Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.
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ABSTRACT:
SUBMITTER: Pasic L
PROVIDER: S-EPMC7711988 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Pneumocystis jirovecii is an atypical fungus responsible for severe respiratory infections, often reported as local outbreaks in immunocompromised patients. Epidemiology of this infection, and transmission risk emphasises the need for developing genotyping techniques. Currently, two methods have emerged: Multilocus Sequence typing (MLST) and microsatellite length polymorphism (MLP). Here we compare an MLST strategy, including 2 nuclear loci and 2 mitochondrial loci, with an MLP strategy including 6 nuclear markers using 37 clinical PCR-positive respiratory samples from two French hospitals. Pneumocystis jirovecii MLST and MLP provided 30 and 35 different genotypes respectively. A higher number of mixed infections was detected using MLP (48.6% vs. 13.5% respectively; p = 0.002). Only one MLP marker (STR279) was statistically associated with the geographical origin of samples. Haplotype network inferred using the available genotypes yielded expanded network for MLP, characterized by more mutational steps as compared to MLST, suggesting that the MLP approach is more resolutive to separate genotypes. The correlation between genetic distances calculated based on MLST and MLP was modest with a R 2 value = 0.32 (p < 0.001). Finally, both genotyping methods fulfilled important criteria: (i) a discriminatory power from 97.5% to 99.5% and (ii) being quick and convenient genotyping tools. While MLP appeared highly resolutive regarding genotypes mixture within samples, using one genotyping method rather than the other may also depend on the context (i.e., MLST for investigation of suspected clonal outbreaks versus MLP for population structure study) as well as local facilities.
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Project description:Pneumocystis jirovecii pneumonia (PCP) is an opportunistic infection with airborne transmission and remains a major cause of respiratory illness among immunocompromised individuals. In recent years, several outbreaks of PCP, occurring mostly in kidney transplant recipients, have been reported. Currently, multilocus sequence typing (MLST) performed on clinical samples is considered to be the gold standard for epidemiological investigations of nosocomial clusters of PCP. However, until now, no MLST consensus scheme has emerged. The aim of this study was to evaluate the discriminatory power of eight distinct loci previously used for the molecular typing of P. jirovecii (internal transcribed spacer 1 [ITS1], cytochrome b [CYB], mitochondrial rRNA gene [mt26S], large subunit of the rRNA gene [26S], superoxide dismutase [SOD], ?-tubulin [?-TUB], dihydropteroate synthase [DHPS], and dihydrofolate reductase [DHFR]) using a cohort of 33 epidemiologically unrelated patients having respiratory samples that were positive for P. jirovecii and who were admitted to our hospital between 2006 and 2011. Our results highlight that the choice of loci for MLST is crucial, as the discriminatory power of the method was highly variable from locus to locus. In all, the eight-locus-based scheme we used displayed a high discriminatory power (Hunter [H] index, 0.996). Based on our findings, a simple and alternative MLST scheme relying on three loci only (mt26S, CYB, and SOD) provides enough discriminatory power (H-index, 0.987) to be used for preliminary investigations of nosocomial clusters of PCP.
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Project description:Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.
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Project description:A multilocus sequence typing (MLST) scheme has been developed for Enterococcus faecium. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene purK and the surface protein gene esp. The MLST results suggest that epidemic lineages of E. faecium emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of E. faecium provides an excellent tool for isolate characterization and long-term epidemiologic analysis.
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Project description:We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.
