Micro 443 Combined PDF

Welcome to Microm 443! Kendall M Gray, Ph.D. (he/him) Rebecca Rashid Achterman, Ph.D. (she/her) (Mira Beins, PhD, winter) Dept. of Microbiology  Microm 443 Purpose of the Course General Course Information Identifying Bacteria Today in Lab ...

Welcome to Microm 443! Kendall M Gray, Ph.D. (he/him) Rebecca Rashid Achterman, Ph.D. (she/her) (Mira Beins, PhD, winter) Dept. of Microbiology  Microm 443 Purpose of the Course General Course Information Identifying Bacteria Today in Lab  Microm 443 Purpose of the Course Learn how to identify the causative agent of a bacterial infection Why bother?? treatment of. back ↳ deff types zepil surveillance ↳ out breaks  General Course Information Two components Class always starts at 11:30am Lectures Lectures meet here. Lab If no lecture, meet in the lab room.  General Course Information Two components Lecture Lab Additional information sources Lab manual - includes the schedule; required; UW bookstore Course web site – schedule, lecture outlines, announcements, grading system, required videos, optional resources Medical microbiology text – (Sherris’ Medical Microbiology – free online, link on Canvas site) Other books: A Photographic Atlas for the Microbiology Laboratory Gram Stain Tutor – link under Resources on the Canvas site Sign in: LoginUW Pwd: UW * * X  Strategy for Success: Course structure 1st half – Meet the Microbes ABC DEF HIJ KLM six Second half - Clinical specimens ster i CSF cerebrallaundStool Sputum ABH AKLM ABDH JCDEH CK From previous student course evaluation: “The structure of this class was excellent. Having a chance to use all the skills we learned in the first half of the course repeatedly in the second half was very useful…”  Grades based on: Quizzes (best 6 out of 7: ~20-25% of total score) Short answer, matching, multiple-choice Know details about each organism covered Identifying characteristics Body site(s) in which it typically causes infection Body site(s), if any, in which it is normal microbiota Cumulative “…excellent that all the quizzes are cumulative. This helped me stay on top of the material through the quarter.” “…The questions on the quizzes required critical thinking, it incorporated all covered material as well as deductive reasoning!” previous Microm 443 students  Grades based on: Quizzes (best 6 out of 7: ~20-25% of total score) Short answer, matching, multiple-choice Know details about each organism covered Identifying characteristics Body site(s) in which it typically causes infection Body site(s), if any, in which it is normal microbiota Cumulative Unknowns (7 + differentiation scheme, 2-3 pts each; ~10-15%) Lots of help for early ones, with progressively less help later on Midterm (~20-25%) Lab work and props/questions Final (~40-50%) Props/questions and short answer questions  Safety Working with pathogens (BSL-2) Standard laboratory safety procedures (listed in lab manual) Proper aseptic technique is essential!!!!!!!! Infectious dose (ID50) of Shigella species: 10 - 100 cells (ingested) Important to avoid aerosols  Safety Use of alcohol-sand bottles in addition to aseptic technique “Lab Safety” section of website for all aspects of safety Electronics Policy … Safety quiz questions are on last page of lab manual find the answers in lab, from discussion, and on ‘safety’ page on-line quiz due Wk2 Monday Where to find these things on the course site…  Microm 443 Purpose of the Course General Course Information Identifying Bacteria  What is a Clinical Specimen?  Microscopic Examination  Basic Microscopy Today in Lab Identifying Bacteria  What is a Clinical Specimen? A biological substance removed from a patient for diagnostic or research purposes.  Identifying Bacteria  What is a Clinical Specimen? A biological substance removed from a patient for diagnostic or research purposes. Example: A person comes to the health clinic complaining of a sore throat. The medical professional swabs the back of the throat and sends the swab to the clinical microbiology lab for analysis. The disease is: sore throat The body site is: throat The clinical specimen is: throatSwab Please do the practice and bring your answers to the next lecture. You aren’t expected to be perfect (yet)!  Identifying Bacteria (in patient specimens) lots of microbiota & Microscopic exam of clinical specimen throat : host cells host cells? (WBC, epithelial) bacterial cells? (size, shape, grouping)  Identifying Bacteria (in patient specimens) Microscopic exam of specimen host cells? (WBC, epithelial) bacterial cells? (size, shape, grouping) Culture  identification cultivating bacteria microscopic exam of pure culture identification Colony morphology is a helpful clue but usually not definitive on its own…  Microscopic Examination Wet mount - very simple; motility  Microscopic Examination Wet mount - very simple; motility Dark field > - enhances light field  Microscopic Examination Wet mount - very simple; motility Gram stain (a differential stain) Gram-positive Gram-negative ② Iodine purple