General Microbiology PDF

General Microbiology Jessica Kafka, PhD, D(ABMM), FCCM Clinical Microbiologist, Public Health Microbiology Lab Dr. L.A. Miller Centre Clinical Assistant Professor, Memorial University Learning Objectives Look out for this icon:...

General Microbiology Jessica Kafka, PhD, D(ABMM), FCCM Clinical Microbiologist, Public Health Microbiology Lab Dr. L.A. Miller Centre Clinical Assistant Professor, Memorial University Learning Objectives Look out for this icon: for key concepts!  Introducing The Microbiology Lab Who We Are Medical laboratory Technologists (MLTs) Medical Laboratory Assistants (MLAs) Microbiologists (MD and PhD) Scientists (MSc and PhD) Administrative Assistants, Clerical Support Staff Storekeepers Where We Are Dr. L.A. Miller Centre, St. John’s – Public Health Microbiology Lab (Reference Lab) 9 microbiology labs located across all zones What We Do…for YOU! After receiving a specimen collected from your sick patient, we work to tell you what is the likely causative micro-organism and what drugs are effective in treating your patient. Micro-organisms: Bacteria Fungi Viruses Parasites  Infectious Agent Infectious agent - something that invades another living thing Infected host - the living entity (human, animal, plant) that is infiltrated/invaded by an infectious agent Most (but not all) infectious agents are microscopic, i.e., cannot be seen by the unaided eye Classes of infectious agents in humans: Bacteria (prokaryote, single cell) Viruses (subcellular, non-living) non.euprokaryoti Fungi (eukaryote, single or multi-cell) Parasites (eukaryote, single or multi-cell) Prions (transmissible, abnormally-folded protein; not covered in lecture) Algae (eukaryote, single cell; not covered in lecture)  Eukaryotes vs. Prokaryotes Distinguished based on presence/absence of nuclear structures and organelles. Eukaryotes (“true-nucleus”) have genetic material inside a nuclear membrane and have membrane- bound organelles. Prokaryotes have no membrane-bound organelles and genetic material is not bound within a nuclear membrane. Viruses are not considered prokaryotes or eukaryotes Not considered living Dependent on host cells for replication Lack cell membranes, cytoplasm and organelles Larger smaller Size Spectrum  Compound Light Microscope Understand generally Most common microscope used in the clinical microbiology lab. not in detail stillimportant Allows us to see micro-organisms not able to be seen by the naked eye (bacteria, yeast, molds, some parasites and their eggs). Light at the base travels upward through the condenser where it is focused and passes through the specimen on a glass slide that sits on the stage. The image is magnified first by the objective lens and then by the ocular lens.  Dark-Field Microscope Only light reflected by a specimen can enter the objective lens. Mainly used to detect Treponema pallidum, a spirochete bacterium that causes syphilis. Less-commonly used in microbiology labs. Mostly replaced by serology tests which looks at anti-syphilis antibodies in serum. Don't need to stain forspecimens that can'tbe stained Image of Treponema pallidum by dark field microscopy  Fluorescence Microscope Uses UV light to illuminate a sample labeled with a fluorescent dye to produce a magnified image. 2nd most common Useful in performing immunofluorescence assays used to either detect organisms themselves or detect host antibodies to organisms. The test uses a primary antibody that binds to a target molecule, and a secondary antibody that binds to the primary antibody. The secondary antibody is combined with a fluorescent dye, which emits light when exposed to UV light. Visible Can beused tovisualizefungi Dyesadhere tofungi emitslight during exposure Scanning Electron Microscope Transmission Electron Microscope (SEM) (TEM) Uses a beam of electrons instead of a beam of light to magnify image An electron source emits a fine, focused beam of electrons A broad beam of electrons is transmitted through towards which scans the surface/topography of a sample. an ultra-thin sample, creating an image that details a sample’s internal structure. The interaction between the electron beam and the sample creates secondary electrons (SE), backscattered electrons After the electrons penetrate the sample, they (BSE), and X-rays, which are captured to create a magnified pass through lenses below it. This data is used to image. create an image directly on a fluorescent screen or onto a computer screen using a camera. Image is lower resolution and 3-D. Image is higher resolution and 2-D. show what inside Bacteria  Bacteria Single-celled, microscopic prokaryotes Most common infectious agents requested by clinicians from just about every specimen type that can be collected from a patient. mwhats.atrotiqi.iie Often ordered as “culture and sensitivity” (C&S). Important to distinguish whether the presence of specific bacteria is INFECTION (causing disease) vs. COLONIZATION (present but causing no harm).  