Phylogenetic Tree of Bacteria PDF

PHYLOGENETIC TREE OF BACTERIA Aquifex pyrophilus Thermotoga maritima Aquificae Thermotogae Deinococcus radiodurans Thermus aquaticus Chloroflexus aurantiacus “Deinococcus-Thermus” Chloroflexi Corynebacterium glutamicum Mycobacterium tuberculosis Micrococcus luteus Streptomyces griseus Frankia sp...

PHYLOGENETIC TREE OF BACTERIA Aquifex pyrophilus Thermotoga maritima Aquificae Thermotogae Deinococcus radiodurans Thermus aquaticus Chloroflexus aurantiacus “Deinococcus-Thermus” Chloroflexi Corynebacterium glutamicum Mycobacterium tuberculosis Micrococcus luteus Streptomyces griseus Frankia sp. Actinobacteria (High G + C Gram-positives) Fusobacteria Fusobacterium ulcerans Staphylococcus aureus Bacillus cereus Enterococcus faecalis Streptococcus pyogenes Firmicutes (Low G + C Gram-positives) Mycoplasma pneumoniae Clostridium perfringens Anabaena “cylindrica” Synechococcus lividus Oscillatoria sp. Chlamydia trachomatis Cyanobacteria Chlamydiae Planctomycetes Chlorobi Planctomyces maris Chlorobium limicola Flexibacter litoralis Cytophaga aurantiaca Flavobacterium hydatis Bacteroides fragilis Fibrobacter succinogenes Treponema pallidum Borrelia burgdorferi Campylobacter jejuni Helicobacter pylori Desulfovibrio desulfuricans Bdellovibrio bacteriovorus Myxococcus xanthus Rickettsia rickettsii Caulobacter crescentus Rhodospirillum rubrum Vibrio cholerae Escherichia coli Pseudomonas aeruginosa Neisseria gonorrhoeae Alcaligenes denitrificans Nitrosococcus mobilis Copyright © The McGraw-Hill Companies, Inc. Bacteroidetes Fibrobacteres Spirochaetes Epsilonproteobacteria Deltaproteobacteria Alphaproteobacteria Proteobacteria Gammaproteobacteria Betaproteobacteria PHYLOGENY • Defined as the evolutionary history of development of a group of organisms (or even of proteins and genes) • Derived from “phylo-”, the Greek word for “clan” or “tribe”, and “genesis” meaning “the origin of ”. • Phylogenetics is the study of evolutionary relationships between organisms and the construction of an accurate and complete “tree of life” that faithfully tells us how all the current species arose is its ultimate goal. An early tree of life drawn by Ernst Haeckel in 1866 TAXONOMY Taxonomy is the study and creation of hierarchical systems for classifying and identifying organisms. How did we build that tree? How did we know where to place each species? What’s a species? PRINCIPLES OF TAXONOMY • Taxonomy is the science that studies organisms to arrange them into groups, or taxa • Three separate but interrelated areas: CLASSIFICATION ➢ Arranging organisms into similar or related groups and defining what set of features defines a group/taxa ➢ NOMENCLATURE ➢ System of assigning names ➢ IDENTIFICATION ➢ Process of characterizing a microbe to determine group where it belongs ➢ STRATEGIES TO CLASSIFY PROKARYOTES • Determining phylogeny of prokaryotes is difficult In higher organisms, species are morphologically similar and capable of interbreeding to produce viable offspring ➢ Prokaryotes, are similar in physical characteristics (size, shape) and do not undergo sexual reproduction ➢ Difficult to apply same classification criteria ➢ Classically relied heavily on phenotypic characteristics for classification ➢ BELONG TO THE SAME TAXA? http://www.dolphinsc.com/ http://room42.wikispaces.com/Oceanic+Animals If you made a classification scheme that includes a category of “large water-dwelling animals with fins that can jump out of water”, then swordfish and dolphins could be considered as “members of the same group”. 7 Identify me! Which animal is more closely related to the one in the middle? 8 Escherichia coli Listeria monocytogenes Which bacteria is more closely related to the one in the middle? For many reasons the rules we use for animals are difficult to apply to microbes!!! 9 STRATEGIES TO CLASSIFYING PROKARYOTES • Goal is to group according to evolutionary relatedness • Classification historically based on phenotypic traits Size, shape, staining, metabolic capabilities ➢ Drawbacks: ➢ Phenotypic differences can be due to few gene products & single mutations may affect organism’s capabilities ➢ Phenotypically similar organisms may be only distantly related; conversely, closely related organisms may appear dissimilar ➢ • New molecular techniques more accurate and less prone to human bias Provide greater insights into evolutionary relatedness ➢ DNA sequences viewed as evolutionary chronometers ➢ • DNA sequencing allows construction of phylogenetic tree PROKARYOTE CLASSIFICATION SYSTEMS Current Classification System – “Three Domain System” • No “official” classification system – change as new information discovered - new ones introduced, others outdated Three-domain system (Carl Woese et. al.) currently favored ➢ Compared ribosomal RNA nucleotide sequences ➢ Bacteria Archaea Eucarya Entamoebae Slime molds Filamentous anoxygenic phototrophic bacteria Methanosarcina GramSpirochetespositive MethanoHalobacteria bacteria bacterium Proteobacteria ThermococcusMethanoThermoplasma Thermoproteuscoccus Cyanobacteria Pyrodictium Flavobacteria Animals Fungi Plants Trypanosoma Trichomonads Thermotoga Aquifex Diplomonads Microsporidia Adapted from G.J. Olsen and C.R. Woese “Ribosomal RNA: A Key to Phylogeny” FASEB Journal, 7:113-123, 1993. Copyright © The McGraw-Hill Companies, Inc. PROKARYOTE CLASSIFICATION SYSTEMS • Three-domain system is based on evolutionary relatedness • Replaces five-kingdom system Plantae, Animalia, Fungi, Protista, Prokaryotae ➢ Based on obvious morphological differences ➢ Does not reflect recent genetic insights of ribosomal RNA data indicating plants, animals more closely related than Archaea, Bacteria ➢ SYSTEMS TO CLASSIFY PROKARYOTES Taxonomic Hierarchies • Species is basic unit of taxonomy ➢ ➢ Species is group of closely related isolates or strains – permits identification The use of “Kingdoms” is still in state of flux - not used for bacteria/ archaea • Informal groupings also used ➢ May be genetically unrelated ➢ Lactic acid bacteria ➢ Anoxygenic phototrophs ➢ Endospore-formers ➢ Sulfate reducers CLASSIFYING PROKARYOTES • Phylogenetic tree shows evolutionary relatedness E.g., Bacterium Thermotoga maritima appears to have acquired ~25% of genes from archaeal species • Some scientists have proposed a shrub with interwoven branches Crenarchaeota Euryarchaeota Plantae Fungi Animalia s ts pla o r olo Ch ria nd o h toc Mi Archaea Archezoa ➢ Eucarya Cyanobacteria • Horizontal (lateral) gene transfer complicates DNA comparisons Bacteria Proteobacteria • But DNA sequencing also highlights obstacle Copyright © The McGraw-Hill Companies, Inc. Gene Content Between Two Strains of E. coli Can Differ More Than Humans and Fish E. coli CFT073 Uropathogenic strain Less than 1 million years of evolution ≈ 65% genes found in CFT073 are found in K12 Approx. 450 MYr ≈ 75% genes found in human are found in fugu Homo sapiens E. coli K12 Non-pathogenic laboratory strain Fugu rubripes 15 http://genome.jgi-psf.org/Takru4Takru4.home.html WHAT ARE STRAINS? • Sub-species and Strains are levels of categorization underneath species. Strains typically identify specific isolates of a given species • There can be considerable variation between different strains of a single species. E. coli MG1655 (also called K12) – Most common lab strain of E. coli – first isolated in 1922 from a patient with diphtheria (E. coli does not cause diphtheria) ➢ E. coli XL1-Blue – a manipulated version of MG1655 to make it easier to get plasmid DNA into and out of ➢ E. coli Sakai (O157:H7) – a cause of severe intestinal disease (Hamburger disease) ➢ E. coli CFT073 – isolated from a urinary tract (bladder) infection ➢ • Strain names are very arbitrary – usually given by an inventory system or the first person to work with that isolate. BERGEY’S MANUAL Bergey’s Manual of Systematic Bacteriology • Describes all known species, including those not yet cultivated • Newest edition in five volumes • Classifies according to genetic relatedness ➢ • Previous edition grouped according to phenotype, so some major differences in newest edition Includes information on ecology, methods of isolation, enrichment, culture, maintenance & preservation METHODS OF PROKARYOTIC CLASSIFICATION TO DETERMINE SPECIES RELATEDNESS • Phenotypic Methods • 16S rDNA sequence analysis (most common) • Whole genome sequence alignment (new) • DNA hybridization* • DNA Base Ratio (G + C Content)** (*outdated and no longer used – **do not need to know) PHENOTYPIC METHODS • The traditional methods relied on predicting relatedness based on phenotypic properties (Gram-stain, size, morphology, biochemical tests, aerobic/anaerobic, etc) • Have been largely replaced by 16S ribosomal nucleic acid sequence methods • Some taxonomists still believe classification should be based on more than just genotypic traits • Phenotypic methods still important since they provide a foundation for prokaryotic identification DNA DNA HYBRIDIZATION SEQUENCING RIBOSOMAL RNA GENES • 16S rRNAs (18S in eukaryotes) make up most of the small subunit of the ribosome • Where to start if you have a complete unknown – good for identifying genus and, mostly, species • Ribosomal RNA (rRNA), or the corresponding DNA (rDNA), can be used to identify prokaryotes and to determine their position on the evolutionary tree of life. • Analysis & comparison of the sequence of the 16S ribosomal RNA (rRNA – coded for by rDNA) have revolutionized classification • 16S rRNA most useful because of its moderate size ~1,500 nucleotides 70S 30S 16S rRNA + 21 polypeptide chains 50S 5S rRNA + 23S rRNA + 34 polypeptide chains Copyright © The McGraw-Hill Companies, Inc. 16S RIBOSOMAL DNA SEQUENCE ANALYSIS • Cutoffs to call something the same genus or species ➢ ➢ Two members of the same genus will typically have >95% identity Two members of the same species generally have >98% identity in their rDNA sequences • May not resolve at species or strain level since closely related prokaryotes have nearly identical 16S rDNA sequences ➢ Multi-locus sequence typing or whole genome sequence better in this circumstance COMPARISON OF WHOLE GENOMES • This is where it’s all headed in the future. Get all the information at once for an unambiguous assignment on the phylogenetic tree. • There is a massive project underway to develop a “Genomic Encyclopedia of Bacteria and Archaea (GEBA)” that pulls together genome sequences of thousands of microbial isolates to generate a high-resolution microbial tree of life including obscure and rarely studied microbes. STRATEGIES TO IDENTIFY PROKARYOTES • Identifying microbes often an important goal Food manufacturer – important to identify food contaminant ➢ Clinical setting – ➢ important to quickly identify agent & treat appropriately ➢ Important to look for and track outbreaks ➢ • Numerous procedures used to identify Microscopy, culture characteristics, biochemical test, nucleic acid analysis ➢ Clinical setting – patient’s symptoms play important role but can be misleading or of limited informational value ➢ Sometimes sufficient to rule out presence of disease-causing organism rather than conclusively identify it ➢ Fecal specimen from patient with diarrhea and fever tested for organisms causing such symptoms ➢ SPECIMEN COLLECTION • Sampling body sites or fluids for suspected infectious agent • Results depend on specimen collection, handling, transport, and storage • Aseptic procedures should be used Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Saliva Sputum Nasopharynx Tamper-evident seal Throat (tonsils) Skin: –– Swab Plastic case Blood Insert Fig 17.1 Spinal tap (Cerebrospinal fluid) Feces Long swab with rayon tip Vaginal swab or stick Clean catch Transport medium Squeeze container to release medium Catheter Skin: Scalped (b) 2 (a) Specimen Collection 3 OVERVIEW OF DIAGNOSTIC TECHNIQUES • Routes taken in specimen analysis ➢ Direct tests (microscopic, immunologic, or genetic) ➢ Cultivation, isolation, and identification (general and specific tests) • Two categories of tests ➢ Presumptive ➢ Confirmatory Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Specimen Direct Testing Culture/Isolation Microscopic Gram stain Acid-fast stain Fluorescent Ab stain Gene probes Tests on isolates Biochemical Serotyping (Slide) Antimicrobic sensitivity PCR analysis Phage typing Animal inoculation Macroscopic Direct antigen DNA typing Patient Immunologic and serological tests (antibody titer) are performed on blood and other fluids Clinical signs and symptoms In vivo test for reaction to microbe 4 USING PHENOTYPIC CHARACTERISTICS TO IDENTIFY CELLULAR PATHOGENS • Microscopic morphology • Culture characteristics • Metabolic capabilities • Serology (immunological methods) • Fatty acid analysis • Mass spectrometry MICROSCOPIC MORPHOLOGY • Important initial step to quickly determine size, shape, staining characteristics ➢ Sometimes enough to diagnose eukaryotic infections & start appropriate treatment • Gram stain may provide enough information to start appropriate therapy • Special stains (e.g., acid-fast, endospore) also useful Gram stain Streptococcus pneumoniae Wet mount Neisseria gonorrhoeae Candida albicans Roundworm egg White blood cell © E. Koneman/Visuals Unlimited © Biophoto Associates/Photo Researchers, Inc. Copyright © The McGraw-Hill Companies, Inc. © George Wilder/Visuals Unlimited Courtesy of Dr. Thomas R. Fritsche, M.D./Ph.D., Clinical Microbiology Division, Univ of Washington, Seattle CULTURE CHARACTERISTICS • Colony morphology can give initial clues that identify organism Streptococci colonies generally fairly small ➢ Serratia marcescens colonies often red at 22°C ➢ Pseudomonas aeruginosa often produces green pigment ➢ Cultures also have distinct fruity odor ➢ • Differential media aids in identification Streptococcus pyogenes (strep throat) yields B-hemolytic colonies on blood agar ➢ E. coli (urinary tract infection) ferments lactose, forms pink colonies on MacConkey agar ➢ METABOLIC CAPABILITIES Biochemical tests • Provide