AutoMLST: an automated web server for generating multi-locus species trees highlighting natural product potential.
Nucleic acids research
Understanding the evolutionary background of a bacterial isolate has applications for a wide range of research. However generating an accurate species phylogeny remains challenging. Reliance on 16S rDNA for species identification currently remains popular. Unfortunately, this widespread method suffers from low resolution at the species level due to high sequence conservation. Currently, there is now a wealth of genomic data that can be used to yield more accurate species designations via modern phylogenetic methods and multiple genetic loci. However, these often require extensive expertise and time. The Automated Multi-Locus Species Tree (autoMLST) was thus developed to provide a rapid 'one-click' pipeline to simplify this workflow at: https://automlst.ziemertlab.com. This server utilizes Multi-Locus Sequence Analysis (MLSA) to produce high-resolution species trees; this does not preform multi-locus sequence typing (MLST), a related classification method. The resulting phylogenetic tree also includes helpful annotations, such as species clade designations and secondary metabolite counts to aid natural product prospecting. Distinct from currently available web-interfaces, autoMLST can automate selection of reference genomes and out-group organisms based on one or more query genomes. This enables a wide range of researchers to perform rigorous phylogenetic analyses more rapidly compared to manual MLSA workflows.
10.1093/nar/gkz282
A decade of genomic history for healthcare-associated Enterococcus faecium in the United Kingdom and Ireland.
Raven Kathy E,Reuter Sandra,Reynolds Rosy,Brodrick Hayley J,Russell Julie E,Török M Estée,Parkhill Julian,Peacock Sharon J
Genome research
Vancomycin-resistant Enterococcus faecium (VREfm) is an important cause of healthcare-associated infections worldwide. We undertook whole-genome sequencing (WGS) of 495 E. faecium bloodstream isolates from 2001-2011 in the United Kingdom and Ireland (UK&I) and 11 E. faecium isolates from a reference collection. Comparison between WGS and multilocus sequence typing (MLST) identified major discrepancies for 17% of isolates, with multiple instances of the same sequence type (ST) being located in genetically distant positions in the WGS tree. This confirms that WGS is superior to MLST for evolutionary analyses and is more accurate than current typing methods used during outbreak investigations. E. faecium has been categorized as belonging to three clades (Clades A1, hospital-associated; A2, animal-associated; and B, community-associated). Phylogenetic analysis of our isolates replicated the distinction between Clade A (97% of isolates) and Clade B but did not support the subdivision of Clade A into Clade A1 and A2. Phylogeographic analyses revealed that Clade A had been introduced multiple times into each hospital referral network or country, indicating frequent movement of E. faecium between regions that rarely share hospital patients. Numerous genetic clusters contained highly related vanA-positive and -negative E. faecium, which implies that control of vancomycin-resistant enterococci (VRE) in hospitals also requires consideration of vancomycin-susceptible E. faecium Our findings reveal the evolution and dissemination of hospital-associated E. faecium in the UK&I and provide evidence for WGS as an instrument for infection control.
10.1101/gr.204024.116
Chewie Nomenclature Server (chewie-NS): a deployable nomenclature server for easy sharing of core and whole genome MLST schemas.
Nucleic acids research
Chewie Nomenclature Server (chewie-NS, https://chewbbaca.online/) allows users to share genome-based gene-by-gene typing schemas and to maintain a common nomenclature, simplifying the comparison of results. The combination between local analyses and a public repository of allelic data strikes a balance between potential confidentiality issues and the need to compare results. The possibility of deploying private instances of chewie-NS facilitates the creation of nomenclature servers with a restricted user base to allow compliance with the strictest data policies. Chewie-NS allows users to easily share their own schemas and to explore publicly available schemas, including informative statistics on schemas and loci presented in interactive charts and tables. Users can retrieve all the information necessary to run a schema locally or all the alleles identified at a particular locus. The integration with the chewBBACA suite enables users to directly upload new schemas to chewie-NS, download existing schemas and synchronize local and remote schemas from chewBBACA command line version, allowing an easier integration into high-throughput analysis pipelines. The same REST API linking chewie-NS and the chewBBACA suite supports the interaction of other interfaces or pipelines with the databases available at chewie-NS, facilitating the reusability of the stored data.
