Mic661 Molecular Diagnostics Lecture 5-1 (Mar 2024) PDF

MIC661 MOLECULAR DIAGNOSTICS Molecular strain typing or genotyping Dr. Muhd Hanis Md Idris MIC661 MOLECULAR DIAGNOSTICS Molecular strain typing or genotyping Dr. Muhd Hanis Md Idris  TABLE OF CONTENTS Introduction of Typing Significance o...

MIC661 MOLECULAR DIAGNOSTICS Molecular strain typing or genotyping Dr. Muhd Hanis Md Idris MIC661 MOLECULAR DIAGNOSTICS Molecular strain typing or genotyping Dr. Muhd Hanis Md Idris  TABLE OF CONTENTS Introduction of Typing Significance of Typing 01 Methods 02 Methods Restriction 03 Fragment Length 04 Sequencing Polymorphism 05 Ribotyping 06 DNA Chips 07 Emerging Technologies 01 Introduction of typing methods  INTRODUCTION Molecular strain typing has become an essential tool for the analysis of bacterial pathogens obtained during investigations of epidemiologic outbreaks, laboratory contamination, and recurrent infection. A wide variety of strain typing methods have been described using contemporary DNA-based technologies. However, developing methods and generating data have proven easier than defining robust approaches for interpreting the results.  PREVALENCE OF HOSPITAL ACQUIRED INFECTIONS IN CENTRAL AND SOUTHERN REGIONS OF PENINSULAR MALAYSIA Source: http://nehapmalaysia.moh.gov.my/wp- content/uploads/2020/12/Prevalence-of-Hospital-Acquired- Infections-in-Central-and-Southern-Regions-of- Peninsular-Malaysia.pdf THE BURDEN OF HEALTHCARE-ASSOCIATED INFECTIONS IN SOUTHEAST ASIA  THE BURDEN OF HEALTHCARE-ASSOCIATED INFECTIONS IN SOUTHEAST ASIA Source: Moi Lin Ling, Anucha Apisarnthanarak, and Gilbert Madriag (2015). The Burden of Healthcare-Associated Infections in Southeast Asia: A Systematic Literature Review and Meta-analysis. Healthcare Epidemiology 60: 1690-1699.  AFFECTED PATIENTS IN US in 2013 Affected patients 2M An estimated 2 million patients in the United States are affected each year Hospitalized 5% approximately 5% of hospitalized patients Death 4.4% estimated 88,000 deaths annually 4,500,000,000 4.5 billion dollars in excess healthcare costs  MULTIDRUG-RESISTANT PATHOGENS Gram +ve Nosocomial Gram –ve Bacilli glycopeptide (vancomycin)- extended spectrum resistant Enterococci, betalactamase-producing strains methicillin-resistant of Escherichia coli and Klebsiella Staphylococcus aureus (MRSA), pneumoniae, and and more recently, glycopeptide fluoroquinolone-resistant strains intermediate and glycopeptide of Pseudomonas aeruginosa and resistant S. aureus E. coli 02 Significance of typing methods  TYPING SYSTEM CHARACTERIZATION Typeability Reproducibility Discriminatory the ability of a technique the ability to yield the the ability to differentiate to assign an unambiguous same result upon repeat among epidemiologically result (type) to each isolate testing of a bacterial strain unrelated isolates  FEATURES OF THE TOPIC Typing by Restriction Ribotyping with Fragment Length Southern Blot Polymorphism Analysis Typing by Typing by Polymerase Sequencing Chain Reaction Analysis 03 Restriction Fragment Length Polymorphism Restriction Fragment Length Polymorphism (RFLP) refers to differences (or variations) among people or bacterial species in their DNA sequences at sites recognized by restriction enzymes. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.  PRINCIPLES : Typing by Restriction Fragment Length Polymorphism RFLP-PFGE Chromosomal DNA is digested with restriction enzymes, resulting in a series of fragments of different sizes that form different patterns (i.e., DNA fingerprinting) when analyzed by agarose gel electrophoresis (PFGE) and can be transferred to a membrane via Southern blotting. Differences in these patterns are referred to as RFLPs. Usually, six nucleotide cutters such as SmaI, XbaI, and SalI, are used to digest DNA in order to generate relatively few DNA fragment for analysis. The product is usually analyzed by pulse field gel electrophoresis (PFGE) which allows the separation of DNA molecules of 20–1,000 kbp in length by periodically changing the direction of the electrical field. Field inversion gel electrophoresis utilizes a conventional electrophoresis chamber in which the orientation of the electric field is periodically inverted by 180.  PROCEDURE : RFLP-PFGE Cell lysis and release of Restriction intact chromosomal endonuclease Bacterial cells are DNA by soaking the gel digestion of embedded in gel block block in lysis solution, chromosomal usually lysozyme, which DNA in gel block digests cell wall Gel block is mounted Analysis of DNA RFLP in agarose gel and Gel is stained by ethidium using a computer DNA fragments are bromide program separated by PFGE at 14C for 22 h  ADVANTAGES : Typing by Restriction Fragment Length Polymorphism RFLP-PFGE  A fast, simple and accurate molecular tool for the profiling and identification of population.  High reliability – it is generated from specific sites via known restriction enzymes and the results are constant over time and location.  Co-dominance, which means investigators are able to distinguish heterozygotes from homozygotes and highly locus specific.  Selective neutrality refers to a situation in which different alleles of a certain gene confer equal fitness.  