more certainty of identification Catalase test ➢ Bacteria that grow in presence of oxygen – catalase positive ➢ Exception – lactic acid bacteria (e.g. Streptococcus) ➢ Many tests rely on color changes (e.g. pH indicators) ➢ Sugar fermentation & urease production ➢ © Denise Anderson © Dennis Strete/Fundamental Photographs Copyright © The McGraw-Hill Companies, Inc. © Dennis Strete/Fundamental Photographs USING PHENOTYPIC CHARACTERISTICS TO IDENTIFY PROKARYOTES Biochemical tests API test strip Courtesy of bioMerieux, Inc. • Commercial kits available allow rapid identification via biochemical tests • Less labour intensive and more consistent Enterotube II Courtesy of Becton, Dickinson and Company Copyright © The McGraw-Hill Companies, Inc. API TEST STRIPS http://www.jlindquist.net/generalmicro/102bactid2.html METABOLIC CAPABILITIES Biochemical tests • Basic strategy relies on dichotomous key Flowchart of tests with positive or negative result ➢ Simultaneous inoculating speeds process ➢ • Some tests accomplished without culturing (e.g., breath test or urease to identify Helicobacter pylori) Gram stain Gram-positive coccus Gram-negative rod Catalase Oxidase test Positive Negative Coagulase Positive Staphylococcus aureus Enterococcus sp. Negative Staphylococcus saprophyticus Negative Positive Pseudomonas aeruginosa Lactose fermentation Positive E. Coli or Other coliform Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Negative Proteus sp. SEROLOGY • The use of antisera (antibodies) from animals to diagnose a particular pathogen. • Animals have been immunized with any one of hundreds of different pathogens such that their blood plasma (sera) contains antibodies specific to that pathogen. These ‘antisera’ can be purchased from a variety of specialty diagnostic companies to facilitate diagnosis and identification. • Proteins, polysaccharides of prokaryotic cells can serve as identifying markers • Most useful include surface structures of cell wall, LPS O-antigen, capsule, flagella, pili FATTY ACID ANALYSIS (FAME) • Prokaryotic species differ in type, quantities of fatty acids in membranes • Fatty acid composition therefore useful in identification Cells grown under standard conditions – lipids can differ if conditions are not kept the same from experiment to experiment ➢ Treated to release fatty acids ➢ Converted to fatty acid methyl ester (FAME) form ➢ Separated and measured via gas chromagraphy ➢ Chromatogram compared to those of known species ➢ DETECTING SPECIFIC NUCLEOTIDE SEQUENCES Nucleic acid amplification tests (NAATs) • Refers to several methods used to increase number of copies of specific DNA sequences in a sample for detection ➢ Polymerase chain reaction (PCR) is the most common NAAT ➢ Allows detection of small numbers of organisms ➢ Often from body fluids, soil, food, water ➢ Detection of organisms that cannot be cultured DETECTING SPECIFIC NUCLEOTIDE SEQUENCES Nucleic acid probes Locate nucleotide sequence characteristic of species or group ➢ Probe is single-stranded nucleic acid (usually DNA) complimentary to sequence of interest – usually with a tag (radioactive or fluorescent) ➢ Most methods first increase DNA in sample ➢ Fluorescence in situ hybridization (FISH) probes for 16S rRNA ➢ Unknown organism (double-stranded DNA) Probe is added to denatured, single-stranded DNA of unknown organism. Organism X DNA Probe Double-stranded DNA sequence unique to organism X DNA is labeled, then denatured to become a probe. Denatured Single-stranded DNA Copyright © The McGraw-Hill Companies, Inc. If probe does not bind to DNA, then unknown organism is not organism X. If probe binds to DNA, then unknown organism is organism X. DETECTING SPECIFIC NUCLEOTIDE SEQUENCES Nucleic acid probes • Limitation is each probe only detects single possible species/strain • Need to run multiple probes if organism being tested could be one of multiple different species or related groups Unknown organism (double-stranded DNA) Probe is added to denatured, single-stranded DNA of unknown organism. Organism X DNA Probe Double-stranded DNA sequence unique to organism X DNA is labeled, then denatured to become a probe. Denatured Single-stranded DNA Copyright © The McGraw-Hill Companies, Inc. If probe does not bind to DNA, then unknown organism is not organism X. If probe binds to DNA, then unknown organism is organism X. ADVANDX QUICKFISH® Contains a fluorescent green labeled DNA probe that binds to a sequence found only in S. aureus and a red fluoresent probe that only binds to “coagulase negative” staphylococci (CoNS). Assay can be done in 20 minutes directly on patient samples without waiting for cultures to grow. CHARACTERIZING