10.1093/nar/gkaa889
SRST2: Rapid genomic surveillance for public health and hospital microbiology labs.
Inouye Michael,Dashnow Harriet,Raven Lesley-Ann,Schultz Mark B,Pope Bernard J,Tomita Takehiro,Zobel Justin,Holt Kathryn E
Genome medicine
Rapid molecular typing of bacterial pathogens is critical for public health epidemiology, surveillance and infection control, yet routine use of whole genome sequencing (WGS) for these purposes poses significant challenges. Here we present SRST2, a read mapping-based tool for fast and accurate detection of genes, alleles and multi-locus sequence types (MLST) from WGS data. Using >900 genomes from common pathogens, we show SRST2 is highly accurate and outperforms assembly-based methods in terms of both gene detection and allele assignment. We include validation of SRST2 within a public health laboratory, and demonstrate its use for microbial genome surveillance in the hospital setting. In the face of rising threats of antimicrobial resistance and emerging virulence among bacterial pathogens, SRST2 represents a powerful tool for rapidly extracting clinically useful information from raw WGS data. Source code is available from http://katholt.github.io/srst2/.
10.1186/s13073-014-0090-6
Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans.
Skurnik David,Clermont Olivier,Guillard Thomas,Launay Adrien,Danilchanka Olga,Pons Stéphanie,Diancourt Laure,Lebreton François,Kadlec Kristina,Roux Damien,Jiang Deming,Dion Sara,Aschard Hugues,Denamur Maurice,Cywes-Bentley Colette,Schwarz Stefan,Tenaillon Olivier,Andremont Antoine,Picard Bertrand,Mekalanos John,Brisse Sylvain,Denamur Erick
Molecular biology and evolution
In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities.
10.1093/molbev/msv280
Whole-genome epidemiology, characterisation, and phylogenetic reconstruction of Staphylococcus aureus strains in a paediatric hospital.
Genome medicine
BACKGROUND:Staphylococcus aureus is an opportunistic pathogen and a leading cause of nosocomial infections. It can acquire resistance to all the antibiotics that entered the clinics to date, and the World Health Organization defined it as a high-priority pathogen for research and development of new antibiotics. A deeper understanding of the genetic variability of S. aureus in clinical settings would lead to a better comprehension of its pathogenic potential and improved strategies to contrast its virulence and resistance. However, the number of comprehensive studies addressing clinical cohorts of S. aureus infections by simultaneously looking at the epidemiology, phylogenetic reconstruction, genomic characterisation, and transmission pathways of infective clones is currently low, thus limiting global surveillance and epidemiological monitoring. METHODS:We applied whole-genome shotgun sequencing (WGS) to 184 S. aureus isolates from 135 patients treated in different operative units of an Italian paediatric hospital over a timespan of 3 years, including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) from different infection types. We typed known and unknown clones from their genomes by multilocus sequence typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), Staphylococcal protein A gene (spa), and Panton-Valentine Leukocidin (PVL), and we inferred their whole-genome phylogeny. We explored the prevalence of virulence and antibiotic resistance genes in our cohort, and the conservation of genes encoding vaccine candidates. We also performed a timed phylogenetic investigation for a potential outbreak of a newly emerging nosocomial clone. RESULTS:The phylogeny of the 135 single-patient S. aureus isolates showed a high level of diversity, including 80 different lineages, and co-presence of local, global, livestock-associated, and hypervirulent clones. Five of these clones do not have representative genomes in public databases. Variability in the epidemiology is mirrored by variability in the SCCmec cassettes, with some novel variants of the type IV cassette carrying extra antibiotic resistances. Virulence and resistance genes were unevenly distributed across different clones and infection types, with highly