-
Current Issues in Molecular Biology 2017Within less than a decade matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial... (Review)
Review
Within less than a decade matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a gold standard for microbial identification in clinical microbiology laboratories. Besides identification of microorganisms the typing of single strains as well as the antibiotic and antimycotic resistance testing has come into focus in order to speed up the microbiological diagnostic. However, the full potential of MALDI-TOF MS has not been tapped yet and future technological advancements will certainly expedite this method towards novel applications and enhancement of current practice. So, the following chapter shall be rather a brainstorming and forecast of how MALDI-TOF MS will develop to influence clinical diagnostics and microbial research in the future. It shall open up the stage for further discussions and does not claim for overall validity.
Topics: Bacterial Typing Techniques; Clinical Laboratory Techniques; Forecasting; Humans; Microbiological Techniques; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 28504240
DOI: 10.21775/cimb.023.017 -
Saudi Medical Journal May 2024The aim of this study was to go through the molecular methods used for typing of carbapenem-resistant (CRAB) isolates for investigating the molecular epidemiology all... (Review)
Review
The aim of this study was to go through the molecular methods used for typing of carbapenem-resistant (CRAB) isolates for investigating the molecular epidemiology all over the world. Multiple typing techniques are required to understand the source and nature of outbreaks caused by () and acquired resistance to antimicrobials. Nowadays, there is gradual shift from traditional typing methods to modern molecular methods to study molecular epidemiology and infection control. Molecular typing of strains has been revolutionized significantly in the last 2 decades. A few sequencing-based techniques have been proven as a breakthrough and opened new prospects, which have not been achieved by the traditional methods. In this review, discussed different pre-existing and recently used typing methods to explore the molecular epidemiology of pertaining in context with human infections.
Topics: Acinetobacter baumannii; Humans; Molecular Epidemiology; Acinetobacter Infections; Molecular Typing; Bacterial Typing Techniques
PubMed: 38734425
DOI: 10.15537/smj.2024.45.5.20230886 -
Indian Journal of Medical Microbiology 2016The viridans group streptococci are a heterogeneous group of organisms which exist as commensals in the oropharynx and the gut. They cause serious infections when they... (Review)
Review
The viridans group streptococci are a heterogeneous group of organisms which exist as commensals in the oropharynx and the gut. They cause serious infections when they gain entry into sterile sites particularly in patients with predisposing conditions. Classification and species differentiation of these organisms has always been a challenge because of phenotypic differences between strains of the same species. Facklam's typing scheme based on six metabolic properties has been the most widely used and many commercial identification systems are based on it. Due to the ambiguity in species differentiation based on phenotypic tests, nucleic acid-based methods have been developed to improve the identification of these organisms. Results using genotypic methods such as 16S rRNA and sodA gene sequencing have been promising. Multilocus sequence analysis of seven house-keeping genes map, pfl, pyk, ppaC, rpoB, soda and tuf amplified by polymerase chain reaction was found to be an accurate alternative to other methods and could be useful in the characterisation of larger collections of isolates.
Topics: Bacterial Proteins; Bacterial Typing Techniques; Humans; Molecular Diagnostic Techniques; Multilocus Sequence Typing; RNA, Bacterial; RNA, Ribosomal, 16S; Streptococcal Infections; Viridans Streptococci
PubMed: 27934818
DOI: 10.4103/0255-0857.195371 -
ACS Infectious Diseases May 2020A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often...
A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.
Topics: Bacterial Typing Techniques; DNA; DNA, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Sequence Analysis, DNA
PubMed: 32294378
DOI: 10.1021/acsinfecdis.9b00464 -
Microbiology Spectrum Dec 2022Over the past decade, whole-genome sequencing (WGS) has overtaken traditional bacterial typing methods for studies of genetic relatedness. Further, WGS data generated...