Host-Bacteria Relationships detective work notblankandwhite Requires Host Bacteria Colonization: wins win Presence of bacteria on a body surface (e.g. skin, mucous membranes) without causing disease or damage to the host Infection: Host Bacteria Invasion of host tissues by disease-causing benefits benefit organisms Routes of Entry: Ingestion/eating Inhalation/breathing Trauma/injury, burns (compromised skin barrier) Inoculation (mosquito or tick bite, needlestick) Sexual transmission  Host-Bacteria Relationships Flora/colonizer Lives on a body surface, but does not cause disease Lives in cooperation with a host; is often beneficial by: Outcompeting pathogens Helping with host metabolism Keeping the host immune system vigilant Opportunist Causes disease in a compromised host Colonizers can become pathogens if the circumstances are right Pathogen Causes disease in a healthy host Pathogenicity - ability of a bacterium to cause disease patitethe Virulence - the disease-producing power of a bacterium The pathogenicity of an organism is determined by its virulence factors  Bacterial Virulence Factors The disease-producing power of an organism, “villain special abilities” Virulence factors help bacteria to: Invade the host Cause disease Evade host defenses Motility Flagella - “whip-like appendages” Adhesion Pilli - “grabbers” slimy Protection 4k Capsules (lipid or protein; mucoid colonies) Biofilm (scaffold that surrounds bacteria; offers protection and allows binding to Piglet medical devices) antibiotic protection  Bacterial Virulence Factors Virulent bacteria cause disease! Toxin production Neurotoxin – effects nerves causing paralysis Enterotoxin – acts on GI tract, causing infectious diarrhea or food poisoning Endotoxin – bound to the surface of bacteria but are released by normal shedding or cell lysis (destruction) Spore formation Some bacteria produce spores when in unfavorable environmental conditions. Dormant spores can later germinate to become active bacteria. Hardy; difficult to get rid of in hospital settings! Bacterial Gene bacteria Transfer between Tools can be tradedmoved bacteriophages fedayat Mating pair Identifying Bacteria in the Clinical Microbiology Lab  Identifying Bacteria in the Clinical Microbiology Lab Microscopic Gram stain (determine size, shape and configuration) Macroscopic Colony size, colour, shape, odour* Colony resistance to antibiotics, use of substrates, ability to lyse blood Bacterial colonies are a group of bacteria derived from a single “mother cell” that have divided to the point where they are seen on an agar plate Metabolic can it survive inoxygen What age Aerobic/anaerobic, growth requirements, enzyme production Antigenic (serotyping) Use of antibodies to distinguish bacteria by their antigens Subtypeidentification Molecular Sequencing of genetic material *Do not smell agar plates!  Key identity of bacteria Oxygen Tolerance When oxygen snatches up electrons, it can form biproducts (radicals) that are toxic to bacteria. These oxygen biproducts can also be produced by our immune cells as a defense mechanism. Obligate aerobes: Produce protective enzymes to break down toxic oxygen biproducts Use oxygen as the final electron receptor during aerobic respiration to generate energy Obligate anaerobes: Have no enzymes to defend against toxic oxygen biproducts Use fermentation to generate energy Anaerobic Facultative anaerobes: Far Prefer oxygen, but can survive without it Use oxygen as electron acceptor, and produce protective enzymes Can also grow in the absence of oxygen by using fermentation for energy  Bacterial Growth Rate Fast growers – E. coli (doubling time is ~20 minutes) Slow growers – Mycobacterium