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Current Protocols in Microbiology Aug 2014Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between...
Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens.
Topics: Bacterial Outer Membrane Proteins; Bacterial Typing Techniques; Borrelia burgdorferi; Molecular Typing; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Bacterial; RNA, Ribosomal
PubMed: 25082003
DOI: 10.1002/9780471729259.mc12c05s34 -
Journal of Clinical Microbiology Nov 2018and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission... (Comparative Study)
Comparative Study
Typing and Species Identification of Clinical Klebsiella Isolates by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.
and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different species. Phylogenetic analysis revealed that the isolates comprised three different species: , , and Genome analysis showed that MALDI-TOF MS can be used to distinguish from due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of and by MALDI-TOF MS.
Topics: Bacterial Typing Techniques; Cluster Analysis; Costs and Cost Analysis; Genome, Bacterial; Humans; Klebsiella; Klebsiella Infections; Polymorphism, Single Nucleotide; Sensitivity and Specificity; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Fourier Transform Infrared; Time Factors
PubMed: 30135233
DOI: 10.1128/JCM.00843-18 -
Journal of Clinical Laboratory Analysis Sep 2021Salmonella Wandsworth is a rare serotype of Salmonella. This study analyzed the genotyping, genome structure, and molecular biological functions of Salmonella Wandsworth...
BACKGROUND
Salmonella Wandsworth is a rare serotype of Salmonella. This study analyzed the genotyping, genome structure, and molecular biological functions of Salmonella Wandsworth based on the results of multilocus sequence typing and next-generation sequencing genome assembly analysis.
METHODS
Serological typing was performed using the slide-agglutination method. The micro broth dilution method was used to test antibiotic susceptibility. Multilocus sequence typing (MLST) was used to perform the homology analysis, while the second-generation sequencing genome analysis was used to analyze the whole genome of the bacteria.
RESULTS
Salmonella Wandsworth is Group Q Salmonella. The MLST of this strain was ST1498. Salmonella Wandsworth was sensitive to antibiotics, such as ceftriaxone, imipenem, chloramphenicol, and colistin, but was resistant to ampicillin, cefalotin, gentamicin, and ciprofloxacin. The second-generation sequencing results showed that the genome sequence length of the bacteria was 5109457bp. Annotated COG library analysis generated 3,746 corresponding genes. After the comparison with the KEGG library, 1,340 genes, which participate in 19 types of metabolic pathways, were obtained. A total of 249 pathogenic factors and 2 disease islands were predicted. 2 CRISPR sites and 8 Cas sites were predicted. It can be seen from the evolutionary tree that Salmonella Wandsworth MLST1498 and Paratyphi B str.SPB7 are gathered together. We identified one resistance gene, namely, aac(6')-Iaa accounting for aminoglycoside resistance.
CONCLUSION
Salmonella Wandsworth isolated in this study is Salmonella group Q. Consequently, it is necessary to strengthen the understanding of clinical infections of Salmonella Wandsworth and carry out continuous monitoring and research.
Topics: Bacterial Proteins; Bacterial Typing Techniques; DNA, Bacterial; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Humans; Multilocus Sequence Typing; Salmonella; Salmonella Infections
PubMed: 34245607
DOI: 10.1002/jcla.23901 -
Euro Surveillance : Bulletin Europeen... Jan 2013Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely... (Review)
Review
Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely phenotypes of aetiological agents, enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that many microbiological typing methods have addressed over the last 100 years or so; indeed, providing large volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites have generated an infrastructure that can exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST website to extract clinically useful, strain-characterisation information that can be provided to physicians and public health professionals in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to instigate public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data obtained in the absence of WGS, providing a flexible infrastructure for WGS-based clinical microbiology.
Topics: Bacterial Typing Techniques; Computational Biology; Genome, Bacterial; Humans; Meningococcal Infections; Molecular Typing; Neisseria meningitidis; Sequence Analysis, DNA
PubMed: 23369391
DOI: 10.2807/ese.18.04.20379-en -
Revista Chilena de Infectologia :... Dec 2006
Topics: Anti-Infective Agents; Bacterial Typing Techniques; Humans; Nocardia Infections; Nocardia asteroides; Trimethoprim, Sulfamethoxazole Drug Combination
PubMed: 17186085
DOI: 10.4067/s0716-10182006000400011 -
Clinical Microbiology and Infection :... Oct 2007For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the...
For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.
Topics: Bacteria; Bacterial Typing Techniques; Communicable Diseases; Humans; Reproducibility of Results
PubMed: 17716294
DOI: 10.1111/j.1469-0691.2007.01786.x -
Scientific Reports Jun 2017Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial...
Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative of metabolic changes within the cells. The FLIM-phasor approach represents a powerful, non-invasive imaging technique to study bacterial metabolism in situ and could provide unique insights into bacterial community behavior, pathology and antibiotic resistance with sub-cellular resolution.
Topics: Bacteria; Bacterial Typing Techniques; Microscopy, Fluorescence; Molecular Typing
PubMed: 28623341
DOI: 10.1038/s41598-017-04032-w -
BMC Genomics Dec 2022Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis,...
BACKGROUND
Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform.
RESULTS
In routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST's discriminatory power.
CONCLUSIONS
A pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.
Topics: Multilocus Sequence Typing; Genotype; Bacteria; Bacterial Typing Techniques; Genome; Escherichia coli
PubMed: 36581824
DOI: 10.1186/s12864-022-08550-4 -
The Veterinary Clinics of North... Mar 2011Gut microbes play a crucial role in the regulation of host health, but the true complexity of the gastrointestinal microbiota has been underestimated using traditional... (Review)
Review
Gut microbes play a crucial role in the regulation of host health, but the true complexity of the gastrointestinal microbiota has been underestimated using traditional culture techniques. Recent molecular-phylogenetic and metagenomic studies have revealed a highly diverse microbial community in the canine and feline gastrointestinal tract of healthy animals, consisting of bacteria, archaea, fungi, protozoa, and viruses. Alterations in microbial communities have also been reported in dogs and cats with chronic enteropathies, notably increases in Proteobacteria and depletions of Firmicutes. This review summarizes the current information about the intestinal microbial ecosystem in dogs and cats.
Topics: Animals; Bacteria; Bacterial Typing Techniques; Cats; Dogs; Gastrointestinal Tract; Phylogeny
PubMed: 21486635
DOI: 10.1016/j.cvsm.2010.12.006 -
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