STRAIN DIFFERENCES • Characterizing strains is important Finding sources of water and foodborne illnesses ➢ Diagnosing certain diseases ➢ Forensic investigations of bioterrorism, biocrimes ➢ Tracking antibiotic resistance ➢ • Summary of Methods: Biochemical Typing ➢ Serological Typing ➢ Molecular Typing (MLST, RFLP) ➢ Phage Typing ➢ Antibiograms ➢ CHARACTERIZING STRAIN DIFFERENCES Biochemical Typing • Identify bacterial species & distinguish strains ➢ Group of strains with characteristic pattern of growth called a biovar or biotype Flagella (H antigen) Example – Agrobacterium Biovar 1 = can grow at 37° C ➢ Biovar 2 = can’t grow at 37° C and need the compound biotin for growth ➢ Biovar 3 = can’t grow at 37° C but can tolerate high salt in growth medium ➢ Capsule (K antigen) Cell wall (O antigen of outer membrane) CHARACTERIZING STRAIN DIFFERENCES Serological Typing • Proteins & carbohydrates that vary among strains can be used as markers E. coli distinguished by antigenic type of flagella, capsules, lipopolysaccharide molecules ➢ E. coli O157:H7 (O antigen is lipopolysaccharide; H antigen is flagella, K antigen is capsule – remember K12?) ➢ Group with characteristic antigens: serovar, or serotype Flagella (H antigen) ➢ Capsule (K antigen) Cell wall (O antigen of outer membrane) CHARACTERIZING STRAIN DIFFERENCES Restriction sites Strain A DNA Molecular Typing • Subtle differences in DNA structure distinguish phenotypically identical strains ➢ Important for tracing epidemics Cut with restriction enzyme 3 kb 6 kb 1 kb Strain B DNA 5 kb 4 kb 1 kb Strain C DNA 3 kb 2 kb 4 kb 1 kb • Restriction Fragment Length Polymorphism (RFLPs) Different RFLPs indicate different strains ➢ PulseNet tracks RFLPs of certain bacterial foodborne pathogens ➢ Courtesy of Patricia Exekiel Moor Copyright © The McGraw-Hill Companies, Inc. MULTI-LOCUS SEQUENCE TYPING (MLST) • A good way of distinguishing strains of a given species apart from one another • Compare the sequences of a set of highly conserved genes (housekeeping genes) – usually 8 to 10 genes • Perform PCR to amplify 500 bp regions from a set of different genes and sequence each of them • Align sequences to a database of known species isolates – there is a different database used for S. aureus than for the fungus Candida albicans (see mlst.net) • Not every species has an MLST database – only a few of the most important pathogens • Highly similar sequences = closely related microbes CHARACTERIZING STRAIN DIFFERENCES 1 An inoculum of Staphylococcus aureus is spread over the surface of agar medium. Phage Typing Inoculum of Staphylococcus aureus strain to be typed Agar medium Petri dish • Relies on differences in bacterial strain susceptibility to bacteriophages • Susceptibility pattern can be determined with bacteria and different bacteriophage suspensions • Largely replaced by molecular methods ➢ Still useful in labs lacking equipment for genomic testing 2 Different bacteriophage suspensions are deposited in a fixed pattern. incubation, different patterns of lysis are seen with 3 After different strains of S. aureus. Dye marker Lysis Copyright © The McGraw-Hill Companies, Inc. © Evans Roberts CHARACTERIZING STRAIN DIFFERENCES Antibiograms (disc diffusion method) • Distinguish bacterial strains Antibiotic susceptibility patterns ➢ Clearing zones around antibiotic discs ➢ Largely replaced by molecular techniques ➢ © Evan Roberts Copyright © The McGraw-Hill Companies, Inc. 16S rRNA gene analysis for bacterial identification in in clinical microbiology Julianne Kus, MSc, PhD, FCCM Assistant Professor, Laboratory Medicine and Pathobiology, U of T Clinical Microbiologist, Public Health Ontario Conflicts of Interest • none PublicHealthOntario.ca Learning Objectives • To gain an understanding of the challenges of bacterial identification in the clinical setting • To be able to understand and describe why the 16S rRNA gene is used for bacterial identification and how this is done PublicHealthOntario.ca 3 Specimen to bacterial isolation Urine Blood Blood CSF Tissue PublicHealthOntario.ca Many different types of media used - Enriched (eg. Sheep Blood Agar) - Selective (eg. CAN Agar) - Differential (eg. MacConkey Agar) 4 Diagnostics - Microorganism Identification • If you know what you are looking for you can use a specific test to “rule in” or “rule out” particular microorganisms • Eg. Neisseria gonnorhea, Methicillin-resistant Staphylococcus aureus, Haemophilus influenzae • If you are not sure of the identity of the organism in question the tests performed for identification will need to be more expansive PublicHealthOntario.ca 5 Methods of Microbial Identification IMMUNOLOGICAL mono-or