tuberculosis (doubling time is 24-48 hours!) Anaerobes can takea longtime to s to 5days  p Bacterial Cell Walls OUTSIDE CELL OUTSIDE CELL INSIDE CELL INSIDE CELL Thin peptidoglycan layer Thick peptidoglycan layer Outer membrane with LPS (endotoxin) No outer membrane  Gram Stain Principle and Procedure flush or carbol fuchsin stool blood urine Sample fixed to slide crystalviolet t espepti.tt on ppi A turnred i Bacteria that cannot be classified by Gram stain include: Mycobacteria – waxy cell wall, so use acid-fast staining Mycoplasma – no peptidoglycan/cell wall; detected by molecular methods (e.g. RT-PCR) Bacterial Morphology (Shape)  Microscopic Arrangements of Cocci Diplococci (pairs) Chains Clusters Streptococcus pneumoniae Streptococcus pyogenes Staphylococcus aureus Don't knownames understand orientation know these images Neisseria meningitidis Micrococcus luteus  Microscopic Arrangements of Bacilli Singles - Escherichia coli Curved - Vibrio vulnificus Pallisade Coryebacterium diptheriae Streptobacilli Fusoform Coccobacilli Streptobacillus moniliformis Fusobacterium nucleatum Haemophilus influenzae  Macroscopic Description “Colony Morphology” Shape (form, elevation, margin) Size (pin-point vs. large) Pigment or sheen Ability to break down blood cells (hemolysis) Mucoid (presence of capsule) Odour*  Manual Biochemical Testing to Identify Bacteria 7-digit profile number is compared to the numerical profile in the API 20 E analytical profile index database to obtain the organism ID. (i.e. 514572 is identified as E. coli)  Automated Biochemical and Antibiotic Susceptibility Testing Commonly used in clinical microbiology labs to identify bacteria and determine which antibiotics can be used to treat infections Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI-TOF) Revolutionized Identification Identification that once took days to weeks now takes minutes! Protein Fragment Signature Analysis by Mass Spectrometry Fungi  Mycology Mycology - Study of fungi Mycoses - Fungal infections i.e. Mucormycosis Eukaryotic Mold Contain cell walls Fungi classification: Molds – multicellular, filamentous (hyphae; “hair-like”) Like growing at 25-30 degrees C skinsurfaceenv withstand coldertemps Yeasts – unicellular, produce by budding internal bodytemps Yeast Like growing at 35-37 degrees C Dimorphic fungi – depending on temperature in the environment, can be either mold OR yeast form “Mold in the cold, yeast in the heat”  Fungal Disease Diagnosis Identification of fungi can be based on: Microscopic characteristics Macroscopic plate features Growth rate Metabolism Calcofluor white stain Protein signature on MALDI-TOF or DNA sequencing Some fungal infections are made clinically; may not need lab testing: Oral thrush (yeast) Oral thrush Vaginal candidiasis (yeast) Ring-worm infection (mold, Dermatophytosis) Fungal infection of hair shafts, skin and nails While superficial fungal infections can be common, invasive fungal infections can be seen in immunocompromised hosts  Yeasts Some common medically important examples: Candida sp. Cryptococcus sp. Yeast colony morphology Cryptococcus neoformans  Molds Some common medically important examples: Aspergillus sp. Penicillium sp. Rhizopus sp. Mucor sp. Fusarium sp. Dermatophytes (Tinea; Trichophyton sp.) Fusarium (LCB stain) Aspergillus (LCB stain) Mucor (LCB stain)  Dimorphic Fungi Common medically important examples include: Blastomycosis dermatitidis Coccidioides immitis Histoplasma capsulatum Paracoccidioides braziliensis “Mold in the cold, yeast in the heat” These are all high-risk group pathogens that if suspected, need to be processed in a biosafety level 3 lab space. Notify the microbiology lab if infection is suspected! Parasites  Parasitology Medical parasite classification: Protozoa – Eukaryote, single cell Need Microscope tosee Needt Intestinal (i.e. Cryptosporidium sp.) remember Blood (i.e. Plasmodium sp.) her Tissue (i.e. Acanthamoeba sp.) Kinetoplastids (i.e. Leishmania sp., Trypanosoma sp.) Plasmodium ring forms on Helminths (worms) – Eukaryote, multicellular blood smear Nematodes (Round worms) (i.e. Ascaris lumbricoides) Cestodes (Segmented worms; tapeworms) (i.e. Taenia solium) alivetheist Trematodes (Flat worms; flukes) (i.e. Fasciola hepatica)  Protozoan Identification Traditional means of identification relied on microscopy: Blood smears: for Malaria (Plasmodium sp.) Stools: for Entamoeba histolytica Vaginal specimens for motility: Trichomonas vaginalis Molecular detection techniques are replacing microscopy Increased sensitivity Stool multiplex PCR detection identifies the most common protozoan infections: condition diarrhea Looks multiph causes of a certain for Giardia lamblia Cryptosporidium parvum Cyclospora cayetanensis Entamoeba histolytica Dientamoeba fragilis Serology – ELISA and Immunofluorescent assays (serum) Culture (rare), i.e. Leishmania sp.  