resistant and lowly virulent clones showing strong association with chronic diseases, and highly virulent strains only reported in acute infections. Antigens included in vaccine formulations undergoing clinical trials were conserved at different levels in our cohort, with only a few highly prevalent genes fully conserved, potentially explaining the difficulty of developing a vaccine against S. aureus. We also found a recently diverged ST1-SCCmecIV-t127 PVL- clone suspected to be hospital-specific, but time-resolved integrative phylogenetic analysis refuted this hypothesis and suggested that this quickly emerging lineage was acquired independently by patients. CONCLUSIONS:Whole genome sequencing allowed us to study the epidemiology and genomic repertoire of S. aureus in a clinical setting and provided evidence of its often underestimated complexity. Some virulence factors and clones are specific of disease types, but the variability and dispensability of many antigens considered for vaccine development together with the quickly changing epidemiology of S. aureus makes it very challenging to develop full-coverage therapies and vaccines. Expanding WGS-based surveillance of S. aureus to many more hospitals would allow the identification of specific strains representing the main burden of infection and therefore reassessing the efforts for the discovery of new treatments and clinical practices.
10.1186/s13073-018-0593-7
STing: accurate and ultrafast genomic profiling with exact sequence matches.
Espitia-Navarro Hector F,Chande Aroon T,Nagar Shashwat D,Smith Heather,Jordan I King,Rishishwar Lavanya
Nucleic acids research
Genome-enabled approaches to molecular epidemiology have become essential to public health agencies and the microbial research community. We developed the algorithm STing to provide turn-key solutions for molecular typing and gene detection directly from next generation sequence data of microbial pathogens. Our implementation of STing uses an innovative k-mer search strategy that eliminates the computational overhead associated with the time-consuming steps of quality control, assembly, and alignment, required by more traditional methods. We compared STing to six of the most widely used programs for genome-based molecular typing and demonstrate its ease of use, accuracy, speed and efficiency. STing shows superior accuracy and performance for standard multilocus sequence typing schemes, along with larger genome-scale typing schemes, and it enables rapid automated detection of antimicrobial resistance and virulence factor genes. STing determines the sequence type of traditional 7-gene MLST with 100% accuracy in less than 10 seconds per isolate. We hope that the adoption of STing will help to democratize microbial genomics and thereby maximize its benefit for public health.
10.1093/nar/gkaa566
BacWGSTdb, a database for genotyping and source tracking bacterial pathogens.
Ruan Zhi,Feng Ye
Nucleic acids research
Whole genome sequencing has become one of the routine methods in molecular epidemiological practice. In this study, we present BacWGSTdb (http://bacdb.org/BacWGSTdb), a bacterial whole genome sequence typing database which is designed for clinicians, clinical microbiologists and hospital epidemiologists. This database borrows the population structure from the current multi-locus sequence typing (MLST) scheme and adopts a hierarchical data structure: species, clonal complex and isolates. When users upload the pre-assembled genome sequences to BacWGSTdb, it offers the functionality of bacterial genotyping at both traditional MLST and whole-genome levels. More importantly, users are told which isolates in the public database are phylogenetically close to the query isolate, along with their clinical information such as host, isolation source, disease, collection time and geographical location. In this way, BacWGSTdb offers a rapid and convenient platform for worldwide users to address a variety of clinical microbiological issues such as source tracking bacterial pathogens.
10.1093/nar/gkv1004
MetaMLST: multi-locus strain-level bacterial typing from metagenomic samples.