purple & ⑤ alcorol clear (decolor ( ① satrain purple & pink counter stain)  Microscopic Examination Wet mount - very simple; motility Gram stain (a differential stain) Gram-positive Gram-negative outer membrane Peptidoglycan Outer membrane defferences Peptidoglycan in perminability Peptidoglycan peptidoglycan Cytoplasmic membrane Cytoplasmic membrane coccus (cocci) rod bacillus Microscopic Examination G cocci G rods Gram-positive cocci (GPC) Gram-negative cocci (diplococci) (GNDC)  Gram-positive rods pleomorphic O endospores Gram-negative rods coccobacillus Gram-negative curved rods Interpreting “real-life” Gram stains  Other microscopic techniques Dark field– described earlier Methylene blue stain - observation of metachromatic granules (volutin, polyphosphate) push awaa > - Capsule stain – observation of capsule via negative staining (stains background) Acid fast stain - detects Mycobacterium species (ex. the pathogen M. tuberculosis) Fluorescence tagged antibody tagged nucleic acid probe – fluorescence in situ hybridization (FISH)  Microm 443 Purpose of the Course General Course Information Identifying Bacteria Today in Lab Find a spot in room T369 or T375 (adjoining labs) and await further info on getting started in lab… Microm 443 Introduction Part 2: General Strategies for Cultivation and Identification of Medically Important Bacteria Clinical Specimens Practice: For each disease described on the left, fill in the table with the appropriate clinical sample from the word cloud on the right Disease Clinical sample - Steril throat swab sore throat throat swab CSF (pharyngitis) (cerebrospinal fluid, the urinary tract infection Urine sample fluid that surrounds the diarrhea fecal sample meninges) fecal sample bacterial pneumonia (stool sample) (infection deep within sputum urine sample the lungs) by microbes meningitis (swelling of be contaminated will through membranes covering [SF - when going blood mouth brain and spinal cord) sputum the area out (liquid from the lungs bacteremia (bacteria blood that is coughed up) in bloodstream) ↳ throat & mouth microbes contaminates  General work-up of clinical specimens 1. Macroscopic 3. Culture to grow pathogens 4. Microscopic exam of exam in pure culture pure culture (Gram Only want to identify the stain) 2. Microscopic exam pathogens Direct Gram stain Consider incubation 5. Biochemical and Host and bacterial conditions and media other tests as indicated cells may be for identification observed  Microscopic Examination Gram stain (a differential stain) – become comfortable troubleshooting this procedure.  General work-up of clinical specimens 1. Macroscopic 3. Culture to grow pathogens 4. Microscopic exam of exam in pure culture pure culture (Gram Only want to identify the stain) 2. Microscopic exam pathogens Direct Gram stain Consider incubation 5. Biochemical and Host and bacterial conditions and media other tests as indicated cells may be for identification observed  Cultivating bacteria Streak plate technique ↑  Cultivating bacteria Streak plate technique  Cultivating bacteria Streak plate technique  Cultivating bacteria Streak plate technique  General work-up of clinical specimens 1. Macroscopic 3. Culture to grow pathogens 4. Microscopic exam of exam in pure culture pure culture (Gram Only want to identify the stain) 2. Microscopic exam pathogens Direct Gram stain Consider incubation 5. Biochemical and Host and bacterial conditions and media other tests as indicated cells may be for identification observed  Cultivating medically-important bacteria Incubation conditions medical idea Temperature emp (37 - Generally 35oC sometime w a b ↳ at not it too Atmosphere Air Increased CO2 (candle jar) atm just ↑ in CO2 ↳ no anaerobic up to~3 % Ina CO2 I still need to be an acrobe to grow works better in more - no oxygen at all ↑ will chemicals dec 02  Cultivating medically-important bacteria Incubation conditions Temperature Generally 35oC Atmosphere Air Increased CO2 (candle jar) Anaerobic  Cultivating medically-important bacteria Incubation conditions Temperature Generally 35oC Atmosphere Air Increased CO2 (candle jar) Anaerobic Microaerophilic > - like certain amount of 02 a little but of air  Cultivating medically-important bacteria Media Routine (support many types of bacteria) TSY, Nutrient agar, Mueller-Hinton Blood agar > - 5% sheep blood > - supports more growth -> able to differenciate Chocolate agar > - richest nutrient used red blood cells&  Cultivating medically-important bacteria Media Routine TSY, Nutrient agar, Mueller-Hinton Blood agar Chocolate agar Selective > - only certain trungs will grow MacConkey - inhibits all but intestinal GNR and many others Differential > - feel the diference between 25Hungs that grow Blood agar - hemolysis > - zone clear/grease MacConkey agar - lactose fermentation > - ferment dec PH = dark pink color Various nutrients + RBC Blood Agar lactose + pH indicator + various nutrients, other things + bile salts + CV selectivea differencial MacConkey Agar  General