results demonstrate the value of using multiple approaches to assess the potential level of pathogenicity of Legionella strains isolated from different environmental matrices.

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Genomic Diversity of Legionella pneumophila Serogroup 1 from Environmental Water Sources and Clinical Specimens Using Pulsed-Field Gel Electrophoresis (PFGE) from 1985 to 2007, Korea: Hae Kyung Lee , Yeon Ho Kang , Jae Yon Yu; J. Microbiol. 2010;48(5):547-553. Published online November 3, 2010; DOI: https://doi.org/10.1007/s12275-010-0031-z

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Abstract: The molecular typing of 202 Legionella pneumophila sg 1 isolates obtained from environmental water sources and clinical specimens from 1985 to 2007 was conducted using pulsed-field gel electrophoresis (PFGE). In this study, a total of 212 isolates were grouped into 35 different PFGE types and Type 1 was the predominant type, accounting for 28.7% in PFGE types. Type 1 and Type 8 were observed continuously from 1985 to 2007. In the analysis of the distribution of PFGE types in six geographic regions (Seoul-Incheon, Gangwon, Chungcheong, Gyeongsang, Jeolla, and Jeju), Type 1 was predominant throughout four regions except for Jeolla and Jeju, and Type 6 was observed in four regions except two regions (Gangwon and Jeju). Six clinical isolates belonged to PFGE Type 1, Type 6, Type 9, and Type 15. Type 1 among these types, was isolated from 3 patients with confirmed nosocomial infection at the hospital and Type 6, Type 9, and Type 15 were isolated 3 patients with suspected community-acquired infection. Type R, PFGE pattern of L. pneumophila sg 1 (ATCC 33152, Philadelphia-1), was not observed in the isolates evaluated in this study. Therefore, our results suggest that PFGE Type 1 was very prevalent in the environmental and clinical isolates in Korea. Type 1 was distributed continuously for many years throughout Korea.

Journal Articles

Characterization of a Spontaneous Avirulent Mutant of Legionella pneumophila Serogroup 6: Evidence of DotA and Flagellin Involvement in the Loss of Virulence: Maria Scaturro , Stefania Meschini , Giuseppe Arancia , Fontana Stefano , Maria Luisa Ricci; J. Microbiol. 2009;47(6):768-773. Published online February 4, 2010; DOI: https://doi.org/10.1007/s12275-009-0103-0

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Abstract

The pathogenesis of Legionella pneumophila mainly resides in its ability to inhibit the phagosome-lysosome fusion, which normally prevents the killing of the host cells. In order to characterize the molecular alterations that occurred in a spontaneous avirulent mutant of Legionella pneumophila serogroup 6, named Vir-, we investigated the ability of the mutant to adhere to and multiply in the WI26VA4 alveolar epithelial cell line and in human macrophages, when compared to its parental strain, Vir+. We also determined the co- localization of bacteria with LAMP-1 to gain an insight into the phagosome-lysosome fusion process. Additionally, we determined the flagellin expression and dotA nucleotide sequencing. We observed a lack of expression of flagellin and an in-frame mutation in the dotA gene. The data obtained strongly suggest the loss of virulence of the mutant could probably be due to the absence of flagellin and the dysfunctional type IV secretion system, resulting from the DotA protein being severely compromised.

Citations

Citations to this article as recorded by

Identification and characterization of genes, encoding the 3‐hydroxybutyrate dehydrogenase and a putative lipase, in an avirulent spontaneous Legionella pneumophila serogroup 6 mutant
Maria Scaturro, Cristina Barello, Melania De Giusti, Stefano Fontana, Federica Pinci, Maria Gabriella Giuffrida, Maria Luisa Ricci
APMIS.2015; 123(4): 330. CrossRef
Evidence of the Presence of a Functional Dot/Icm Type IV-B Secretion System in the Fish Bacterial Pathogen Piscirickettsia salmonis
Fernando A. Gómez, Jaime A. Tobar, Vitalia Henríquez, Mariel Sola, Claudia Altamirano, Sergio H. Marshall, Riccardo Manganelli
PLoS ONE.2013; 8(1): e54934. CrossRef

Prevalence of Antibodies in Response to Legionella Species, Analysis of a Healthy Population from Jeollanam-do Province, Korea: Hae Kyung Lee , Mi Kyeong Woo , Yong In Ju , Soo Jin Baek , Hyeon Je Song , Jin Su Choi , Sun Seog Kweon , Doo Young Jeon , Yeon Ho Kang; J. Microbiol. 2008;46(2):160-164. Published online June 11, 2008; DOI: https://doi.org/10.1007/s12275-007-0181-9

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Abstract: Seroepidemological investigation of antibodies to Legionella species in 500 healthy individuals from a single geographical location in Korea was conducted by indirect fluorescent antibody assay (IFA). Considering an antibody titer of ≥1:128 as positive reaction, 15.2% of total sera were positive. In males and females older than 40 years old, levels of IgM and IgG were 1.2% and 14%, respectively. The sera with antibody titers of ≥1:128 to Legionella species accounted for 85 sera, and 9 sera of these were reacted to more than one Legionella species. Reactivity to L. bozemanii, L. micdadei, L. longbeachae, L. pneumophila sg 6, and L. gormanii were 32.9%, 20%, 15%, 10.6%, and 8%, respectively. However, L. pneumophila sg 1, sg 2, and sg 3 did not react to any sera. Serological analysis revealed that the level of antibody in response to L. bozemanii was more prevalent than L. pneumophila. Our results suggest that the antibodies of non-L. pneumophila species, such as L. bozemanii, may be highly prevalent in healthy population within Korea. Although conclusions based on the findings of this study must be cautiously considered given that the population sampled were sourced from a single province, we have added to the knowledge base of serodiagnosis of infections due to non-L. pneumophila species in Korea.

Cloning and Sequencing of the rph Gene Encoding RNase PH from Legionella pneumophila: Se Jin Kim , Jong-Seok Lim , Nicholas P. Cianciotto , Yong-Kyung Choe; J. Microbiol. 1999;37(4):218-223.

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Abstract: Legionella pneumophila, the cause of Legionnaires disease, is able to survive intracellularily in eukaryotic cells such as monocytes, macorphages, and protozoan ogranisms. During protein biosynthesis, the rph gene encodes ribonuclease (RNase) PH which functions as a phosphorolytic nuclease that removes nucleotides following the CCA terminus of tRNA and as a nucleotidyl-transferase which adds nucleotides to the ends of RNA molecules by usingnucelside diohosphates as substrates.In this sutdy, the rph gene was screened in pUC19 library employing a DNA probe whcich was constructed from PCR based on a consensus pattern of multiple alignment of RNas PH. The encoded protein consists of 235 amino acid residues with a calculated molecualr weight of 26,112 Daltons. The RNase PH signature domains are completely conserved.

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