Zolfo Moreno,Tett Adrian,Jousson Olivier,Donati Claudio,Segata Nicola
Nucleic acids research
Metagenomic characterization of microbial communities has the potential to become a tool to identify pathogens in human samples. However, software tools able to extract strain-level typing information from metagenomic data are needed. Low-throughput molecular typing schema such as Multilocus Sequence Typing (MLST) are still widely used and provide a wealth of strain-level information that is currently not exploited by metagenomic methods. We introduce MetaMLST, a software tool that reconstructs the MLST loci of microorganisms present in microbial communities from metagenomic data. Tested on synthetic and spiked-in real metagenomes, the pipeline was able to reconstruct the MLST sequences with >98.5% accuracy at coverages as low as 1×. On real samples, the pipeline showed higher sensitivity than assembly-based approaches and it proved successful in identifying strains in epidemic outbreaks as well as in intestinal, skin and gastrointestinal microbiome samples.
10.1093/nar/gkw837
A Dual Barcoding Approach to Bacterial Strain Nomenclature: Genomic Taxonomy of Klebsiella pneumoniae Strains.
Molecular biology and evolution
Sublineages (SLs) within microbial species can differ widely in their ecology and pathogenicity, and their precise definition is important in basic research and for industrial or public health applications. Widely accepted strategies to define SLs are currently missing, which confuses communication in population biology and epidemiological surveillance. Here, we propose a broadly applicable genomic classification and nomenclature approach for bacterial strains, using the prominent public health threat Klebsiella pneumoniae as a model. Based on a 629-gene core genome multilocus sequence typing (cgMLST) scheme, we devised a dual barcoding system that combines multilevel single linkage (MLSL) clustering and life identification numbers (LINs). Phylogenetic and clustering analyses of >7,000 genome sequences captured population structure discontinuities, which were used to guide the definition of 10 infraspecific genetic dissimilarity thresholds. The widely used 7-gene multilocus sequence typing (MLST) nomenclature was mapped onto MLSL SLs (threshold: 190 allelic mismatches) and clonal group (threshold: 43) identifiers for backwards nomenclature compatibility. The taxonomy is publicly accessible through a community-curated platform (https://bigsdb.pasteur.fr/klebsiella), which also enables external users' genomic sequences identification. The proposed strain taxonomy combines two phylogenetically informative barcode systems that provide full stability (LIN codes) and nomenclatural continuity with previous nomenclature (MLSL). This species-specific dual barcoding strategy for the genomic taxonomy of microbial strains is broadly applicable and should contribute to unify global and cross-sector collaborative knowledge on the emergence and microevolution of bacterial pathogens.
10.1093/molbev/msac135
Genomic surveillance of multidrug-resistant organisms based on long-read sequencing.
Genome medicine
BACKGROUND:Multidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only. METHODS:Genomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli, 15 Enterobacter cloacae complex, 10 Citrobacter freundii, 34 Pseudomonas aeruginosa, 16 Acinetobacter baumannii, and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate). RESULTS:Comparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa. The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae, E. coli, E. cloacae complex, C. freundii, A. baumannii complex, and MRSA. For P. aeruginosa, differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0-10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92-100%/99-100%). Long-read sequencing enabled analysis of an MRSA outbreak. CONCLUSIONS:We demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.
10.1186/s13073-024-01412-6
Investigating Clinical Issues by Genotyping of Medically Important Fungi: Why and How?
Alanio Alexandre,Desnos-Ollivier Marie,Garcia-Hermoso Dea,Bretagne Stéphane
Clinical microbiology reviews
Genotyping studies of medically important fungi have addressed elucidation of outbreaks, nosocomial transmissions, infection routes, and genotype-phenotype correlations, of which secondary resistance has been most intensively investigated. Two methods have emerged because of their high discriminatory power and reproducibility: multilocus sequence typing (MLST) and microsatellite length polymorphism (MLP) using short tandem repeat (STR) markers. MLST relies on single-nucleotide polymorphisms within the coding regions of housekeeping genes. STR polymorphisms are based on the number of repeats of short DNA fragments, mostly outside coding regions, and thus are expected to be more polymorphic and more rapidly evolving than MLST markers. There is no consensus on a universal typing system. Either one or both of these approaches are now available for spp., spp., spp., spp., , , and endemic mycoses. The choice of the method and the number of loci to be tested depend on the clinical question being addressed. Next-generation sequencing is becoming the most appropriate method for fungi with no MLP or MLST typing available. Whatever the molecular tool used, collection of clinical data (e.g., time of hospitalization and sharing of similar rooms) is mandatory for investigating outbreaks and nosocomial transmission.
