New Multilocus Sequence Typing Scheme for Enterococcus faecium Based on Whole Genome Sequencing Data.
Microbiology spectrum
The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.
10.1128/spectrum.05107-22
Multilocus sequence typing (MLST) of clinical and environmental isolates of Cryptococcus neoformans and Cryptococcus gattii in six departments of Colombia reveals high genetic diversity.
Revista da Sociedade Brasileira de Medicina Tropical
INTRODUCTION:The average annual incidence of cryptococcosis in Colombia is 0.23 cases per 100,000 inhabitants in the general population, and 1.1 cases per 1000 in inhabitants with Acquired Immune Deficiency Syndrome (AIDS). In addition, the causal fungus has been isolated from the environment, with serotypes A-B and C in different regions. This study aims to determine the genetic association between clinical and environmental isolates of C. neoformans/C. gattii in Colombia. METHODS:Multilocus sequence typing (MLST) was used to identify possible clones, providing information about the epidemiology, ecology, and etiology of this pathogen in Colombia. RESULTS:A total of 110 strains, both clinical (n=61) and environmental (n=49), with 21 MLST sequence types (ST) of C. neoformans (n=14STs) and C. gattii (n=7STs) were identified. The STs which shared clinical and environmental isolate sources were grouped in different geographical categories; for C. neoformans, ST93 was identified in six departments, ST77 in five departments; and for C. gattii, ST25 was identified in three departments and ST79 in two. CONCLUSIONS:High genetic diversity was found in isolates of C. neoformans/gattii by MLST, suggesting the presence of environmental sources harboring strains which may be sources of infection for humans, especially in immunocompromised patients; these data contribute to the information available in the country on the distribution and molecular variability of C. neoformans and C. gattii isolates recovered in Colombia.
10.1590/0037-8682-0422-2019
Sequencing Independent Molecular Typing of Staphylococcus aureus Isolates: Approach for Infection Control and Clonal Characterization.
Dufkova Kristyna,Bezdicek Matej,Cuprova Kristina,Pantuckova Dagmar,Nykrynova Marketa,Brhelova Eva,Kocmanova Iva,Hodova Silvie,Hanslianova Marketa,Juren Tomas,Lipovy Bretislav,Mayer Jiri,Lengerova Martina
Microbiology spectrum
Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations. The main aim of the presented study was to determine and optimize a novel sequencing independent approach that enables molecular typing of S. aureus isolates and elucidates the transmission of emergent clones between patients. In total, 987 S. aureus isolates including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates were used to evaluate the novel typing approach combining high-resolution melting (HRM) analysis of multilocus sequence typing (MLST) genes (mini-MLST) and gene (spa-HRM). The novel approach's discriminatory ability was evaluated by whole-genome sequencing (WGS). The clonal relatedness of tested isolates was set by the BURP and BURST approach using spa and MLST data, respectively. Mini-MLST classified the S. aureus isolates into 38 clusters, followed by spa-HRM classifying the isolates into 101 clusters. The WGS proved HRM-based methods to effectively differentiate between related S. aureus isolates. Visualizing evolutionary relationships among different spa-types provided by the BURP algorithm showed comparable results to MLST/mini-MLST clonal clusters. We proved that the combination of mini-MLST and spa-HRM is rapid, reproducible, and cost-efficient. In addition to high discriminatory ability, the correlation between spa evolutionary relationships and mini-MLST clustering allows the variability in population structure to be monitored. Rapid and cost-effective molecular typing tools for Staphylococcus aureus epidemiological applications such as transmission tracking, source attribution and outbreak investigations are highly desirable. High-resolution melting based methods are effective alternative to those based on sequencing. Their good reproducibility and easy performance allow prospective typing of large set of isolates while reaching great discriminatory power. In this study, we established a new epidemiological approach to S. aureus typing. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.
10.1128/spectrum.01817-21
MentaLiST - A fast MLST caller for large MLST schemes.
