Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis.
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ABSTRACT:
SUBMITTER: Coscolla M
PROVIDER: S-EPMC2738094 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Inadequate discriminatory power to distinguish between L. pneumophila isolates, especially those belonging to disease-related prevalent sequence types (STs) such as ST1, ST36 and ST47, is an issue of SBT scheme. In this study, we developed a multilocus sequence typing (MLST) scheme based on two non-virulence loci (trpA, cca) and three virulence loci (icmK, lspE, lssD), to genotype 110 L. pneumophila isolates from various natural and artificial water sources in Guangdong province of China, and compared with the SBT. The isolates were assigned to 33 STs of the SBT and 91 new sequence types (nSTs) of the MLST. The indices of discrimination (IODs) of SBT and MLST were 0.920 and 0.985, respectively. Maximum likelihood trees of the concatenated SBT and MLST sequences both showed distinct phylogenetic relationships between the isolates from the two environments. More intragenic recombinations were detected in nSTs than in STs, and they were both more abundant in natural water isolates. We found out the MLST had a high discriminatory ability for the disease-associated ST1 isolates: 22 ST1 isolates were assigned to 19 nSTs. Furthermore, we assayed the discrimination of the MLST for 29 reference strains (19 clinical and 10 environmental). The clinical strains were assigned to eight STs and ten nSTs. The MLST could also subtype the prevalent clinical ST36 or ST47 strains: eight ST36 strains were subtyped into three nSTs and two ST47 strains were subtyped into two nSTs. We found different distribution patterns of nSTs between the environmental and clinical ST36 isolates, and between the outbreak clinical ST36 isolates and the sporadic clinical ST36 isolates. These results together revealed the MLST scheme could be used as part of a typing scheme that increased discrimination when necessary.
| S-EPMC5794064 | biostudies-literature
Project description:Sequence type from Brazilian Legionella pneumophila isolates
| PRJEB71865 | ENA
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.
| S-EPMC3877988 | biostudies-literature
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.
| S-EPMC10226664 | biostudies-literature
Project description:A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.
| S-EPMC1153775 | biostudies-literature
Project description:Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.
| S-EPMC165343 | biostudies-literature
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.
| S-EPMC5068164 | biostudies-literature
Project description:Legionella pneumophila is an important human pathogen causing Legionnaires' disease. In this study, whole genome sequencing (WGS) was used to study the characteristics and population structure of L. pneumophila strains. We sequenced and compared 53 isolates of L. pneumophila covering different serogroups and sequence-based typing (SBT) types (STs). We found that 1,896 single-copy orthologous genes were shared by all isolates and were defined as the minimum core genome (MCG) of L. pneumophila. A total of 323,224 single-nucleotide polymorphisms (SNPs) were identified among the 53 strains. After excluding 314,059 SNPs which were likely to be results of recombination, the remaining 9,165 SNPs were referred to as MCG SNPs. Population Structure analysis based on MCG divided the 53 L. pneumophila into nine MCG groups. The within-group distances were much smaller than the between-group distances, indicating considerable divergence between MCG groups. MCG groups were also supplied by phylogenetic analysis and may be considered as robust taxonomic units within L. pneumophila. Among the nine MCG groups, eight showed high intracellular growth ability while one showed low intracellular growth ability. Furthermore, MCG typing also showed high resolution in subtyping ST1 strains. The results obtained in this study provided significant insights into the evolution, population structure and pathogenicity of L. pneumophila.
| S-EPMC4766850 | biostudies-other
Project description:An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.
| S-EPMC2884934 | biostudies-literature
Project description:We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.
| S-EPMC3697553 | biostudies-literature