10.1128/CMR.00043-16
The evolution of Campylobacter jejuni and Campylobacter coli.
Sheppard Samuel K,Maiden Martin C J
Cold Spring Harbor perspectives in biology
The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.
10.1101/cshperspect.a018119
Historic methicillin-resistant Staphylococcus aureus: expanding current knowledge using molecular epidemiological characterization of a Swiss legacy collection.
Genome medicine
BACKGROUND:Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes. METHODS:We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes. RESULTS:Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally. CONCLUSIONS:The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time.
10.1186/s13073-024-01292-w
Microbiological features, epidemiology, and clinical presentation of Clostridioidesdifficile strains from MLST Clade 2: A narrative review.
Badilla-Lobo Adriana,Rodríguez César
Anaerobe
Clostridioides difficile is an emerging One Health pathogen and a common etiologic agent of diarrhea, both in healthcare settings and the community. This bacterial species is highly diverse, and its global population has been classified in eight clades by multilocus sequence typing (MLST). The C. difficile MLST Clade 2 includes the NAP1/RT027/ST01 strain, which is highly recognized due to its epidemicity and association with severe disease presentation and mortality. By contrast, the remaining 83 sequence types (STs) that compose this clade have received much less attention. In response to this shortcoming, we reviewed articles published in English between 1999 and 2020 and collected information for 27 Clade 2 STs, with an emphasis on STs 01, 67, 41 and 188/231/365. Our analysis provides evidence of large phenotypic differences that preclude support of the rather widespread notion that ST01 and Clade 2 strains are "hypervirulent". Moreover, it revealed a profound lack of (meta)data for nearly 70% of the Clade 2 STs that have been identified in surveillance efforts. Targeted studies aiming to relate wet-lab and bioinformatics results to patient and clinical parameters should be performed to gain a more in-depth insight into the biology of this intriguing group of C. difficile isolates.
10.1016/j.anaerobe.2021.102355
Comparative analysis of IR-Biotyper, MLST, cgMLST, and WGS for clustering of vancomycin-resistant in a neonatal intensive care unit.
Microbiology spectrum
Healthcare-associated infections caused by vancomycin-resistant (VREFM) pose a significant threat to healthcare. Confirming the relatedness of the bacterial isolates from different patients is challenging. We aimed to assess the efficacy of IR-Biotyper, multilocus sequencing typing (MLST), and core-genome MLST (cgMLST) in comparison with whole-genome sequencing (WGS) for outbreak confirmation in the neonatal intensive care unit (NICU). Twenty VREFM isolates from four neonates and ten control isolates from unrelated patients were analyzed. Genomic DNA extraction, MLST, cgMLST, and WGS were performed. An IR-Biotyper was used with colonies obtained after 24 h of incubation on tryptic soy agar supplemented with 5% sheep blood. The optimal clustering cutoff for the IR-Biotyper was determined by comparing the results with WGS. Clustering concordance was assessed using the adjusted Rand and Wallace indices. MLST and cgMLST identified sequence types (ST) and complex types (CT), revealing suspected outbreak isolates with a predominance of ST17 and CT6553, were confirmed by WGS. For the IR-Biotyper, the proposed optimal clustering cut-off range was 0.106-0.111. Despite lower within-run precision, of the IR-Biotyper, the clustering concordance with WGS was favorable, meeting the criteria for real-time screening. This study confirmed a nosocomial outbreak of VREFM in the NICU using an IR-Biotyper, showing promising results compared to MLST. Although within-run precision requires improvement, the IR-Biotyper demonstrated high discriminatory power and clustering concordance with WGS. These findings suggest its potential as a real-time screening tool for the detection of VREFM-related nosocomial outbreaks. IMPORTANCE:In this study, we evaluated the performance of the IR-Biotyper in detecting nosocomial outbreaks caused by vancomycin-resistant , comparing it with MLST, cgMLST, and WGS. We proposed a cutoff that showed the highest concordance compared to WGS and assessed the within-run precision of the IR-Biotyper by evaluating the consistency in genetically identical strain when repeated in the same run.
