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The Journal of Molecular Diagnostics :... Feb 2022Hospital-acquired infections pose significant costly global challenges to patient care. Rapid and sensitive methods to identify potential outbreaks are integral to...
Hospital-acquired infections pose significant costly global challenges to patient care. Rapid and sensitive methods to identify potential outbreaks are integral to infection control measures. Whole-genome sequencing (WGS)-based bacterial strain typing provides higher discriminatory power over standard nucleotide banding pattern-based methods such as repetitive sequence-based PCR (rep-PCR). However, integration of WGS into clinical epidemiology is limited by the lack of consensus in methodology and data analysis/interpretation. In this study, WGS was performed on genomic DNA extracted from 22 multidrug-resistant Pseudomonas aeruginosa (MDR-PA) isolates using next-generation sequencing. Resulting high-quality reads were analyzed for phylogenetic relatedness using a whole-genome multilocus sequence typing (wgMLST)-based software program and single-nucleotide variant phylogenomics (SNVPhyl). WGS-based results were compared with conventional MLST and archived rep-PCR results. Rep-PCR identified three independent clonal clusters of MDR-PA. Only one clonal cluster identified by rep-PCR, an endemic strain within the pediatric cystic fibrosis population at Texas Children's Hospital, was concordantly identified using wgMLST and SNVPhyl. Results were highly consistent between the three sequence-based analyses (conventional MLST, wgMLST, and SNVPhyl), and these results remained consistent with the addition of 74 MDR-PA genomes. These WGS-based methods provided greater resolution for strain discrimination than rep-PCR or standard MLST classification, and the ease of use of wgMLST software renders it clinically viable for analysis, interpretation, and reporting of WGS-based strain typing.
Topics: Bacterial Typing Techniques; Child; Humans; Multilocus Sequence Typing; Phylogeny; Polymerase Chain Reaction; Pseudomonas aeruginosa; Repetitive Sequences, Nucleic Acid; Whole Genome Sequencing
PubMed: 34775029
DOI: 10.1016/j.jmoldx.2021.10.004 -
Clinical Microbiology and Infection :... Nov 2010Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for the species and subspecies classification of a broad... (Review)
Review
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a powerful tool for the species and subspecies classification of a broad spectrum of bacteria, including Gram-positive bacteria such as Staphylococcus, Streptococcus and Listeria, and Gram-negative bacteria such as Neisseria, Salmonella, Aeromonas, Campylobacter and Helicobacter. MALDI-TOF MS has also been used for the rapid identification and typing of potential bioterrorism agents, including Coxiella burnetii, Francisella tularensis and Bacillus anthracis.
Topics: Bacteria; Bacterial Proteins; Bacterial Typing Techniques; Bioterrorism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 20825435
DOI: 10.1111/j.1469-0691.2010.03364.x -
Clinical Microbiology and Infection :... Nov 2010Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained... (Review)
Review
Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1-93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications.
Topics: Bacteria; Bacterial Proteins; Bacterial Typing Techniques; Blood-Borne Pathogens; Clinical Laboratory Techniques; Cost-Benefit Analysis; Fungal Proteins; Fungi; Humans; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 20636422
DOI: 10.1111/j.1469-0691.2010.03311.x -
FEMS Microbiology Ecology May 2004The spatial distribution of the tremendous bacterial diversity in soil partially depends on the broad range of scales of soil physical structures and the size of... (Review)
Review
The spatial distribution of the tremendous bacterial diversity in soil partially depends on the broad range of scales of soil physical structures and the size of bacteria. The aim of this article is to collect information on spatial distribution of bacteria, the genetic structure of bacterial populations and communities, and on spatial constraints that operate in soil. This has been addressed by studying the spatial pattern of micro-habitats for various bacterial types and the spatial spread of clones in soil environment. The clones were considered as the units of genetic population structure. Experimental findings from a number of studies provide evidence that in soils a clone and a micro-colony are not necessarily identical. For some bacterial types, members of the same clone have been found far apart. Besides, micro-colonies of a few cells have also been reported. Short-range cell movements seem to be common in soil, in agreement with the observation of high small-scale diversity (millimetre scale). The mechanisms for the spread of clones are complex and probably operate at different spatial scales, even for soil bacteria with no specific vectors. The hypothesis underlying the study of the spatial dimension of diversity is that it can reveal mechanisms of diversity maintenance and contribute to their evaluation, complementing available knowledge of genetic processes.