A good repeatability in the typing of Salmonella gallinarum.  An effective tool for distinguishing very closely related strain subpopulations within Lactobacillus delbrueckii  LIMITATIONS : Typing by Restriction Fragment Length Polymorphism RFLP-PFGE  RFLP–PFGE requires large amounts of genomic DNA; the process is somewhat time-consuming and technically demanding, thus limiting the laboratory’s ability to process large numbers of organisms simultaneously.  RFLP–PFGE analysis provides relatively global chromosomal overview, scanning more than 90% of the chromosome (the sum of the restriction fragment sizes), but it has only moderate sensitivity, since minor genetic changes may go undetected.  The most common problem associated with this assay is incomplete digestion, resulting in difficulty in the interpretation of band patterns. The complex profiles which consist of hundreds of bands that may be overlapping make it difficult to interpret.  APPLICATIONS : Typing by Restriction Fragment Length Polymorphism RFLP-PFGE  Conducting an outbreak investigation and certain false-positive culture investigations  Clustered patients with possible epidemiologic links  Detection of bacterial contamination in food (e.g. milk etc.)  04 Sequencing “DNA sequencing is a process of determining or identifying the order of nucleotides present in a DNA sequence.”  PRINCIPLES : Typing by Sequencing Analysis Sequence-based molecular epidemiology is attractive in offering the promise of reproducible typing profiles that are highly amenable to standardization, uniform interpretation, and database cataloging, since they are based on simple quaternary data (A, T, G, and C). Universal sequences can be used to genotype bacteria, including 16S rRNA genes, 16S–23S rRNA gene interspacer region, (and heat shock protein genes (i.e., hsp65). Sequence variation in a specific gene (i.e., gene for virulence, pathogenicity, drug resistance, etc.) at single nucleotide level or short repeats can be resolved by sequencing analysis. Therefore, the sequence data for specific loci/gene from different strains of the same species can be used for molecular epidemiologic application. TYPES OF SEQUENCE TYPING SINGLE The single locus sequence typing (SLST) 1 LOCUS involves analysis of a SEQUENCE particular region of TYPING gene MULTILOCUS MLST utilizes a larger, 2 and potentially more SEQUENCE representative, portion TYPING of the genome WHOLE Recent advancement of 3 high-throughput deep GENOME sequencing SEQUENCING technologies  TYPES OF SEQUENCE TYPING SINGLE SLST is a method that targets a single gene or locus for LOCUS sequencing. SEQUENCE It is a relatively simple and inexpensive method but may TYPING not provide the same level of resolution as MLST or WGS. MULTILOCUS SEQUENCE TYPING WHOLE GENOME SEQUENCING  TYPES OF SEQUENCE TYPING SINGLE LOCUS MLST is a widely used method for typing bacteria, SEQUENCE particularly for epidemiological studies. TYPING It involves sequencing of several housekeeping genes, typically 7-8, and comparing the sequences to assign MULTILOCUS unique allelic profiles or sequence types (STs) to each isolate. SEQUENCE The advantage of MLST is that it provides high-resolution TYPING data, and the results can be easily shared and compared between laboratories. However, the method can be time-consuming and WHOLE expensive. GENOME SEQUENCING  TYPES OF SEQUENCE TYPING SINGLE LOCUS SEQUENCE TYPING MULTILOCUS WGS involves sequencing the entire genome of the SEQUENCE microorganism and comparing the sequences to identify TYPING variations between isolates. WGS provides the highest level of resolution and can reveal detailed information about the genetic makeup of WHOLE the organism. GENOME It can also identify novel mutations and antibiotic resistance genes. SEQUENCING However, WGS can be expensive, and data analysis can be complex. PROCEDURES : Typing by Sequencing Analysis  ADVANTAGES & LIMITATION : Typing by Sequencing Analysis The advantages The limitations of sequencing-based approach is include high cost for sequencing, its ability in detecting phylogenetic limited throughput, requirement informative genetic variations, of extensive bioinformatics for thus, offering broad coverage of data analysis and interpretation, sequence, enhanced sensitivity, and technical complexity. specificity, reproducibility, and discrimination power. It also offers the better ability for special distribution analysis. APPLICATIONS : Typing by Sequencing Analysis It is can be used in the microbial identification and study of the new bacterial species via metagenomics study. The sequencing technique advances microbial identification by eliminating the traditional and time- consuming culturing methods. With the advancement of technology, microbial identification and characterization have become more rapidly and accurately done using sequencing. By comparing the sequence of the target microbes with the available data, scientists can identify new mutations and new strains. THANKS! Do you have any questions? [email protected] +6 016 666 4359 A616, FSG UiTM Shah Alam CREDITS: This presentation template was created by Slidesgo, including icons by Flaticon, infographics and images by Freepik Please keep this slide for attribution