Dataset Information

Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis.

ABSTRACT: We have developed a procedure to test the efficiency and reliability of sequencing of Legionella pneumophila genes directly from respiratory samples and have compared the results with those derived from cultured isolates. We tried to obtain the nucleotide sequences of six protein-coding loci included in the sequence-based typing scheme for Legionella pneumophila and three intergenic regions from 132 samples corresponding to 106 patients positive for urine antigen. A seminested PCR approach was used to amplify and sequence these nine loci directly from respiratory samples. Nucleotide sequences were directly obtained for 23 Legionella isolates and also for 66 respiratory secretions from a total of 69 patients. The efficiency of sequencing from respiratory secretions was higher than that of sequencing after the isolation of the Legionella isolates. Moreover, the perfect match between the sequences obtained by both approaches when respiratory samples and cultured isolates from the same patient were available corroborates the suitability of the direct sequencing approach for the identification of Legionella species and molecular epidemiology studies with Legionella species.

SUBMITTER: Coscolla M

PROVIDER: S-EPMC2738094 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis.

Coscollá Mireia M González-Candelas Fernando F

Journal of clinical microbiology 20090715 9

We have developed a procedure to test the efficiency and reliability of sequencing of Legionella pneumophila genes directly from respiratory samples and have compared the results with those derived from cultured isolates. We tried to obtain the nucleotide sequences of six protein-coding loci included in the sequence-based typing scheme for Legionella pneumophila and three intergenic regions from 132 samples corresponding to 106 patients positive for urine antigen. A seminested PCR approach was u ...[more]

PMID: 19605573

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Project description:BACKGROUND: Legionella pneumophila is an opportunistic pathogen of humans where the source of infection is usually from contaminated man-made water systems. When an outbreak of Legionnaires' disease caused by L. pneumophila occurs, it is necessary to discover the source of infection. A seven allele sequence-based typing scheme (SBT) has been very successful in providing the means to attribute outbreaks of L. pneumophila to a particular source or sources. Particular sequence types described by this scheme are known to exhibit specific phenotypes. For instance some types are seen often in clinical cases but are rarely isolated from the environment and vice versa. Of those causing human disease some types are thought to be more likely to cause more severe disease. It is possible that the genetic basis for these differences are vertically inherited and associated with particular genetic lineages within the population. In order to provide a framework within which to test this hypothesis and others relating to the population biology of L. pneumophila, a set of genomes covering the known diversity of the organism is required. RESULTS: Firstly, this study describes a means to group L. pneumophila strains into pragmatic clusters, using a methodology that takes into consideration the genetic forces operating on the population. These clusters can be used as a standardised nomenclature, so those wishing to describe a group of strains can do so. Secondly, the clusters generated from the first part of the study were used to select strains rationally for whole genome sequencing (WGS). The data generated was used to compare phylogenies derived from SBT and WGS. In general the SBT sequence type (ST) accurately reflects the whole genome-based genotype. Where there are exceptions and recombination has resulted in the ST no longer reflecting the genetic lineage described by the whole genome sequence, the clustering technique employed detects these sequence types as being admixed, indicating their mixed inheritance. CONCLUSIONS: We conclude that SBT is usually a good proxy for the genetic lineage described by the whole genome, and therefore utility of SBT is still suitable until the technology and economics of high throughput sequencing reach the point where routine WGS of L. pneumophila isolates for outbreak investigation is feasible.

Project description:Sequence-based typing (SBT) of Legionella pneumophila is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires' disease. In the L. pneumophila SBT scheme, mompS2 is one of seven genes that determine the sequence type (ST). The Legionella genome typically contains two copies of mompS (mompS1 and mompS2). When they are non-identical it can be challenging to determine the mompS2 allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 L. pneumophila genomes, there were 62 STs, 18 of which carried non-identical mompS copies. Using short-reads, the mompS2 allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried mompS1 allele 7 and mompS2 allele 15, were assigned an incorrect mompS2 allele and/or mompS gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical mompS copies, short-read assemblers occasionally failed to resolve the structure of the mompS-region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the mompS2 allele from short-reads, assess if other short-read methods were able to resolve the mompS-region, and investigate the possibility of using long-reads to obtain the mompS alleles, and thereby perform L. pneumophila SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the mompS-region and thus SBT from short-reads, but no method consistently solved the mompS2 allele. By using Oxford Nanopore Technology (ONT) sequencing together with Trycycler and Medaka for long-read assembly and polishing we were able to resolve the mompS copies and correctly identify the mompS2 allele, in accordance with Sanger sequencing/EQA results for all tested isolates (n=35). The remaining six genes of the SBT profile could also be determined from the ONT-only reads. The STs called from ONT-only assemblies were also consistent with hybrid-assemblies of Illumina and ONT reads. We therefore propose ONT sequencing as an alternative method to perform L. pneumophila SBT to overcome the mompS challenge observed with short-reads. To facilitate this, we have developed ONTmompS (https://github.com/marithetland/ONTmompS), an in silico approach to determine L. pneumophila ST from long-read or hybrid assemblies.

Project description:The aim of this study is to explore the dispersion, clonality, and virulence of Legionella pneumophila serogroups 2 to 14 in the Greek environment. Eighty L. pneumophila serogroup 2 to 14 strains isolated from water distribution systems of hotels, hospitals, athletic venues, and ferries in Greece were tested by monoclonal antibodies (MAbs) for serogroup discrimination and molecularly by amplified fragment length polymorphism (AFLP) for genetic diversity. Fifty-six of 80 strains were also typed by the sequence-based typing (SBT) method. ?ll strains were further analyzed for detection of two pathogenicity loci: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). Thirty-seven strains (46.2%) belonged to serogroup 6, 26 strains (32.5%) to serogroup 3, and 7 (8.8%) to other serogroups (4, 5, 8, and 10). Ten strains (12.5%) were nontypeable (NT) into the known serogroups. Thirty-nine different AFLP types were found among the 80 L. pneumophila serogroup 2 to 14 strains, and 24 different SBT types were found among the 56 strains tested. Among the 80 strains, the lvh locus was present in 75 (93.8%), the rtxA locus was found in 76 (95%), and both loci were found in 73 (91.3%) strains. This study showed that there is genetic variability of L. pneumophila serogroups 2 to 14 in the Greek environment as well as a high percentage of the pathogenicity loci. ?ntroducing an effective diagnostic test for L. pneumophila serogroups 2 to 14 in urine and promoting the examination of respiratory specimens from patients hospitalized for pneumonia in Greek hospitals are essential.In this study, the dispersion, clonality, and virulence of environmental isolates of Legionella pneumophila serogroups 2 to 14 (Lp2-14) in Greece were investigated. Genetic variability of Lp2-14 in the Greek environment was identified together with the presence of the pathogenicity loci in a high percentage of the isolates. Despite the high prevalence of Lp2-14 in the Greek environment, no clinical cases were reported, which may be due to underdiagnosis of the disease. Almost all the legionellosis cases are diagnosed in Greece by using the urine antigen test, which is specific for Lp1. There is an urgent need to improve the clinical diagnosis of legionellosis by introducing an effective diagnostic test for Lp2-14 in urine and by promoting the PCR examination of respiratory specimens from patients with compatible clinical symptoms.

Dataset Information

Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis.

Publications

Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis.

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