work-up of clinical specimens 1. Macroscopic 3. Culture to grow pathogens 4. Microscopic exam of exam in pure culture pure culture (Gram Only want to identify the stain) 2. Microscopic exam pathogens Direct Gram stain Consider incubation 5. Biochemical and Host and bacterial conditions and media other tests as indicated cells may be for identification observed  Identification Genus species (strain) I riy important for surveilance , etc Staphylococcus aureus Staphylococcus epidermidis Staphylococcus aureus MRSA (methicillin-resistant Staphylococcus aureus) USA-300 - Strain  Identification Earlier steps can yield clues On plates - size, shape and elevation, consistency, color  Identification Earlier steps can yield clues On plates - size, shape and elevation, consistency, color Presence/absence on selective media Characteristics on differential media Identification -numolosis zone double numolosis of  Identification Biochemical tests Ability to degrade various sugars, urea, etc Antibiotic (or other) susceptibility  Identification Biochemical tests Ability to degrade various sugars, urea, etc Antibiotic (or other) susceptibility find AB body Serological tests > - In see if reconizes M body by AB antigens recognized E. coli O157:H7 Bact. AB -  Identification Biochemical tests Ability to degrade various sugars, urea, etc Antibiotic (or other) susceptibility Serological tests Nucleic acid probes/PCR at early stage. Generates used & - proteins profile MALDI-TOF compares to database Matrix assisted laser desorption ionization – time of flight  MALDI-TOF: Matrix-assisted Laser Desorption/Ionization- Time of Flight Antimicrobial Drugs Intro Determining susceptibility of an organism:  MIC (minimum inhibitory concentration)  Kirby-Bauer Disk Diffusion Practice Today in Lab Microbiology 443  So far in lab: Microbes are everywhere (pretty much) Some cause disease Identification of microbes is aided by: Colony morphology and cellular size/shape Differential stains and special stains Growth characteristics on differential and/or selective media and results of biochemical tests Clinical aim: identification of etiological agent as a means to inform treatment. Learning Outcome: Perform basic antimicrobial susceptibility testing and interpretation. Understand the principles used in antimicrobial susceptibility testing.  Antimicrobial Drugsredact is generaland bactivaperitics historical cre antimicrobial drugs/medications vs. antibiotics vs. chemotherapeutic agents Penicillium notatum * (mold) Zone of inhibition Staphylococcus (bacteria) Alexander Fleming Dorothy Crowfoot Hodgkin 1928 1945 Common Targets of Antimicrobial Drugs want have selective toxcity to hust & Carger pathogen not Inhibit an essential function of pathogen Antimicrobial Drugs Intro - Determining susceptibility of an organism:  MIC (minimum inhibitory concentration) (broth-dilution)  Kirby-Bauer Disk Diffusion Practice KBDD Today in Lab Microbiology 443  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) method broth dilution 0 12 turpidno growth MIC =. 2 told dilution not => iii - : - drug : : ↑ no growth E lower concentration havesentration > - look at lowest of AM that will inbit growth by visual observation Preparing the dilutions of drug  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) broth Preparing the dilutions of drug Add 1 ml sterile broth to all but the tube labeled “16 g/ml” Make a 32 g/ml solution of the drug Add 1 ml of drug to first three tubes  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) - 16 32 16 Preparing the dilutions of drug Add 1 ml sterile broth to all but the tube labeled “16 g/ml” Make a 32 g/ml solution of the drug Add 1 ml of drug to first three tubes Starting with the tube labeled “8 g/ ml”, make serial 2-fold dilutions  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) 16 32 16 8 4 2 1 0.5 0.25 0.12 0.06 Preparing the dilutions of drug Add 1 ml sterile broth to all but the tube labeled “16 g/ml” Make a 32 g/ml solution of the drug Add 1 ml of drug to first three tubes Starting with the tube labeled “8 g/ ml”, make serial 2-fold dilutions Preparing the inoculum Should be 105-106 cells/ml 5 x 109 cells/ml  5 x 107 cells/ml  5 x 105 cells/ml  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) 16 32 16 8 4 2 1 0.5 0.25 0.12 0.06 Preparing the dilutions of drug Add 1 ml sterile broth to all but the tube labeled “16 g/ml” Make a 32 g/ml solution of the drug Add 1 ml of drug to first three tubes Starting with the tube labeled “8 g/ ml”, make serial 2-fold dilutions Preparing the inoculum 105-106 cells/ml 5 x 109 cells/ml  5 x 107 cells/ml  5 x 105 cells/ml Doing the test Add 1 ml of bacterial suspension to each tube except first  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) Resistant vs intermediate vs susceptible Breakpoint: cutoff MIC that determines if bacteria is S, I, R  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) S I R Resistant vs intermediate vs susceptible? Breakpoint: cutoff MIC that determines if bacteria is S, I, R  