New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene
ABSTRACT The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021-12-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/Spectrum.01161-21 |
| _version_ | 1828126807049633792 |
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| author | Maria Rosaria Pascale Silvano Salaris Marta Mazzotta Luna Girolamini Giulia Fregni Serpini Laura Manni Antonella Grottola Sandra Cristino |
| author_facet | Maria Rosaria Pascale Silvano Salaris Marta Mazzotta Luna Girolamini Giulia Fregni Serpini Laura Manni Antonella Grottola Sandra Cristino |
| author_sort | Maria Rosaria Pascale |
| collection | DOAJ |
| description | ABSTRACT The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes. IMPORTANCE Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields. |
| first_indexed | 2024-04-11T15:39:58Z |
| format | Article |
| id | doaj.art-19f0bd8d84e64e3b811375aa1f93c329 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T15:39:58Z |
| publishDate | 2021-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-19f0bd8d84e64e3b811375aa1f93c3292022-12-22T04:15:50ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-12-019310.1128/Spectrum.01161-21New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB GeneMaria Rosaria Pascale0Silvano Salaris1Marta Mazzotta2Luna Girolamini3Giulia Fregni Serpini4Laura Manni5Antonella Grottola6Sandra Cristino7Department of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, ItalyDepartment of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, ItalyDepartment of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, ItalyDepartment of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, ItalyRegional Reference Laboratory for Clinical Diagnosis of Legionellosis, Molecular Microbiology and Virology Unit, University Hospital-Policlinico Modena, Modena, ItalyRegional Reference Laboratory for Clinical Diagnosis of Legionellosis, Molecular Microbiology and Virology Unit, University Hospital-Policlinico Modena, Modena, ItalyRegional Reference Laboratory for Clinical Diagnosis of Legionellosis, Molecular Microbiology and Virology Unit, University Hospital-Policlinico Modena, Modena, ItalyDepartment of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna, ItalyABSTRACT The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes. IMPORTANCE Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.https://journals.asm.org/doi/10.1128/Spectrum.01161-21Legionella non-pneumophila speciesLegionella identificationmip and rpoB gene sequencingnucleotide identity percentageinterspecies and intraspecies variation intervalclassification scheme |
| spellingShingle | Maria Rosaria Pascale Silvano Salaris Marta Mazzotta Luna Girolamini Giulia Fregni Serpini Laura Manni Antonella Grottola Sandra Cristino New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene Microbiology Spectrum Legionella non-pneumophila species Legionella identification mip and rpoB gene sequencing nucleotide identity percentage interspecies and intraspecies variation interval classification scheme |
| title | New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene |
| title_full | New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene |
| title_fullStr | New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene |
| title_full_unstemmed | New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene |
| title_short | New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene |
| title_sort | new insight regarding legionella non pneumophila species identification comparison between the traditional mip gene classification scheme and a newly proposed scheme targeting the rpob gene |
| topic | Legionella non-pneumophila species Legionella identification mip and rpoB gene sequencing nucleotide identity percentage interspecies and intraspecies variation interval classification scheme |
| url | https://journals.asm.org/doi/10.1128/Spectrum.01161-21 |
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