Feijao Pedro,Yao Hua-Ting,Fornika Dan,Gardy Jennifer,Hsiao William,Chauve Cedric,Chindelevitch Leonid
Microbial genomics
MLST (multi-locus sequence typing) is a classic technique for genotyping bacteria, widely applied for pathogen outbreak surveillance. Traditionally, MLST is based on identifying sequence types from a small number of housekeeping genes. With the increasing availability of whole-genome sequencing data, MLST methods have evolved towards larger typing schemes, based on a few hundred genes [core genome MLST (cgMLST)] to a few thousand genes [whole genome MLST (wgMLST)]. Such large-scale MLST schemes have been shown to provide a finer resolution and are increasingly used in various contexts such as hospital outbreaks or foodborne pathogen outbreaks. This methodological shift raises new computational challenges, especially given the large size of the schemes involved. Very few available MLST callers are currently capable of dealing with large MLST schemes. We introduce MentaLiST, a new MLST caller, based on a k-mer voting algorithm and written in the Julia language, specifically designed and implemented to handle large typing schemes. We test it on real and simulated data to show that MentaLiST is faster than any other available MLST caller while providing the same or better accuracy, and is capable of dealing with MLST schemes with up to thousands of genes while requiring limited computational resources. MentaLiST source code and easy installation instructions using a Conda package are available at https://github.com/WGS-TB/MentaLiST.
10.1099/mgen.0.000146
Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA).
Lüdeke Catharina H M,Gonzalez-Escalona Narjol,Fischer Markus,Jones Jessica L
Frontiers in microbiology
Vibrio parahaemolyticus is a leading cause of seafood-borne infections in the US. This organism has a high genetic diversity that complicates identification of strain relatedness and epidemiological investigations. However, sequence-based analysis methods are promising tools for these identifications. In this study, Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was performed on 58 V. parahaemolyticus isolates (28 of oyster and 30 of clinical origin), to identify differences in phylogeny. The results obtained by both methods were compared to Pulsed-Field Gel Electrophoresis (PFGE) patterns determined in a previous study. Forty-one unique sequence types (STs) were identified by MLST among the 58 isolates. Almost half of the isolates (22) belonged to a new ST and added to the MLST database. A ST could not be generated for 5 (8.6%) isolates, primarily due to an untypable recA locus. Analysis with eBURST did not identify any clonal complex among the strains analyzed and revealed 37 singeltons with 4 of them forming 2 groups (1 of them SLV, and the other a DLV). An established MLVA assay, targeting 12 total genes through three separate 4-plex PCRs, was successfully adapted to high resolution melt (HRM) analysis with faster and easier experimental setup; resulting in 58 unique melt curve patterns. HRM-MLVA was capable of differentiating isolates within the same PFGE cluster and having the same ST. Conclusively, combining the three methods PFGE, MLST, and HRM-MLVA, for the phylogenetic analysis of V. parahaemolyticus resulted in a high resolution subtyping scheme for V. parahaemolyticus. This scheme will be useful as a phylogenetic research tool and as an improved method for outbreak investigations for V. parahaemolyticus.
10.3389/fmicb.2015.00564
Transmission patterns of Streptococcus mutans demonstrated by a combined rep-PCR and MLST approach.
Momeni Stephanie S,Whiddon Jennifer,Moser Stephen A,Childers Noel K
Clinical oral investigations
OBJECTIVE:Clinical typing methods of the oral pathogen Streptococcus mutans with molecular analysis can be very specific, but expensive. Repetitive extragenic palindromic PCR (rep-PCR) is a relatively inexpensive pre-screening alternative for isolate selection for additional analyses. This study evaluated the prediction accuracy of using rep-PCR to identify S. mutans multilocus sequence typing (MLST) sequence types (ST) among children and their family members. Potential S. mutans strain sources were evaluated for evidence of transmission. MATERIAL AND METHODS:Ten dendrograms (rep-PCR), with 20 isolates each of the 10 most common S. mutans genotypes, were generated from different subjects. Using a cut-off of 98% similarity, 7-11 isolates of each genotype were selected for MLST analysis to determine ST match/no-match. RESULTS:Overall, rep-PCR was 75% effective at determining MLST ST match/no-match and 90% effective when applied to related individuals. Most genotypes were further differentiated by MLST. MLST ST diversity was greatest for one genotype (genotype 12, G12) and evidence of transmission among children and their family members was identified by rep-PCR and MLST. Younger children (6 months to 4 years old) shared ST with their mothers but 50% of older children (5-9 years old) had ST not identified in their mother. Six ST were shared between different families and probable source members were identified. CONCLUSION:This study confirms that rep-PCR offers an affordable option to predict diverse isolates for downstream applications. CLINICAL RELEVANCE:Using a combined rep-PCR and MLST approach, it is possible to track probable transmission and strain sources for S. mutans genotypes.