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Bactericidal Concen. (MBC) C? ( bactenasutal : kills battend bactena still them bacteristanc = grow in the absence of drug MBC: lowest conc of antimicrobial that kills 99.9% of the bacteria  Determining the susceptibility of a bacterial strain to an antimicrobial drug - Minimum Inhibitory Concen. (MIC) Broth dilution tests: Minimum Inhibitory Concentration (MIC) ”macro”dilution tests Microdilution tests Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Sherris, Turck - Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Sherris, Turck of inhibition zone 201 # Mueller-Hinton agar most non-fastidious pathogens can grow on it “loose” agar lets antibiotics diffuse away from the disks addition of starch allows absorption of some toxic metabolites that can interfere with antibiotics less likely to inactivate some antibiotics (lower concentration of certain ingredients) Provides a reproducible standard Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Sherris, Turck Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Sherris, Turck The zone of inhibition is measured in millimeters (mm) Need additional information to determine clinical significance Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Determining the susceptibility of a bacterial strain to an antimicrobial drug - Disk diffusion (Kirby-Bauer) test Determining the susceptibility of a bacterial strain to an ? antimicrobial drug - E test grows thinasd to For na are a Godthat higher AB ↓ = MIC Determining the susceptibility of a bacterial strain to an antimicrobial drug – automated systems Vitek ASC (Ab susceptibility card) Other methods: PCR for antibiotic resistance genes, chromogenic media (MRSA, VRE) Antimicrobial Drugs Intro Determining susceptibility of an organism:  MIC (minimum inhibitory concentration) (broth-dilution)  Kirby-Bauer Disk Diffusion Practice Today in Lab Microbiology 443 Antimicrobial Drugs Intro Determining susceptibility of an organism:  MIC (minimum inhibitory concentration) (broth-dilution)  Kirby-Bauer Disk Diffusion Practice Today in Lab Microbiology 443 Today in Lab: MIC day 1 -> prepare culture and Ampicillin stock, then set up the MIC test -> work with your lab partner -> Ampicillin (160 ug/mL) on front bench -> choose Proteus, E. coli, or Enterobacter carefully follow instructions in lab manual Blue pipettors = for 1mL pipettes Green pipettors = for 5mL pipettes Bring a rack to do your inner lab pickup -> Gram stain cultures you haven’t seen before Gram-Positive Cocci: Staphylococcus Streptococcus Enterococcus Members of the genus Staphylococcus Cells tend to form clusters; may be single, in pairs, or short chains  Members of the genus Staphylococcus Cells tend to form clusters; may be single, in pairs, or short chains Common skin microbiota of mammals (also found on mucous membranes) Generally hardy and not nutritionally fastidious Facultative anaerobes (w/ O2 à respire; w/o O2 à ferment) Catalase-positive (2 H2O2 à 2 H2O + O2) Species differentiation: Coagulase test Novobiocin susceptibility Biochemical tests  Members of the genus Streptococcus Cells tend to form chains Common oral microbiota May be nutritionally fastidious Aerotolerant anaerobes / obligate fermenters Some are capnophiles (CO2 loving) Catalase-negative > - use trus first - usually Species differentiation: Type of hemolysis Cell wall carbohydrate group (A, B, etc.) Other tests  Members of the genus Enterococcus Cells tend to form short chains Common normal fecal microbiota Hardy and not nutritionally fastidious Aerotolerant anaerobes / obligate fermenters Catalase-negative (may show "pseudocatalase" activity) Grow in 6.5% NaCl & Bile-esculin positive Species differentiation: Biochemical tests Staphylococcus species Staphylococus aureus Staphylococus saprophyticus Staphylococus epidermidis  Staphylococcus aureus happen transeaut( ? ) does Pathogen/opportunistic pathogen (carried in the nose/on the skin of 10 - 40% of people) larger boils skin/hair follicle infections - furuncles (boils), carbuncles I wound infections - surgical wounds (a nosocomial infection) - hospital infection & ↳ get own pneumonia moderate curonic , etc Healthcare-associated (clearable) , travesent infection (HAI) , be > - might bacteremia, sepsis spread bacterima in ↳ Chronic wide ↳ bacteria hits hard blood stream & Immune response Toxin-producing strains: food poisoning workines I staphylococcal toxic shock scalded skin syndrome  Notable characteristics of Staphylococcus aureus good indicator Colony morphology on blood agar > - Medium to large colonies Cream to golden color Typically β hemolytic, may show double zone (particularly after refrigeration)  Notable characteristics of Staphylococcus aureus Colony morphology on blood agar Medium to large colonies Cream to golden color Typically β hemolytic, may show double zone (particularly after refrigeration) Distinguishing S. aureus from other Staph. species β hemolysis Ter [ Mannitol fermentation (positive) > - yellow * Coagulase / clumping factor (positive) > - fibrin( ? ) production clumps cells coagulase pos always (Slide coagulase test) trait definitive  Coagulase-negative Staph. Staphylococcus saprophyticus Urinary tract infections Colony morphology on blood agar non-remolytic Medium, gray to white colonies (Similar to other coagulase-negative Staphylococci) Distinguishing S. saprophyticus from other species of Staph. Coagulase (negative) * Novobiocin susceptibility (resistant) - KB test W/ AB (novobin) Coagulase-negative Staph. Staphylococcus saprophyticus  Coagulase-negative Staph. Staphylococcus saprophyticus Urinary tract infections Colony morphology on blood agar Medium, gray to white colonies (Similar to other coagulase-negative Staphylococci) Distinguishing S. saprophyticus from other species of Staph. Coagulase (negative) Novobiocin susceptibility (resistant)  Coagulase-negative Staph. Staphylococcus epidermidis Common normal skin microbiota / occasional opportunist Hospitalized patients (particularly those with indwelling catheters) Colony morphology on blood agar Small to medium, white colonies Characteristics that distinguish Staph. epidermidis from Staph. aureus and Staph. saprophyticus Coagulase (negative) * Novobiocin susceptibility (sensitive) opp of staph. Saprophyticus  Novobiocin Test (Wednesday) ween each of the (seeareas, p. 20 foras diagrammed half-plate below. streak instructions) B · NB NB 20  Streptococcus species Beta-hemolytic Streptococcus pyogenes (Group A strep) on serovant Streptococcus agalactiae (Group B strep) Alpha-hemolytic Streptococcus pneumoniae Istrange) Viridans streptococci Non-hemolytic (gamma-hemolytic) Other streptococci  Streptococcus pyogenes (Group A strep = GAS) Pathogen Pharyngitis (strep throat) Wound infections Pneumonia Bacteremia/sepsis Toxin-producing strains Scarlet fever Streptococcal toxic shock  Notable characteristics of GAS Colonies on blood agar Small, β-hemolytic * Isolated from separated sources ↑ (isolate Grown anaerobically, all strains are β-hemolytic * Grown aerobically, 85% of strains are β-hemolytic, 15% of strains are non-hemolytic Inactivated by O2 Two hemolysins (streptolysins) - O and S A sensitive O - encoded by 100% of strains; oxygen labile S - encoded by 85% of strains; oxygen stable To reliably see hemolysis of GAS, you must incubate cultures anaerobically > - will be incorrect 15 % of time If aerobic For throat cultures, the first job is to "R/O GrpA"  Notable characteristics of GAS Distinguishing GAS from other β-hemolytic strep Bacitracin susceptibility (generally sensitive) Pyrase (positive) Latex agglutination using Ab to Group A carbohydrate antigen AB clumping right AB ↳ = If ↓ a cells  Notable characteristics of GAS Distinguishing GAS from other β-hemolytic strep Bacitracin susceptibility (generally sensitive) Pyrase (positive) Latex agglutination using Ab to Group A carbohydrate antigen Fluorescent Ab test  Streptococcus agalactiae Group B strep Pathogen (primarily in neonates; transmitted via maternal genital tract) Neonatal meningitis brith candl Neonatal sepsis infection through > - baby gets Post-partum infections, bacteremia  Characteristics of Group B strep all oxygen stable Colony morphology on blood agar smaller ↳ Small, β-hemolytic - ("softer" hemolysis vs Grp A) Less obvious hemolysis when incubated aerobically Colonies may be orange Distinguishing Group B from other b-hemolytic strep Bacitracin susceptibility (generally resistant) cleave Sodium hippurate (positive) Latex agglutination using Ab to group B antigen Carrot Broth (test for orange pigment) POS Streptococcus pneumoniae (pneumococcus) Streptococcus pneumoniae (pneumococcus) capsies in & auptococci  Streptococcus pneumoniae (pneumococcus) Opportunist (commonly carried in the throat) pneumonia, meningitis Colonial morphology on blood agar Small, α-hemolytic, often mucoid on primary isolation Colonies will flatten over time due to autolysin production - luse wi age goop do of olaea center  Streptococcus pneumoniae (pneumococcus) Opportunist (commonly carried in the throat) pneumonia, meningitis Colonial morphology on blood agar Small, α-hemolytic, often mucoid on primary isolation Colonies will flatten over time due to autolysin production Distinguish pneumococcus from other α-hemolytic strep Optochin susceptibility (sensitive) > - activates autolysis alsome Bile solubility (soluble) & see colome will > - add bile If  Viridans streptococci (a group of α-hemolytic streptococci) Normal microbiota of the throat; occasional opportunists Must be distinguished from pneumococcus in a sputum specimen Colony morphology on blood agar Tiny, α-hemolytic Distinguishing viridans strep. from pneumococcus autolysin Optochin susceptibility (resistant) > - be no Bile solubility (insoluble)  Enterococcus Normal fecal microbiota; opportunist Urinary tract infections (UTI) Bacteremia, endocarditis * Colony morphology on blood agar Small, gray, sometimes slightly hemolytic small nemolytic - very similar +8 Distinguish