polyclonal antibodies MORPHOLOGY Microscopy, Gram stain, colour, growth on differential media MOLECULAR - specific-PCR, probes - 16S rDNA sequencing, WGS BIOCHEMICAL metabolic capacities and resistance to antibiotics CHEMOTAXONOMIC Lipids Glycans Proteins PublicHealthOntario.ca Julianne V. Kus What is 16S ribosomal RNA? PublicHealthOntario.ca 7 Ribosomes are made up RNA + Protein Complexes Composed of 2 subunits 80S à EUKARYOTES 70S à PROKARYOTES 2 subunits: 60S & 40S 2 subunits: 50S and 30S 60S = 28S, 5.8S, 5S rRNAs + 49 proteins 50S = 23S rRNA + 5S rRNA + 34 proteins 40S = 18S rRNA + 33 proteins 30S = 16S rRNA + 21 proteins 18S rRNA Part of the small subunit PublicHealthOntario.ca 16S rRNA Part of the small subunit Ribosomal rRNA gene Structure DNA Prokaryote (Bacteria) 16S rDNA tRNA 23S rDNA Eukaryote (Fungi, Parasites, Mammals) 5.8S 18S rDNA 5S 25-28S rDNA tRNA 5S Internal transcribed regions (ITS) – most diversity One transcribed unit • Ribosomal genes are multi-copy genes tandemly organised in the genome PublicHealthOntario.ca Julianne V. Kus The genes that encode the 16S rRNA can be sequenced in order to: • identify an organism's taxonomic group • calculate relatedness between groups • describe new species, and those that have never been successfully cultured PublicHealthOntario.ca Why is the 16S rRNA gene such a good target for bacterial identification/classification? 1. universal - in all bacteria 2. multi-copy 3. with variable regions • • species-specific sequences determine organism relatedness 4. highly function, highly conserved regions 5. can design “universal” primers to amplify any type of bacteria PublicHealthOntario.ca http://www.accugenix.com/microbial-identification-bacteria-fungus-knowledge-center/micro-id-basics/16s-rna-bacterial/ Universal Primers for Organism Identification F R 16S rRNA gene CONSERVED REGIONS VARIABLE REGIONS • Conserved regions, flanking variable regions, allow for “universal” amplification of the target • Sequences of variable regions allow for organism identification PublicHealthOntario.ca Julianne V. Kus How is this accomplished in the lab? Bacteria Extract DNA Run gel If no band – “not detected” PCR Day 1 Sequence PCR product Day 2 Analyse Sequence PublicHealthOntario.ca Sequence Analysis – database comparison GenBank 17618 deposited sequences 16S database rRNA gene PCR at Public Health Ontario • 16S rRNA gene PCR and sequencing • Routinely used for Bacterial Reference Identification when we are unable to assign a genus and/or species level identification using traditional methods • “Research Use Only” test on direct specimens when traditional culture-based methods are negative PublicHealthOntario.ca Use on direct specimens PublicHealthOntario.ca 15 Early Success Stories Use on direct specimens – Pathogen discovery • 1990: Bartonella henselae - the agent of bacillary angiomatosis, was discovered and identified using 16S rDNA amplification and sequencing from tissue sections obtained from patients with HIV infection • Relman DA, Loutit JS, Schmidt TM, et al. The agent of bacillary angiomatosis: an approach to the identification of uncultured pathogens N Engl J Med 1990;323:1573-80. • 1992: Tropheryma whipplei - a previously uncultured bacterium, was first detected with 16S rDNA primers in a small bowel biopsy sample obtained from a patient with Whipple disease • Relman DA, Schmidt TM, MacDermott RP, et al. Identification of the uncultured bacillus of Whipple's disease. N Engl J Med 1992;327:293-301. • both cases, the pathogen was unable to be cultivated and was only able to be detected using staining methods PublicHealthOntario.ca 16 16S PCR on Clinical Specimens at PHO • Run on specimens in which an infection is suspected but no microorganism can be identified using traditional means • Beginning July 2013, I began collecting clinical information on each case/specimen to pair with our results to try to identify: • • • how many tests we run how the test is performing (positives and negatives, bacteria identified) what types of specimens we get • Will enable us to • • • Make more informed decisions about what specimens to test Better understand and interpret the results Identify areas that we can improve the test PublicHealthOntario.ca 17 Results • 116 samples tested, from 105 patients PublicHealthOntario.ca 18 Specimen # run Blood - EDTA 5 Blood Culture 4 CSF 29 Abscess fluid 6 Synovial fluid 11 Other fluid 6 Bone 5 Tissue – not heart 30 Heart valve tissue 20 OVERALL 116 PublicHealthOntario.ca 19 Detection of a Probable Pathogen Specimen PCR positive Blood - EDTA 2/5 = 40% 0/5 = 0% Blood Culture 0/4 = 0% 0/4 = 0% CSF 3/29 = 10% 2/29 = 7% Abscess fluid 2/6 = 