10.1007/s00784-018-2371-8
Comparison of MultiLocus Sequence Typing (MLST) and Microsatellite Length Polymorphism (MLP) for genotyping.
Gits-Muselli Maud,Campagne Pascal,Desnos-Ollivier Marie,Le Pape Patrice,Bretagne Stéphane,Morio Florent,Alanio Alexandre
Computational and structural biotechnology journal
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. 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; = 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 value = 0.32 ( < 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.
10.1016/j.csbj.2020.10.005
Failure of phylogeny inferred from multilocus sequence typing to represent bacterial phylogeny.
Tsang Alan K L,Lee Hwei Huih,Yiu Siu-Ming,Lau Susanna K P,Woo Patrick C Y
Scientific reports
Although multilocus sequence typing (MLST) is highly discriminatory and useful for outbreak investigations and epidemiological surveillance, it has always been controversial whether clustering and phylogeny inferred from the MLST gene loci can represent the real phylogeny of bacterial strains. In this study, we compare the phylogenetic trees constructed using three approaches, (1) concatenated blocks of homologous sequence shared between the bacterial genomes, (2) genome single-nucleotide polymorphisms (SNP) profile and (3) concatenated nucleotide sequences of gene loci in the corresponding MLST schemes, for 10 bacterial species with >30 complete genome sequences available. Major differences in strain clustering at more than one position were observed between the phylogeny inferred using genome/SNP data and MLST for all 10 bacterial species. Shimodaira-Hasegawa test revealed significant difference between the topologies of the genome and MLST trees for nine of the 10 bacterial species, and significant difference between the topologies of the SNP and MLST trees were present for all 10 bacterial species. Matching Clusters and R-F Clusters metrics showed that the distances between the genome/SNP and MLST trees were larger than those between the SNP and genome trees. Phylogeny inferred from MLST failed to represent genome phylogeny with the same bacterial species.
10.1038/s41598-017-04707-4
Multilocus sequence typing (MLST) analysis reveals many novel genotypes and a high level of genetic diversity in Candida tropicalis isolates from Italy and Africa.
Mycoses
BACKGROUND:Candida tropicalis is a human pathogenic yeast frequently isolated in Latin America and Asian-Pacific regions, although recent studies showed that it is also becoming increasingly widespread throughout several African and south-European countries. Nevertheless, relatively little is known about its global patterns of genetic variation as most of existing multilocus sequence typing (MLST) data come from Asia and there are no genotyped African isolates. OBJECTIVES:We report detailed genotyping data from a large set of C. tropicalis isolates recovered from different clinical sources in Italy, Egypt and Cameroon in order to expand the allele/genotype library of MLST database (https://pubmlst.org/ctropicalis), and to explore the genetic diversity in this species. METHODS:A total of 103 C. tropicalis isolates were genotyped using the MLST scheme developed for this species. All isolates were also tested for in vitro susceptibility to various antifungals to assess whether certain genotypes were associated with drug-resistance. RESULTS AND CONCLUSIONS:A total of 104 different alleles were detected across the MLST-loci investigated. The allelic diversity found at these loci resulted in 51 unique MLST genotypes of which 36 (70.6%) were novel. Global optimal eBURST analysis identified 18 clonal complexes (CCs) and confirm the existence of a specific Italian-cluster (CC36). Three CCs were also statistically associated with fluconazole resistance, which was elevated in Cameroon and Egypt. Our data show high genetic diversity in our isolates suggesting that the global population structure of C. tropicalis is still poorly understood. Moreover, its clinical impact in Italy, Egypt and Cameroon appears to be relevant and should be carefully considered.
10.1111/myc.13483
Multilocus Sequence Typing (MLST) of Chlamydiales.
Jelocnik Martina,Polkinghorne Adam,Pannekoek Yvonne
Methods in molecular biology (Clifton, N.J.)