10.1128/spectrum.04119-23
[Multilocus sequence typing (MLST) analysis].
Matsumura Yasufumi
Rinsho byori. The Japanese journal of clinical pathology
Multilocus sequence typing (MLST) analysis has been emerging as a powerful tool for genotyping specific bacterial species. MLST utilizes internal fragments of multiple housekeeping genes and the combination of each allele defines the sequence type for each isolate. MLST databases contain reference data and are freely accessible via internet websites. The standard method for investigating short-term hospital outbreaks is still pulse-field gel-electrophoresis and MLST analysis is not a substitute. However, analysis of sequence types and clonal complexes (closely related sequence types) enables identification and understanding of a specific clone that is widely spreading among drug-resistant organisms, or a key clone that is important for evolution of the organism. In the case of Escherichia coli, CTX-M-15 or CTX-M-14 extended-spectrum beta-lactamase producing ST131 clone has emerged and spread globally in the last 10 years. MLST analysis is an unambiguous procedure and is becoming a common typing method to characterize isolates.
A genomic overview of the population structure of Salmonella.
PLoS genetics
For many decades, Salmonella enterica has been subdivided by serological properties into serovars or further subdivided for epidemiological tracing by a variety of diagnostic tests with higher resolution. Recently, it has been proposed that so-called eBurst groups (eBGs) based on the alleles of seven housekeeping genes (legacy multilocus sequence typing [MLST]) corresponded to natural populations and could replace serotyping. However, this approach lacks the resolution needed for epidemiological tracing and the existence of natural populations had not been independently validated by independent criteria. Here, we describe EnteroBase, a web-based platform that assembles draft genomes from Illumina short reads in the public domain or that are uploaded by users. EnteroBase implements legacy MLST as well as ribosomal gene MLST (rMLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) and currently contains over 100,000 assembled genomes from Salmonella. It also provides graphical tools for visual interrogation of these genotypes and those based on core single nucleotide polymorphisms (SNPs). eBGs based on legacy MLST are largely consistent with eBGs based on rMLST, thus demonstrating that these correspond to natural populations. rMLST also facilitated the selection of representative genotypes for SNP analyses of the entire breadth of diversity within Salmonella. In contrast, cgMLST provides the resolution needed for epidemiological investigations. These observations show that genomic genotyping, with the assistance of EnteroBase, can be applied at all levels of diversity within the Salmonella genus.
10.1371/journal.pgen.1007261
Genomes-based MLST, cgMLST, wgMLST and SNP analysis of Salmonella Typhimurium from animals and humans.
Comparative immunology, microbiology and infectious diseases
Salmonella Typhimurium (S. Typhimurium) is an important food-borne and zoonotic pathogen that causes salmonellosis. With the development of whole genome sequencing (WGS), genome-based typing has been widely applied to bacteriology. In this study, we investigated genotyping and phylogenetic clusters of S. Typhimurium isolates from humans and animals in different provinces (including Beijing, Shandong, Guangxi, Shaanxi, Henan, and Shanghai) of China during 2009-2018 using multi locus sequence typing (MLST), core genome MLST (cgMLST), whole genome MLST (wgMLST) and single nucleotide polymorphism (SNP) based on WGS. 29 S. Typhimurium isolates from chicken (n = 22), sick pigeon (n = 2), patients (n = 4) and sick swine (n = 1) were tested. MLST analysis showed S. Typhimurium strains were divided into four STs, namely ST19 (n = 14), ST34 (n = 12), ST128 (n = 2) and ST1544 (n = 1). cgMLST and wgMLST divided 29 strains into 27 cgSTs and 29 wgST, respectively. Phylogenetic clustering showed that all isolates were divided into 4 clusters and 4 singletons. SNP analysis was used to examine MLST, cgMLST, wgMLST analysis. Finally, comparisons of MLST, cgMLST, wgMLST, and SNP were analyzed and the results showed their precision increased in order. In summary, genomic typing and phylogenetic relationships of 29 S. Typhimurium strains from different sources in China were analyzed. These findings were beneficial to investigate molecular pathogenesis, bacterial diversity, and traceability analysis of Salmonella.