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Project description:Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
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Project description:Giardia intestinalis is an intestinal protozoan most commonly found in humans. It has been grouped into 8 assemblages (A-H). Markers such as the glutamate dehydrogenase gene, triose phosphate isomerase and beta-giardin (β-giardin) have been widely used for genotyping. In addition, different genetic targets have been proposed as a valuable alternative to assess diversity and genetics of this microorganism. Thus, our objective was to evaluate new markers for the study of the diversity and intra-taxa genetic structure of G. intestinalis in silico and in DNA obtained from stool samples. We analysed nine constitutive genes in 80 complete genome sequences and in a group of 24 stool samples from Colombia. Allelic diversity was evaluated by locus and for the concatenated sequence of nine loci that could discriminate up to 53 alleles. Phylogenetic reconstructions allowed us to identify AI, AII and B assemblages. We found evidence of intra- and inter-assemblage recombination events. Population structure analysis showed genetic differentiation among the assemblages analysed.
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Project description:Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.
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Project description:Streptococcus mutans is one of the primary pathogens responsible for the development of dental caries. Recent whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) approaches have been employed in epidemiological studies of specific human pathogens. However, this approach has not been reported in studies of S. mutans Here, we therefore developed a cgMLST scheme for S. mutans We surveyed 199 available S. mutans genomes as a means of identifying cgMLST targets, developing a scheme that incorporated 594 targets from the S. mutans UA159 reference genome. Sixty-eight sequence types (STs) were identified in this cgMLST scheme (cgSTs) in 80 S. mutans isolates from 40 children that were sequenced in this study, compared to 35 STs identified by multilocus sequence typing (MLST). Fifty-six cgSTs (82.35%) were associated with a single isolate based on our cgMLST scheme, which is significantly higher than in the MLST scheme (11.43%). In addition, 58.06% of all MLST profiles with ≥2 isolates were further differentiated by our cgMLST scheme. Topological analyses of the maximum likelihood phylogenetic trees revealed that our cgMLST scheme was more reliable than the MLST scheme. A minimum spanning tree of 145 S. mutans isolates from 10 countries developed based upon the cgMLST scheme highlighted the diverse population structure of S. mutans This cgMLST scheme thus offers a new molecular typing method suitable for evaluating the epidemiological distribution of this pathogen and has the potential to serve as a benchmark for future global studies of the epidemiological nature of dental caries.IMPORTANCEStreptococcus mutans is regarded as a major pathogen responsible for the onset of dental caries. S. mutans can transmit among people, especially within families. In this study, we established a new epidemiological approach to S. mutans classification. This approach can effectively differentiate among closely related isolates and offers superior reliability relative to that of the traditional MLST molecular typing method. As such, it has the potential to better support effective public health strategies centered around this bacterium that are aimed at preventing and treating dental caries.
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Project description:Enterococcus faecium, a common inhabitant of the human gut, has emerged in the last 2 decades as an important multidrug-resistant nosocomial pathogen. Since the start of the 21st century, multilocus sequence typing (MLST) has been used to study the molecular epidemiology of E. faecium. However, due to the use of a small number of genes, the resolution of MLST is limited. Whole-genome sequencing (WGS) now allows for high-resolution tracing of outbreaks, but current WGS-based approaches lack standardization, rendering them less suitable for interlaboratory prospective surveillance. To overcome this limitation, we developed a core genome MLST (cgMLST) scheme for E. faecium. cgMLST transfers genome-wide single nucleotide polymorphism(SNP) diversity into a standardized and portable allele numbering system that is far less computationally intensive than SNP-based analysis of WGS data. The E. faecium cgMLST scheme was built using 40 genome sequences that represented the diversity of the species. The scheme consists of 1,423 cgMLST target genes. To test the performance of the scheme, we performed WGS analysis of 103 outbreak isolates from five different hospitals in the Netherlands, Denmark, and Germany. The cgMLST scheme performed well in distinguishing between epidemiologically related and unrelated isolates, even between those that had the same sequence type (ST), which denotes the higher discriminatory power of this cgMLST scheme over that of conventional MLST. We also show that in terms of resolution, the performance of the E. faecium cgMLST scheme is equivalent to that of an SNP-based approach. In conclusion, the cgMLST scheme developed in this study facilitates rapid, standardized, and high-resolution tracing of E. faecium outbreaks.
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