Developed two decades ago as a molecular method to provide definite characterization of a bacterial isolate, Multilocus Sequence Typing (MLST) is today globally adopted as a universal fine-detailed molecular typing tool and has been applied to numerous pathogenic and nonpathogenic bacterial as well eukaryotic organisms. MLST utilizes DNA sequence of several conserved housekeeping (HK) genes which are assigned an allelic number, which then collectively constitute an allelic profile or sequence type (ST), a "molecular barcode" of the interrogated bacterial strain or a eukaryotic organism. Here, we describe the principles and molecular approaches for generating MLST data for an analysis of a bacteria in the order Chlamydiales, using a Chlamydia pecorum-specific MLST scheme as an example.
10.1007/978-1-4939-9694-0_7
Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology: Comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013-2015.
Lytsy Birgitta,Engstrand Lars,Gustafsson Åke,Kaden Rene
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
Vancomycin-resistant enterococci (VRE) are a challenge to the health-care system regarding transmission rate and treatment of infections. VRE outbreaks have to be controlled from the first cases which means that appropriate and sensitive genotyping methods are needed. The aim of this study was to investigate the applicability of whole genome sequencing based analysis compared to Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) in epidemiological investigations as well as the development of a user friendly method for daily laboratory use. Out of 14,000 VRE - screening samples, a total of 60 isolates positive for either vanA or vanB gene were isolated of which 38 were from patients with epidemiological links from three suspected outbreaks at Uppsala University Hospital. The isolates were genotypically characterised with PFGE, MLST, and WGS based core genome Average Nucleotide Identity analysis (cgANI). PFGE was compared to WGS and MLST regarding reliability, resolution, and applicability capacity. The PFGE analysis of the 38 isolates confirmed the epidemiological investigation that three outbreaks had occurred but gave an unclear picture for the largest cluster. The WGS analysis could clearly distinguish six ANI clusters for those 38 isolates. As result of the comparison of the investigated methods, we recommend WGS-ANI analysis for epidemiological issues with VRE. The recommended threshold for Enterococcus faecium VRE outbreak strain delineation with core genome based ANI is 98.5%. All referred sequences of this study are available from the NCBI BioProject number PRJNA301929.
10.1016/j.meegid.2017.06.010
Classification and prediction of strains with different MLST allelic profiles SERS spectral analysis.
PeerJ
The Gram-negative non-motile is currently a major cause of hospital-acquired (HA) and community-acquired (CA) infections, leading to great public health concern globally, while rapid identification and accurate tracing of the pathogenic bacterium is essential in facilitating monitoring and controlling of outbreak and dissemination. Multi-locus sequence typing (MLST) is a commonly used typing approach with low cost that is able to distinguish bacterial isolates based on the allelic profiles of several housekeeping genes, despite low resolution and labor intensity of the method. Core-genome MLST scheme (cgMLST) is recently proposed to sub-type and monitor outbreaks of bacterial strains with high resolution and reliability, which uses hundreds or thousands of genes conserved in all or most members of the species. However, the method is complex and requires whole genome sequencing of bacterial strains with high costs. Therefore, it is urgently needed to develop novel methods with high resolution and low cost for bacterial typing. Surface enhanced Raman spectroscopy (SERS) is a rapid, sensitive and cheap method for bacterial identification. Previous studies confirmed that classification and prediction of bacterial strains SERS spectral analysis correlated well with MLST typing results. However, there is currently no similar comparative analysis in strains. In this pilot study, 16 strains with different sequencing typings (STs) were selected and a phylogenetic tree was constructed based on core genome analysis. SERS spectra (N = 45/each strain) were generated for all the strains, which were then comparatively classified and predicted six representative machine learning (ML) algorithms. According to the results, SERS technique coupled with the ML algorithm support vector machine (SVM) could achieve the highest accuracy (5-Fold Cross Validation = 100%) in terms of differentiating and predicting all the strains that were consistent to corresponding MLSTs. In sum, we show in this pilot study that the SERS-SVM based method is able to accurately predict MLST types, which has the application potential in clinical settings for tracing dissemination and controlling outbreak of in hospitals and communities with low costs and high rapidity.
10.7717/peerj.16161