Topics: Bacteria; Bacterial Typing Techniques; Biodiversity; DNA Fingerprinting; DNA, Bacterial; Soil Microbiology
PubMed: 19712395
DOI: 10.1016/j.femsec.2004.01.010 -
Genome Biology Feb 2013The majority of people in the developed world spend more than 90% of their lives indoors. Here, we examine our understanding of the bacteria that co-inhabit our... (Review)
Review
The majority of people in the developed world spend more than 90% of their lives indoors. Here, we examine our understanding of the bacteria that co-inhabit our artificial world and how they might influence human health.
Topics: Bacteria; Bacterial Typing Techniques; Environment, Controlled; Environmental Microbiology; Microbiota
PubMed: 23514020
DOI: 10.1186/gb-2013-14-2-202 -
BMC Veterinary Research Mar 2021Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of...
BACKGROUND
Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database.
RESULTS
Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009).
CONCLUSIONS
Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.
Topics: Animals; Bacterial Typing Techniques; Blood Culture; Bronchoalveolar Lavage Fluid; DNA, Bacterial; Dog Diseases; Dogs; Female; High-Throughput Nucleotide Sequencing; Male; Microbiota; Pneumonia, Bacterial; RNA, Ribosomal, 16S; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 33757515
DOI: 10.1186/s12917-021-02841-w -
Indian Journal of Medical Microbiology 2011The major impetus for bacterial identification came after the advent of solid culture media. Morphological appearance of bacterial colonies was often sufficient for... (Review)
Review
The major impetus for bacterial identification came after the advent of solid culture media. Morphological appearance of bacterial colonies was often sufficient for their identification in the laboratory. Even in modern times, preliminary identification of most cultivable bacteria is based on such morphological characters. Advances have been made media for the presumptive identification of common organisms encountered in clinical samples. Phenotypic characterisation of bacteria with, physiological tests with a battery of biochemical tests differentiate related bacterial genera as well as confirm their identity. . Each laboratory can select its own method(s) of identification, provided they are based on scientific / epidemiological evidence; clinical laboratory and standards institute (CLSI) is a widely accepted organization and laboratories in many parts of the world follow its recommendations for bacterial identification. Some of the latest advances in identification include Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectroscopy (MALDI-TOF) is a state of art facility used for fast and reliable species-specific identification of bacteria including Mycobacteria and fungi including yeasts. However the single most important factor that decides the method of bacterial identification in any laboratory is the cost involved. In the final analysis, selection of tests for bacterial identification should be based on their standardization with proper scientific basis. Considering the cost and lack of easy availability of commercial kits, we have put forward a simplified and rapid method of identification for most commonly encountered bacterial pathogens causing human infection in India.
Topics: Bacteria; Bacterial Infections; Bacterial Typing Techniques; Bacteriological Techniques; Genotype; Health Care Costs; Humans; India; Molecular Diagnostic Techniques; Phenotype; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors
PubMed: 22120791
DOI: 10.4103/0255-0857.90156 -
Journal of Clinical Microbiology Mar 2019Among enterococci, occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of complicates both molecular...
Among enterococci, occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of 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 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 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.
Topics: Animals; Bacterial Proteins; Bacterial Typing Techniques; Enterococcus faecalis; Environmental Microbiology; Genome, Bacterial; Genotype; Gram-Positive Bacterial Infections; Humans; Molecular Epidemiology; Multilocus Sequence Typing; One Health; Phylogeny; Polymorphism, Single Nucleotide
PubMed: 30651394
DOI: 10.1128/JCM.01686-18 -
Microbiology Spectrum Oct 2021Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution...
Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies. Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.
Topics: Alleles; Animals; Bacterial Typing Techniques; Bacteriological Techniques; Clostridium Infections; Clostridium perfringens; Disease Outbreaks; Genome, Bacterial; Genotype; Humans; Multilocus Sequence Typing; Phylogeny; Polymorphism, Single Nucleotide; United Kingdom; Whole Genome Sequencing
PubMed: 34704797
DOI: 10.1128/Spectrum.00533-21 -
Annali Di Igiene : Medicina Preventiva... 2017Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of... (Review)
Review
High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.
INTRODUCTION
Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools.
OBJECTIVE
To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health.
MATERIAL AND METHODS
A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available.
RESULTS
HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations.
CONCLUSIONS
Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.
Topics: Bacteria; Bacterial Typing Techniques; Genotype; Humans; Molecular Epidemiology; Multilocus Sequence Typing; Polymorphism, Single Nucleotide; Reproducibility of Results; Sensitivity and Specificity; Transition Temperature
PubMed: 28569339
DOI: 10.7416/ai.2017.2153