Helminth Identification Visualization of the worm by the naked eye Size Morphology Microscopy of eggs (ova) or larva in stool or tissue Serology – ELISA or IFA (serum) Recommend contacting the microbiology lab prior to sample collection Specialized collection containers Discuss most appropriate specimen and collection In-house testing vs. referred out to reference lab Viruses  Virology Non-living particles composed of nucleic acid (RNA or DNA) and a protein coat (capsid) with or without an envelope Require a host cell to reproduce – this is why they are not considered “living” Viruses invade cells (eukaryotic or prokaryotic) and take over enzyme and energy production to reproduce more viruses, often killing the host cell Viral Structure Viral Replication  Virus Classification Genome DNA RNA Single or double-stranded Capsid Shape Polyhedral (i.e. Herpesvirus) Polyhedral Helical Complex Helical/linear (i.e. Ebola) Complex (i.e. Bacteriophage; don’t infect humans) Presence of Envelope Enveloped (i.e. Herpesviruses, HIV) Non-enveloped (i.e. Adenovirus, Parvovirus) Sites of the body they infect i.e. hepatitis viruses Source of viral transmission i.e. arboviruses Mosquitos (i.e. West Nile Virus, Dengue, Zika) Ticks (i.e. Powassan Virus)  Detection/Identification of Viruses Mainly PCR Molecular real-time polymerase chain reaction Most common, sensitive Antigen detection immunofluorescent assays (IFA) Fairly rapid RT-PCR amplification curve Electron microscopy Expensive, not common in clinical lab Not very sensitive Viral culture Add virus to host cells to look for cytopathic effects (CPE) Very time-consuming! Detection of Rabies virus in Syncytia caused by animal brain tissue, CDC Herpes Simplex Virus  Detection/Identification of Viruses Isolating antibodies specific for viruses in patient serum or other bodily fluids Enzyme-linked immunosorbent assay (ELISA) Immunoglobulin M (IgM) and Immunoglobulin G (IgG) Commonly used in public health labs Chemiluminescent Microparticle Immunoassay Immunochromatographic Assay Tying it all Together: Clinical Application  Tying it all Together: Clinical Application 1. Does the patient have an infectious disease? Clinical signs and symptoms Epidemiology of pathogen Patient risk factors, immune status 2. Is the infection bacterial, viral, fungal or parasitic? 3. How do I sample the patient to make the diagnosis? Which specimen is the most appropriate? 4. How can I be sure that what the lab reports is the pathogen and not a colonizer?  Key Points Detection of specific microorganisms is ALWAYS associated with infectious disease; never colonizers! Salmonella, Shigella, Yersinia sp. Mycobacterium tuberculosis Histoplasma capsulatum, Blastomyces dermatitidis Plasmodium sp. (Malaria) Neisseria gonorrhoeae Human Immunodeficiency Virus Type 1 (HIV-1)  Key Points Growth of bacteria from NON-STERILE sources does not necessarily mean infection and disease. Urine Stool Sputum Swabs Growth from a sterile site is INFECTION unless there is contamination somewhere between collection to processing in the laboratory. Specimen quality and aseptic technique are important to prevent contamination. Collection, storage and transportation of specimens to the lab IMPACTS quality of the lab result.  Non-Sterile Site Sources Expected to contain large numbers of colonizing bacteria. Pathogens may or may not be present. Upper respiratory tract Stool Skin Ear Mouth Eye Urogenital tract “Dirty sites”  Non-Sterile Site Interpretation Usually reported as mixed growth. Abdominal abscess fluid Swab of superficial wound Urine from indwelling catheter Endotracheal tube aspirate Growth represents flora unless clinical evidence of infection. Signs and symptoms/clinical context Pure or predominant growth of one type of bacteria is a clue this organism is causing disease. Correlation of Gram stain with growth on agar plates can help with interpretation.  