10.1016/j.cimid.2023.101973
Comparing core-genome MLST with PFGE and MLST for cluster analysis of carbapenem-resistant Acinetobacter baumannii.
Journal of global antimicrobial resistance
OBJECTIVES:Carbapenem-resistant Acinetobacter baumannii (CRAB) is a prevalent pathogen contributing to hospital infections. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and core-genome MLST (cgMLST) are frequently used methods to illuminate the nosocomial transmission of CRAB. In this study, we compared the discriminatory power of the three typing methods. METHODS:Antimicrobial susceptibility tests were performed by the broth microdilution and Vitek2 methods. PFGE, MLST and cgMLST were conducted to determine the clonality and phylogenetic relationship of the strains. Whole-genome sequence data were acquired by an Illumina HiSeq 2000, and cgMLST was analysed by the Ridom SeqSphere+ v.7.2.3 software. RESULTS:A total of 149 carbapenem-resistant A. baumannii isolates had 15 different PFGE profiles (A-O type), and 73 of the isolates had related subtypes (A1 and A2), accounting for the majority of type A isolates. The maximum-likelihood phylogenetic analysis based on the cgMLST genes grouped the same PFGE clonal pattern A into nine different clusters. ST_Pasteur grouped all the strains into ST2, whereas ST_Oxford grouped the PFGE clonal pattern A isolates into six STs. In addition, the gdhB allele in the ST_Oxford scheme had two copies in five strains, which complicated the ST_Oxford typing. CONCLUSIONS:cgMLST was more discriminant than PFGE and MLST. CgMLST is the most suitable and comprehensive method for genotyping A. baumannii in surveillance and epidemiological research.
10.1016/j.jgar.2022.06.014
Pathogen typing in the genomics era: MLST and the future of molecular epidemiology.
Pérez-Losada Marcos,Cabezas Patricia,Castro-Nallar Eduardo,Crandall Keith A
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
Multi-locus sequence typing (MLST) is a high-resolution genetic typing approach to identify species and strains of pathogens impacting human health, agriculture (animals and plants), and biosafety. In this review, we outline the general concepts behind MLST, molecular approaches for obtaining MLST data, analytical approaches for MLST data, and the contributions MLST studies have made in a wide variety of areas. We then look at the future of MLST and their relative strengths and weaknesses with respect to whole genome sequence typing approaches that are moving into the research arena at an ever-increasing pace. Throughout the paper, we provide exemplar references of these various aspects of MLST. The literature is simply too vast to make this review comprehensive, nevertheless, we have attempted to include enough references in a variety of key areas to introduce the reader to the broad applications and complications of MLST data.
10.1016/j.meegid.2013.01.009
Phylogenomics of species and analysis of antimicrobial resistance genes.
Frontiers in microbiology
Introduction:Non- species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 spp. isolates, which included 798 non- genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies. Methods:Phylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment. Results:The Pasteur MLST scheme was able to identify and genotype 72 species in the genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non- genomes assigned to , , , and (heterotypic synonym of ), which were the most clinically relevant non- species of the group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in , respectively, and one each in . Furthermore, genomes were grouped into one cluster within genomes. The SNP phylogeny of genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several species, was found in 503 isolates assigned to 35 species. Conclusion:In conclusion, Pasteur MLST phylogeny of non- isolates coupled with detection of antimicrobial resistance makes it important to study the population structure and epidemiology of spp. isolates.