enterococci from other catalase-negative GPC same not bile sensitive > - medium turns black esculin Bile-esculin (positive) bile & cleare - you grow inNir of > - can If POS Growth in 6.5% NaCl (positive) Pyrase (positive) Today MIC (day 3) Examine your plates (if any) to determine the antibiotic's mode of action (bactericidal vs bacteriostatic) Disc Diffusion Assay (day 2) Measure zones of inhibition for each disc and use the chart to determine susceptibility vs resistance Staphylococcus (day 1) Gram stain and streak onto BA (one plate per pair)  GPC staph. strept. entero - clusters - chains Idiplo-chains/diplo Catalase (t) Catalase (t) Catalase (-) I remology T · tupe alpha gamma -est jactaseI, IGNORE group D beta (t) crit enterococcus strep. · Morin bitain (k) (+ ) (S) staph awers strep. virdans Pneumoniae - IS) (R) Eit GAS CR) strep. pyogenes (s) staph. Sapro staph epi. IGNORE Strep , agalactial  Gram-negative diplococci (GNDC) ↓ S Neisseria and Moraxella General Characteristics Diseases and Normal Microbiota Isolation and Identification Microbiology 443​​ Neisseria and Moraxella General Characteristics purplepoints Gram-negative diplococci (GNDC) somewhat resistant to decolorization Cells typically occur in pairs: kidney beans, coffee beans PHIL #14855 Pneumoniae 00 = Neisseria and Moraxella General Inhabit mucous membranes Characteristics Growth -between Obligate respirers (some are capnophiles) do NOT ferment Fastidious (to varying degrees) “Fragile” (to varying degrees) (warm, humid environ) Typical characteristics Catalase positive * Oxidase positive Non-motile (can have “twitching motility”) changes the donates a reagent pink - Purple color becomes cytochrome c oxidase - enzyme of SOME electron-transport chains  Gram-negative diplococci (GNDC) Neisseria and Moraxella General Characteristics Diseases and Normal Microbiota Isolation and Identification Microbiology 443​​  Neisseria gonorrhoeae (GC) Diseases and “gonococcus” Normal Microbiota always a pathogen STD – Gonorrhoea Infects mucous membranes of reproductive tract (cervix, uterus, fallopian tubes), urethra, mouth, throat, rectum harder to grow in lab -white blood May be asymptomatic or have purulent prity cells ( ?( discharge (more likely symptomatic if urethritis in person AMAB) Can lead to Pelvic Inflammatory Disease, ↑ immunodenna Epididymitis, Disseminated Gonococcal infection Can be transferred to baby during birth (opthalma neonatorum) * Antibiotic Resistance is a concern! Complex nutritional requirements! Will grow on CA but not BA. Specimen handling is critical  moSonoma Diseases and Normal Microbiota : -O bacteria cytoplasm & Public Health Image Library #4085, Gram-stained urethral smear with N. gonorrhoeae endo Diseases and marpetnota Normal Microbiota Public Health Image Library #2976, N. gonorrhoeae inside a neutrophil, with other bacterial microbiota also visible  meningsaround of -infection spinal collom Diseases and Neisseria meningitidis (MC) ↑ brain “meningococcus” Normal Microbiota ~10% are carriers in back of nose and throat Can cause meningitis (and other invasive disease) 6 main serogroups with different capsule types associated with nearly all invasive disease worldwide Bacteria penetrate mucosal cells, enter bloodstream, and cross BBB into the& CSF to cause meningitis. Small hemorrhagic lesions (petechiae) on the skin are · ( characteristic of meningitis caused by Meningococcus. Transmitted person to person via droplet transmission in crowded conditions (example: dormitories) monovalent & Vaccines available vaccines wi capsle type quadral( ) J ? combine We will not be working with N. meningitidis. We will work with a stand-in that is not considered pathogenic but performs the same on the tests we will use for identification.  Diseases and Normal Microbiota Neisseria lactamica Normal microbiota Commensal in upper respiratory tract, frequently isolated from children Moraxella catarrhalis Opportunist 1-5% of adults are carriers in upper respiratory tract Can cause otitis media, sinusitis, bronchitis  Gram-negative diplococci (GNDC) Neisseria and Moraxella General characteristics Diseases and Normal Microbiota Isolation and identification Challenge: Isolate GC or MC and distinguish from normal microbiota Microbiology 443​​ Neisseria gonorrhoeae (GC) Isolation and “gonococcus” Identification Specimen handling: g keepabst no Growta just , Transport swabs in non-nutritive medium (no refrigeration, no dry swabs) If possible inoculate into medium in the exam room and incubate at 35C/CO2 right away Specimens from sites with normal microbiota – use VCN (Thayer-Martin) medium selective Vancomycin – kills G+ · I Colistin – kills G- (but GC can still grow) Nystatin – kills yeast Urethral exudate – don’t expect normal microbiota * I Endocervical smear – has lots of normal microbiota Colony morphology: small to medium grayish white, convex (raised); colony type varies, al Na even in a single strain, due to antigenic variation http://www.microbiologyinpictures.com/ Neisseria