Sterile Site Sources Cross a “dirty boundary” to collect a clean sample. Sterile fluids: Blood Cerebrospinal fluid (CSF) Tissue collected in the OR Pleural, pericardial, peritoneal fluid Suprapubic urine Often invasively collected (OR) Specimens less likely to be rejected Partially sterile fluids: Cystoscopy urine Bronchoalveolar lavage (BAL) Nature of sampling makes collecting a sterile sample difficult  Sterile Site Interpretation Growth represents INFECTION unless poor collection or contamination after collection Improper disinfection of skin prior to blood or tissue collection Using non-sterile collection containers Always make sure your collection container is sterile! Low quality specimen collection clues: 1/4 blood culture bottles growing a skin colonizer Large number of epithelial cells in sputum seen on Gram stain Small number polymorphonuclear cells (PMNs; neutrophils) in Gram stain Mixed growth of organisms Pre-analytics and Specimen Quality Jerry Maguire, 1996  Specimen Quality Variable # 1 COLLECTION Collect from infected area (pain, pus, redness?) Collect the best quality specimen for the infection. Collect urine from a straight catheter rather than indwelling. Use aseptic technique to prevent contamination. Properly disinfect “dirty” areas before collection. Tissues and fluids collected invasively are always Eat.it preferred over superficial collection, i.e. swabs. forgrowthand “Send us the blob, not the swab” detection Specimens for the microbiology lab must not be Bacteria togrowonceit submitted in formalin. Toxic!! beenis very formalin difficult has placed in Collect separate specimens for pathology and microbiology.  Specimen Quality Variable # 2 CONTAINER Based on suspicion of bacterial, viral, fungal or parasitic infection, send specimen in an appropriate container. Often contains antiviral, antibacterial and/or antifungal agents to promote the growth of the target organism. i.e. do not send a swab in viral transport media tube for bacterial culture Collect sufficient specimen volume. The more the better to improve sensitivity and reduce false negatives Volume is the most important variable for recovery of bacteria from patients with bloodstream infections! Low volume with multiple test requests? Expect a call from the lab. Ensure container is properly sealed. Many specimens cannot be processed because they leak, introducing contamination. Containers need to be sterile. In contrast to chemistry and hematology.  Specimen Quality Variable # 3 PRESERVATION AND TRANSPORTATION Place specimen in appropriate container to preserve it until it arrives to the microbiology lab. Ensure specimen is stored/transported according to pathogen detection criteria if cannot be shipped immediately. Ship ASAP – specimens often sit around for too long. Some non-pathogenic bacteria are overgrowing and bacteria of interest are dying within minutes of collection. Tissues can dry out in their containers if no saline is added and not shipped promptly. Specimen Rejection and Recollection Some specimen types are deemed precious (you have only one opportunity to collect; invasive) and must be handled diligently and expeditiously. Cerebrospinal fluid (CSF) Intraoperative tissue or fluid Other specimens can usually be easily recollected. For this reason, it is rejected if the sample is of poor quality or is delayed arriving at the lab. Urine (indwelling catheter, not in preservative/delayed) Stool Sputum (lots of epithelial cells)  Physician-Lab Communication Notify the lab if suspecting infection with a biosafety level 3 agent Mycobacterium tuberculosis Francisella tularensis (Tularemia, Rabbit fever) Bacillus anthracis (Anthrax, woolsorters’ disease) Coccidioides immitus (Coccidiodomycosis, valley fever) Properly label specimens and provide relevant clinical details Not enough detail: “Wound swab” or “abdominal fluid” or “hepatitis” Avoids unnecessary testing (i.e. anaerobic culture needed?) i.e. Lyme disease serology requires clinical/travel history If you collect blood cultures, be prepared to get a call from the lab at any hour to communicate a Gram stain result. Contact the microbiologist on call or the lab with any questions about specimen collection or test result interpretation. Additional Resources