10.3389/fmicb.2023.1264030
Multilocus sequence typing schemes for the emerging swine pathogen Mycoplasma hyosynoviae.
Veterinary microbiology
Mycoplasma (M.) hyosynoviae is a commensal of the upper respiratory tract in swine, which has the potential to spread systemically, usually resulting in arthritis in fattening pigs and gilts. To date, very little is known about the epidemiology of M. hyosynoviae, mainly due to a lack of suitable typing methods. Therefore, this study aimed to develop both a conventional multi locus sequence typing (MLST) and a core genome (cg) MLST scheme. The development of the cgMLST was based on whole genome sequences of 64 strains isolated from pigs and wild boars during routine diagnostics as well as nine publicly available genomes. A cgMLST scheme containing 390 target genes was established using the Ridom© SeqSphere+ software. Using this scheme as a foundation, seven housekeeping genes were selected for conventional MLST based on their capability to reflect genome wide relatedness and subsequently, all 73 strains were typed by applying both methods. Core genome MLST results revealed a high diversity of the studied strain population and less than 100 allele differences between epidemiologically unrelated strains were only detected for four isolates from the US. On the other hand, seven clonal clusters (≤ 12 allele differences) comprising 20 isolates were identified. Comparison of the two typing methods resulted in highly congruent phylogenetic trees and an Adjusted Rand Coefficient of 0.893, while cgMLST showed marginally higher resolution when comparing closely related isolates, indicated by a slightly higher Simpson's ID (0.992) than conventional MLST (Simpson's ID = 0.990). Overall, both methods seem well suited for epidemiological analyses for scientific as well as diagnostic purposes. While MLST is faster and cheaper, cgMLST can be used to further differentiate closely related isolates.
10.1016/j.vetmic.2024.109997
Clustering of Isolates Using MLST and Whole-Genome Phylogenetics and Protein Motif Fingerprinting.
Jesser Kelsey J,Valdivia-Granda Willy,Jones Jessica L,Noble Rachel T
Frontiers in public health
is a ubiquitous and abundant member of native microbial assemblages in coastal waters and shellfish. Though is predominantly environmental, some strains have infected human hosts and caused outbreaks of seafood-related gastroenteritis. In order to understand differences among clinical and environmental strains, we used high quality DNA sequencing data to compare the genomes of isolates ( = 43) from a variety of geographic locations and clinical and environmental sample matrices. We used phylogenetic trees inferred from multilocus sequence typing (MLST) and whole-genome (WG) alignments, as well as a novel classification and genome clustering approach that relies on protein motif fingerprints (MFs), to assess relationships between strains and identify novel molecular targets associated with virulence. Differences in strain clustering at more than one position were observed between the MLST and WG phylogenetic trees. The WG phylogeny had higher support values and strain resolution since isolates of the same sequence type could be differentiated. The MF analysis revealed groups of protein motifs that were associated with the pathogenic MLST type ST36 and a large group of clinical strains isolated from human stool. A subset of the stool and ST36-associated protein motifs were selected for further analysis and the motif sequences were found in genes with a variety of functions, including transposases, secretion system components and effectors, and hypothetical proteins. DNA sequences associated with these protein motifs are candidate targets for future molecular assays in order to improve surveys of pathogenic in the environment and seafood.
10.3389/fpubh.2019.00066
Revisiting Bartonella bacilliformis MLST.
Ruiz Joaquim,Pons Maria J
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
All the studies published including Bartonella bacilliformis MLST data, as well as all B. bacilliformis genomes present in GenBank were analyzed. Overall 64 isolates and their geographical distribution were analyzed, and 14 different MLST patterns were observed. The results highlight the need for expanding the MLST studies and adding a higher number of isolates from all endemic areas.
10.1016/j.meegid.2018.05.032