gonorrhoeae (GC) Isolation and “gonococcus” Identification Biochemical tests CTA slants (cystine tryptic digest agar) w/1% sugar and pH indicator Acid detection from oxidation of glucose not lactose, maltose, or sucrose use heavy inoculum (GC doesn’t (NOT fermentation) grow on CTA) yelow(pos GLMS G M L S Neisseria gonorrhoeae (GC) Isolation and “gonococcus” Identification Biochemical tests CTA slants (cystine tryptic digest agar) w/1% sugar and pH indicator Acid detection from oxidation of glucose not mal, suc, lac (NOT fermentation) Nucleic Acid Amplification Test (NAAT) Can screen for Chlamydia at the same time G M L S  Isolation and Neisseria meningitidis (MC) Identification “meningococcus” Specimen: Cerebrospinal Fluid (CSF) and Blood Is this aE sterile or non-sterile site? Culture and Incubate as per N. gonorrhoeae Less fastidious. Will grow on CA and BA (but not NA). web.mst.edu  G & - S G LM & -  Isolation and Identification Neisseria lactamica - Can grow on CA and BA but not NA CTA slant results: Glu + Lac + - Mal + Suc - Moraxella catarrhalis Grows on CA, BA, AND nutrient, TSY Med-large, whitish gray to pinkish brown, “hockey puck” consistency CTA slant results: no acid detected Microbeonline.com CHA SLANT nothing yellow , will grow , least tedius  Today in Lab: Neisseria/Moraxella day 1 Streak GC on: VCN/CA biplate Incubate 35C, CA/BA biplate candle jar ¼ of a NA plate Streak MC and N. lactamica on: Incubate 35C, CA/BA biplates candle jar ¼ of the NA plate Streak Moraxella on: Incubate 35C CA/BA biplate ¼ of the NA plate Gram stain each of the pure cultures Also continuing the Gram-positive cocci labs Corynebacterium, Listeria Gram positive rods  General Characteristics Listeria, Corynebacterium GPR Non-sporeforming Gram-positive rods, sometimes pleiomorphic (pleio-, "more"; -morphic, "formed") > more than - I form Facultative anaerobes Catalase-positive Characteristics used to differentiate: sugar fermentation pattern motility nitrate reduction urease  Corynebacterium species Club-shaped; V-formations, palisades, Gram-stain irregularly ↓ ↓ v form palisades undergo snapping division  Corynebacterium species Club-shaped; V-formations, palisades, Gram-stain irregularly A Non-motile Widely distributed; mucous membranes/skin of humans and other animals >45 species Corynebacterium diphtheriae lookcapth they like not - but Others; normal microbiota (coryneforms, diphtheroids) diseasething Corynebacterium pseudodiphtheriticum & non pathogene  Corynebacterium diphtheriae Pathogen - causes diphtheria; colonizes upper respiratory tract through aresols coughing , Virulence is due to an AB exotoxin ↳ receptor, ataeves & enters cel  Corynebacterium diphtheriae Pathogen - causes diphtheria; colonizes upper respiratory tract Virulence is due to an AB exotoxin Inhibits eukaryotic protein synthesis à kills cells Causes necrosis (death) of cells in the upper respiratory tract, resulting in formation of a "pseudomembrane" mass of dead In issue throat can enter treakia & cause deatoration  Corynebacterium diphtheriae Pathogen - causes diphtheria; upper respiratory tract Virulence is due to an exotoxin Inhibits eukaryotic protein synthesis à kills cells Causes necrosis (death) of cells in the upper respiratory tract pseudomembrane Toxin can be distributed systemically, causing life-threatening damage to other organs (esp. myocarditis) Inflammation & death > - of membrane surrounding heart AB toxin is encoded by a prophage (lysogenic conversion) infecte bartena bacteraphage &  Assaying for toxin production Modified Elek test (2022) Antitoxin (IgG antibodies) servi against toxIn wI AB Antitoxin disc Dimmuno persipitate happens when toxins are produced  Treatment of diphtheria thats been I raised to toxins Antitoxin (antibody serum) Antibiotics Vaccine – toxoid (formalin-inactivated toxin) own Serum carves and toxin  Culture and identification of Corynebacterium diphtheriae Diagnosis is primarily clinical; physician must suspect that the patient has diphtheria and request that the lab "R/O C. diphtheriae" Colony morphology on blood agar: small, opaque white; may be β hemolytic or non hemolytic  Culture and identification of Corynebacterium diphtheriae Diagnosis is primarily clinical; physician must suspect that the patient has diphtheria and request that the lab "R/O C. diphtheriae" Colony morphology on blood agar: small, opaque white; may be β hemolytic Selective/differential medium - often contains tellurite; ex. Cystine tellurite blood agar (aka Tinsdale agar) kills normal micro Corynebacterium species reduce tellurite to tellurium, producing a black colony (differential) Most normal microbiota are inhibited by tellurite (selective) Loeffler's slant - Growth of C. diphtheriae is favored so at 16 hrs it predominates wI methulme stain red-purple dot you get in blue cell  Throat swab specimen on: Tellurite (Tinsdale) Blood ↓  Culture and identification of Corynebacterium diphtheriae Gram stain Long pleomorphic, club-shaped rods, often in a V-formation, may stain