33% 2/6 = 33% Synovial fluid 0/11 = 0% 0/11 = 0% Other fluid 2/6 = 33% 2/6 = 33% Bone 0/5 = 0% 0/5 = 0% Tissue – not heart 2/30 = 7% 1/30 = 3% Heart valve tissue 10/20 = 50% 10/20 = 50% OVERALL 21/116 = 18% 17/116 = 15% PublicHealthOntario.ca 12% - 21% 20 Pathogens Identified CSF Heart valve tissue Fusobacterium necrophorum Streptococcus species Streptococcus pneumoniae Aggregatibacter actinomycetemcomitans x2 Abscess fluid Bartonella quintana x3 Aerococcus urinae Streptococcus pneumoniae Staphylococcus aureus Other fluid (vitreous and pleural) Tropheryma whipplei Achromobacter species Streptococcus pyogenes (“Group A Strep”) Haemophilus species Coxiella burnetii Tissue – Brain abscess Prevotella species PublicHealthOntario.ca 21 Summary • Based on ~1 year of data, when provided an adequate amount of specimen • We have identified pathogens from tissues, pleural fluids, abscess fluid, CSF • Heart valve tissue have the highest positivity rateà50%; variety of bacteria identified • We have not had any positives from blood, bone, synovial fluids • Almost all positive samples were collected from patients receiving antibiotics and no organisms were seen on the direct Gram stains for most samples PublicHealthOntario.ca 22 Caveats • Because we only get culture negative specimens cannot truly determine the sensitivity of the assay (are our negatives true negatives?) • From the lab end, cannot always determine what is a “contaminant” and what is a “pathogen” • Certain organisms are always considered pathogens if isolated from what should be sterile tissue/fluid (eg. Staphylococcus aureus) • The presence of other bacteria are more difficult to interpret (eg. common skin colonizing bacteria like Corynebacteria species or Staphylococcus epidermidis) - need to discuss with ordering physician PublicHealthOntario.ca 23 16S PCR on Clinical Specimens Advantages • May provide an answer when no other method was able to • Do not need the bacteria to be alive (eg. previous treatment with antibiotics) Disadvantages • Do not have the actual organism so cannot perform susceptibility testing to determine if the organism is resistant to any antibiotics • Cannot use if more than one bacterial species present – mixed sequence • The finding of 16S rDNA alone is not sufficient to infer causation of infection; contaminating DNA in reagents, or upon specimen collection/handling • A negative does not necessarily mean no bacteria present/no infection • Only on tissue/fluid from sterile sites PublicHealthOntario.ca 24 Conclusions • The 16S rRNA gene is an excellent target for bacterial identification and classification • 16S rRNA gene PCR on direct specimens is a good complement to traditional microbiological workup when results are culture negative • The PHO assay has had some good success with identifying bacteria from fluids and tissues, especially heart valves, even in patients receiving antibiotics and with no bacteria seen on direct Gram stain PublicHealthOntario.ca 25 ADVANCED METHODS IN MICROBIAL IDENTIFICATION Ruwandi Kariyawasam, PhD Candidate Institute of Medical Sciences NEW METHODS OF MICROBIAL IDENTIFICATION ARE CONSTANTLY BEING DEVELOPED KEY FEATURES IN DEVELOPING NEW DIAGNOSTICS • Cost-effectiveness and • Shorter turn-around times CLINICAL MICROBIOLOGY LABORATORY 1. Pre-Analytical (few hours) • Specimen collection • Specimen transportation • Specimen sorting, labeling 2. Analytical (days to months) • Diagnostic testing 3. Post-Analytical (few minutes) • Laboratory reporting CLINICAL MICROBIOLOGY LABORATORY 1. Pre-Analytical (few hours) • Specimen collection • Specimen transportation • Specimen sorting, labeling 2. Analytical (days to months) • Diagnostic testing 3. Post-Analytical (few minutes) • Laboratory reporting CLINICAL MICROBIOLOGY LABORATORY 1. Pre-Analytical (few hours) • Specimen collection • Specimen transportation • Specimen sorting, labeling 2. Analytical (days to months) • Diagnostic testing 3. Post-Analytical (few minutes) • Laboratory reporting CLINICAL MICROBIOLOGY LABORATORY 1. Pre-Analytical (few hours) • Specimen collection • Specimen transportation • Specimen sorting, labeling 2. Analytical (days to months) • Diagnostic testing 3. Post-Analytical (few minutes) • Laboratory reporting ANALYTICAL STAGE • The goal of clinical microbiology laboratories is to speed up the analytical process by implementing novel technologies that allow for rapid and low-cost diagnostic testing Day 0 1 2 3-7 Real-Time PCR Clinical Sample Culture Whole Genome Sequencing MALDI-TOF MS // 21 + ADVANCED METHODS OF MICROBIAL IDENTIFICATION • MALDI-ToF MS • Real-Time PCR • Whole Genome Sequencing Ø Illumina MiSeq MALDI-TOF-MS C ASE 1 • Clinical Picture: SP is a 12 year old female presenting with a sore throat • Differential Diagnosis: Acute Group A Streptococcal Pharyngitis Ø Streptococcus pyogenes • Specimen: Throat Swab • Diagnostic Test Platform: MALDI-ToF MS WHY MALDI-TOF MS? • Rapid identification of microorganisms • Advantages: Cheap (~ $1/sample) Ø Fast Ø • Disadvantages Ø Limited to database with known organisms (~5000) MALDI-TOF MS • Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry Ø Ø Ø Analyzes protein composition from intact cells or nucleic acid extract Protein profile compared to database containing unique fingerprints specific to organism Genus and species level identification Unknown profile 100% match to known profile in database C ASE 1: MALDI-TOF MS • Results: Match to Streptococcus pyogenes Ø Time: 1-2 days faster than conventional methods Ø Ø Cost: cheap (~$1/sample) Streptococcus pyogenes REAL-TIME PCR C ASE 2 • Clinical Picture: DF is a 26 year old male presenting with eye, joint and muscle pain; headache, rash and fever • Differential Diagnosis: dengue fever, chikungunya Ø Dengue virus (DENV), chikungunya virus (CHIKV) • Specimen: whole-blood • Diagnostic Test platform: Real-Time PCR WHY REAL TIME PCR? • Rapid, specific results in less than a few hours • Advantages: • Rapid (< 3 hours) • Cheap (few dollars/sample) • Disadvantages • Requires prior sequence knowledge REAL-TIME PCR • Also known as quantitative PCR (qPCR) • Uses fluorescent dyes and reporters that allows for real-time monitoring of amplification • Amplification curve depicts the rate at which DNA is amplified at each cycle High Concentration Fluorescence Low Concentration Threshold Time (Cycle Number) REAL-TIME PCR • Technology: Ø 1) Non-Specific Fluorescent Dyes-interact with any DNA Ø 2) Sequence-Specific DNA Probes-bind to complimentary DNA sequence of a known target C ASE 2: REAL-TIME PCR • Result: Positive DENV result Ø Negative CHIKV result Ø Time – few hours compared to days for a serological test Ø Cost-$2-$5/sample Ø -DENV -CHIKV WHOLE-GENOME SEQUENCING C ASE 3 • Clinical Case: LA is 27 year old pregnant from the state of Paraiba, Brazil. She presented with a skin rash involving itchiness of the hands and back at 18 weeks gestation. Ø At 21 weeks of gestation, her ultrasound indicated a fetal microcephaly diagnosis. A third ultrasound at 27 weeks confirmed the microcephaly • Differential Diagnosis: Zika virus Ø ZIKV • Specimen: Amniotic fluid samples • Diagnostic Test Platform : WGS WHOLE GENOME SEQUENCING: CLINIC DIAGNOSTIC UTILITY • Used for microbial identification, strain typing, outbreak investigation and antimicrobial resistance surveillance • Advantages: Fast Ø Reliable Ø • Disadvantages: Ø Requires strong laboratory infrastructure WHOLE-GENOME SEQUENCING • Also known as deep sequencing or massively parallel sequencing • Sequencing of millions of fragments simultaneously SEQUENCING PIPELINE Library Preparation PCR Sequencing Data Analysis SEQUENCING PIPELINE-LIBRARY PREPARATION Library Preparation PCR Sequencing Data Analysis SEQUENCING PIPLELINE-PCR Library Preparation PCR Sequencing Data Analysis SEQUENCING PIPELINE-SEQUENCING Library Preparation PCR Sequencing Data Analysis SEQUENCING PIPELINE-DATA ANALYSIS Library Preparation PCR Sequencing Data Analysis WHOLE GENOME SEQUENCING PLATFORMS • 2nd Generation Sequencing Platforms: Roche 454 Ø Illumina MiSeq Ø • 3rd Generation Sequencing Platforms (no PCR step): PacBio Ø Oxford Nanopore • ILLUMINA MISEQ • Based on sequencing by synthesis: primers add fluorescently tagged nucleotides to DNA strands attached to flow cell C ASE 3: ILLUMINA MISEQ • Result: Zika virus genome Ø 97-100% genomic identity shared with French Polynesian strain (green) Ø 87-90% identity with Senegal (blue) and Ugandan (red) strains SUMMARY MALDI-TOF MS Real-Time PCR WGS Specimen Type Culture Nucleic Acid Nucleic Acid Time 1-2 minutes 45 minutes – 3 hours Up to 56 hours for a whole genome Cost $1/sample $2-5/sample $280-1200/sample Advantage Very fast identification Identify multiple pathogens Identify new and emerging pathogens Disadvantage Identify known organisms based on database Must know target sequence Costly; requires strong laboratory and computer infrastructure CONCLUSION • Advances in microbial identification are changing the landscape of clinical microbiology laboratories • Conventional methods are not obsolete, but more sensitive, specific and rapid methods are becoming more easily available • Single-pathogen detection assays are slowly being replaced by more robust technologies such as WGS where multiple pathogens can be detected • Faster reliable diagnosis = faster appropriate care THANK YOU!