PHA112 Micro 7 (ID)2020.pdf

MPharm Programme Microbial Identification Dr Callum Cooper [email protected] Learning Objectives Describe different microbiological techniques to identify bacterial contaminants Classical identification Vs molecular identification Highlight limitations and advantages Link lecture togeth...

MPharm Programme Microbial Identification Dr Callum Cooper [email protected] Learning Objectives Describe different microbiological techniques to identify bacterial contaminants Classical identification Vs molecular identification Highlight limitations and advantages Link lecture together with laboratory work What is the contaminant? OR What is causing the infection? What is Diagnostic Microbiology? Identification USUALLY to species level Typing Distinguishing between strains within a species Antimicrobial susceptibility testing Pathogenicity profiling Clinical applications, but also… Veterinary microbiology Plant (crop) pathology Food microbiology Pharmaceutical microbiology (characterising contaminants) Microbial Identification Culture-based methods – Colony morphology – Selective media e.g. Mannitol salt agar – Chromogenic media e.g. MacConkey agar Microscopic examination – Gram’s staining – Electron microscopy Biochemical profiling Molecular analysis – Immunologic – Genomic – Proteomic Colony morphology Microbes are nearly everywhere and they often live in complex communities NOT as individual species Highly inaccurate to identify species by colony morphology Colony morphology To increase accuracy, individual colonies have to be purified Done by sub culturing them What does this tell us about the microorganism? NOTHING Selective and Differential media Selective media: Allows for the growth of particular species Suppresses other species Antibiotic /nutrient supplements MacConkey Agar; inhibits G+ organisms due to bile salts and crystal violet Neutral red dye turns pink in acid Sabouraud Dextrose Agar; selective for certain fungi due to low pH(5.6) and high glucose concentration (3-4%) Baird-Parker Agar: selective for Staphylococci, Pyruvate and glycine promote Staph growth Lithium chloride and tellurite inhibit nonStaph growth Tellurite reduced by S. aureus S. aureus shows shiny black colonies with clear zone around them Selective and Differential media Differential media: enables bacterial species to be distinguished from one another on the same medium Mannitol salt agar (also selective); Inhibits G- organisms (and some G+) due to high salt content (7.5-10%) Mannitol content and phenol red differentiate between Staphylococci Eosin methylene blue (EMB) agar Selective for G- (e.g. E. coli) organisms Contains 2 stains, eosin and methylene blue Toxic to G+ Lactose fermentation causes dye uptake and stains G- purple E. coli develops a metallic green sheen Microscopy Microscopy Light Fluorescence Electron Cryo Transmission Dark field Phase contrast Scanning Transmission Visible light Radiation source Electron beam Air Medium High Vacuum >1000 x Magnification >100000 x 0.2µm Resolution 0.5 nm Microscopy Light Microscopy- Sample preparation In order to see microorganisms more clearly they are often stained Common stains include; Bacteria Gram Stain (Gram Positive Vs Gram Negative) Ziehl–Neelsen stain (Acid fast bacteria; mainly Mycobacteria) Malachite Green Stain (Endospore staining) Fungi Periodic acid-Schiff stain Used to detect polysaccharides Only works on living fungi India ink staining (also used for bacterial capsules) Calcofluor White binds chitin and cellulose Protozoa Direct microscopy (e.g Trypanosoma brucei in blood) Giemsa stain Most viruses cannot be visualised with light microscopy Light Microscopy- Gram Staining 1st step in identifying pure bacterial cultures Differentiates between Gram positive and Gram negative bacteria based on cell wall composition Electron Microscopy Preparation method will vary by type of Electron microscope used Sample Spread over a support grid Chemical Fixation TEM Dehydration Embedding Critical Drying Ultramicrotomy SEM Metal Coating Immunolabelling Contrast staining Negative staining Biochemical Profiling Can identify organisms that have been isolated in pure culture Requires Gram state to be known Individual tests available; Oxidase test (tests is bacteria is able to produce certain cytochrome oxidase or indophenol oxidase) TMPD or DMPD as redox indicators Ox + Pseudomonadaceae Ox - Enterobacteriacae Commercially available kits; Miniaturised biochemical tests API strips (bioMerieux, 1970) BD BBL Crystal Produces a numerical code which identifies bacterium from a database What about in practice: Classical Non-sterile Pharmaceutical Micro TVC Sample TYMC Streak to purity Gram Stain Biochemical Assay Definitive ID Biochemical Profiling: ATP determination Adenosine Triphosphate (ATP) one of the main sources of energy in bacterial cells ATP quantified by the amount of light released following breakdown by firefly luciferase Provides a direct measure of bacterial number Does not require/provide any other information about contaminant Susceptible to contamination Molecular Identification Immunological Identification Genomic Identification ELISA PCR Whole Genome Sequencing 16s rRNA Proteomic Identification MALDI TOF Molecular Identification: ELISA Enzyme-linked immunosorbent assay (ELISA) Used in medical diagnostics (e.g. HIV, TB etc) 2 main types used in diagnostics Direct ELISA (non specific coating of antigens) Sandwich ELISA (allows for capture of specific antigens) Requires specific monoclonal antibodies to work Molecular Identification: Genomic PCR: Amplifies a short sequence of DNA Sequences amplified determined by primers Efficacy dependant on how selective primers are Provides positive/negative results Used for identification of antibiotic resistance e.g. mecA Need to know what you are looking for Molecular Identification: Genomic DNA Sequencing: can sequence whole genome or targeted regions (e.g 16s rRNA) Can identify multiple changes at once (presence/absence of genes, SNPs etc) Multi locus sequence typing (MLST): Amplify housekeeping genes, then sequence 7 genes used in S. aureus; carbamate kinase (arcC) shikimate dehydrogenase (aroE) glycerol kinase (glpF) guanylate kinase (gmk) phosphate acetyltransferase (pta) triosephosphate isomerase (tpi) acetyl coenzyme A acetyltransferase (yqiL) Databases exist for different bacteria species http://www.mlst.net/ https://pubmlst.org/ Need for balance between turnaround time and sensitivity Used less frequently as WGS costs decreases Molecular Identification: MALDI TOF Increasingly used due to speed of identification and lower cost compared to conventional methods Identifies samples based on ionized samples Spectra peaks compared to reference database Can be used to identify bacterial species from selective media Still needs specialist oversight Can also provide information about resistance Only in certain cases E. coli Summary Identified different ways of identifying microbial contaminants Morphology Biochemistry Immunologic Genetic The ideal diagnostic should be; ACCURATE SENSITIVE COST EFFECTIVE FAST Extra Reading Hugo and Russell’s Pharmaceutical Microbiology: Chapter 3 Section 8; Chapter 25 Section 4.1.2 Singhal et al 2015. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Frontiers in microbiology