Over the past decade, whole-genome sequencing (WGS) has overtaken traditional bacterial typing methods for studies of genetic relatedness. Further, WGS data generated during epidemiologic studies can be used in other clinically relevant bioinformatic applications, such as antibiotic resistance prediction. Using commercially available software tools, the relatedness of 38 clinical isolates of multidrug-resistant Pseudomonas aeruginosa was defined by two core genome multilocus sequence typing (cgMLST) methods, and the WGS data of each isolate was analyzed to predict antibiotic susceptibility to nine antibacterial agents. The WGS typing and resistance prediction data were compared with pulsed-field gel electrophoresis (PFGE) and phenotypic antibiotic susceptibility results, respectively. Simpson's Diversity Index and adjusted Wallace pairwise assessments of the three typing methods showed nearly identical discriminatory power. Antibiotic resistance prediction using a trained analytical pipeline examined 342 bacterial-drug combinations with an overall categorical agreement of 92.4% and very major, major, and minor error rates of 3.6, 4.1, and 4.1%, respectively. Multidrug-resistant Pseudomonas aeruginosa isolates are a serious public health concern due to their resistance to nearly all or all of the available antibiotics, including carbapenems. Utilizing molecular approaches in conjunction with antibiotic susceptibility prediction software warrants investigation for use in the clinical laboratory workflow. These molecular tools coupled with antibiotic resistance prediction tools offer the opportunity to overcome the extended turnaround time and technical challenges of phenotypic susceptibility testing.
Topics: Multilocus Sequence Typing; Pseudomonas aeruginosa; Anti-Bacterial Agents; Bacterial Typing Techniques; Whole Genome Sequencing; Genome, Bacterial
PubMed: 36350158
DOI: 10.1128/spectrum.03920-22 -
Research in Microbiology May 2015For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to... (Review)
Review
For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current Clostridium botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity.
Topics: Bacterial Typing Techniques; Botulinum Toxins; Clostridium botulinum; Genetic Variation; History, 20th Century; History, 21st Century; Molecular Typing; Serotyping
PubMed: 25312020
DOI: 10.1016/j.resmic.2014.09.007 -
Microbial Genomics Aug 2017Multi-locus sequence typing (MLST) is a widely used method for categorizing bacteria. Increasingly, MLST is being performed using next-generation sequencing (NGS) data... (Review)
Review
Multi-locus sequence typing (MLST) is a widely used method for categorizing bacteria. Increasingly, MLST is being performed using next-generation sequencing (NGS) data by reference laboratories and for clinical diagnostics. Many software applications have been developed to calculate sequence types from NGS data; however, there has been no comprehensive review to date on these methods. We have compared eight of these applications against real and simulated data, and present results on: (1) the accuracy of each method against traditional typing methods, (2) the performance on real outbreak datasets, (3) the impact of contamination and varying depth of coverage, and (4) the computational resource requirements.
Topics: Bacteria; Bacterial Typing Techniques; Databases, Factual; Genome, Bacterial; Multilocus Sequence Typing; Software
PubMed: 29026660
DOI: 10.1099/mgen.0.000124 -
PloS One 2012Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments...
Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.
Topics: Australia; Bacterial Typing Techniques; Base Composition; Chromosome Mapping; Computational Biology; DNA Primers; Genotype; Klebsiella pneumoniae; Multilocus Sequence Typing; Phylogeny; Polymorphism, Single Nucleotide; Real-Time Polymerase Chain Reaction; Transition Temperature
PubMed: 22428067
DOI: 10.1371/journal.pone.0033530 -
Clinical Microbiology Reviews Jul 2011Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing... (Review)
Review
Review and international recommendation of methods for typing neisseria gonorrhoeae isolates and their implications for improved knowledge of gonococcal epidemiology, treatment, and biology.
Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding.
Topics: Bacterial Typing Techniques; DNA, Bacterial; Gonorrhea; Humans; Multilocus Sequence Typing; Neisseria gonorrhoeae
PubMed: 21734242
DOI: 10.1128/CMR.00040-10 -
Clinical Microbiology Reviews Jan 2015Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous... (Review)
Review
Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic "blind spot" for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.
Topics: Bacterial Typing Techniques; Disease Outbreaks; Humans; Legionella pneumophila; Legionnaires' Disease
PubMed: 25567224
DOI: 10.1128/CMR.00029-14