irregularly Methylene blue stain some strains do it well , Polyphosphate granules (particularly from Loeffler's) & others not much Suspect colonies are confirmed with biochemical tests Sugar fermentation pattern against listeria Motility (–) I Nitrate reduction (+) Urease (–) Sugar fermentation à acidic end products, sometimes gas Motility Nitrate reduction Urease O NC A AG pink : acid bubble : gas Sugar fermentation Motility - Is the organism motile? Nitrate reduction Urease growing , just no motility red = growing neg motility dispension moving = of red -  Sugar fermentation Motility Nitrate reduction - Nitrate as a terminal electron acceptor and erobe Urease NO3 à NO2 à N2 or NH3 nitrate nitrite ammonia ultruger gas ? + use nitrate ability to Nz pos for N2 dk anything Sugar fermentation Motility Nitrate reduction - Nitrate as a terminal electron acceptor Urease NO3 à NO2 à N2 or NH3 look for nitrite & Add nitrate reduction test reagents red = pos Sugar fermentation Motility Nitrate reduction - Nitrate as a terminal electron acceptor Urease NO3 à NO2 à N2 or NH3 ? + could hae a dK ↳ or = nitrite pos Add nitrate reduction nothing test reagents Sugar fermentation Motility Nitrate reduction - Nitrate as a terminal electron acceptor Urease NO3 à NO2 à N2 or NH3 ? + – If nitrate POS = Nitrite = Fink Add zinc If ammonia = neg dust yellow zinc catalyse reaction Sugar fermentation Motility Nitrate reduction - Nitrate as a terminal electron acceptor Urease NO3 à NO2 à N2 or NH3 NO2 NH3 NOs( ) + + – ? ? N2 ? + Add Add zinc nitrate dust to reduction “?” reagents to “?” Sugar fermentation Motility Nitrate reduction Urease - Urea hydrolysis à ammonia (basic) ø nothing added + + –  Other Corynebacterium species Normal microbiota Usually ignored, unless special circumstances Diphtheroids (diphtheria-like), coryneforms  Listeria monocytogenes Very common environmental organism food consumption I contamination Rare cause of sepsis and meningitis (Listeriosis) ↑ crossed blood brain bamer Outbreaks have been foodborne (deli meats, soft cheeses, fruit, etc.) Lab must always R/O Listeria when GPR is seen in blood or CSF Populations at highest risk: Treatment: Pregnant women Intravenous antibiotics Neonates Elderly Immunocompromised destroys vacuole before they can kill bacteria primam vinience factor ↑ (Our strain is ∆actA) membrane More slender, less pleiomorphic than Corynebacterium; still some palisades formed thinner  Culture and identification of Listeria monocytogenes Colony morphology on blood agar Small, translucent; narrow zone of β hemolysis (our strain is less hemolytic) (Can be mistaken for Strep. or Enterococcus) ↳ GBS Catalase and Gram stain readily distinguishes between Listeria and Streptococcus/ Enterococcus Listeria (Bacitracin resistant) Hippurate positive (Just like Group B Strep) Bile-esculin Salt-tolerant positive (Just like Enterococcus)  Culture and identification of Listeria monocytogenes Colony morphology on blood agar Small, translucent; narrow zone of hemolysis + Catalase and Gram stain readily distinguishes between Listeria and Streptococcus / Enterococcus GNDC CGND Motile Tumbling, end-over-end motility Biochemical tests used for confirmation Sugar ferments ⑫ ⑭ Motility (+; "umbrella motility") * ( Nitrate reduction (–) Urease (–) Haemophilus > - GNR  Haemophilus Haemophilus species are Gram-negative pleiomorphic rods. They are non-motile facultative of -anaerobes that are catalase and oxidase positive. Most Haemophilus sp. are normal residents of the upper respiratory tract of humans or animals The most common species of Haemophilus causing disease in humans is H. influenzae URT H. parainfluenzae is part of the human upper respiratory tract microbiota and only occasionally causes infection  Haemophilus influenzae What’s in a name? Haemophilus = "blood loving" In 1892 during an outbreak of influenza, Richard Pfeiffer found the organism in the sputum of patients and proposed it to be the cause. In 1933, Wilson Smith et al. showed that a virus caused influenza and H. influenzae was a secondary infection.  H. influenzae – Clinical Significance est n Causes respiratory infections, bacteremia, and crosse a meningitis, most commonly in infants and young children (≤ 5 years old) Spread by inhalation of aerosolized respiratory droplets Major virulence factor of strains causing invasive disease is the polysaccharide capsule Six serogroups (a - f) of encapsulated strains are known as "typeable strains" Unencapsulated strains, referred to as "non- typeable", can cause noninvasive disease H. influenzae – Clinical Significance 90% of invasive strains are "type b" serogroup Vaccine raised against capsule component PRP (polyribitol phosphate) w/ protein conjugate was developed in 1987 & add protein so immune system would react Vaccination against type b H. influenzae (Hib vaccine) resulted in 99% decrease in disease Estimated Annual Incidence* of Invasive Hib Disease, 1980-2012 *Rate per 100,000 children < 5 years of age *Rate per 100,000 children