PHM242 Midterm Notes PDF

Lec 1: Intro to Medical Microbiology Medical microbio: study of microorganisms (E.g. bacteria, virus, fungi, parasite) + role in health/disease. Importance: plays role in prevention, diagnosis, treatment of infectious disease. Scope of medical microbio: pathoge...

Lec 1: Intro to Medical Microbiology Medical microbio: study of microorganisms (E.g. bacteria, virus, fungi, parasite) + role in health/disease. Importance: plays role in prevention, diagnosis, treatment of infectious disease. Scope of medical microbio: pathogenesis, epidemiology, antimicrobial resistance, developing new diagnostic tools + therapy. Applications: ID management, public health surveillance, food safety, env microbio. History of Medical Microbiology Aristotle Proposed theory of spontaneous generation of infection. Girolamo Fracastoro (1546) Proposed theory of contagious disease: believed disease could be transmitted b/w hosts. Anton van Leeuwenhoek Observed first microorganisms w/microscope. (1676) Theodor Schwann + Justus Established germ theory of disease. von Liebig (1835) Disease doesn’t spontaneously form: is caused by invisible (to human eye) microorganisms. Semmelweis (1847) Proposed hand washing to decrease puerperal sepsis. Observed women who delivered baby w/midwife had lower sepsis rate than w/doctor. ○ Bec midwives were cleaning + washing hands b/w pts. Louis Pasteur (1857) Disproved theory of spontaneous generation. Also demonstrated microorganisms cause fermentation + disease. Developed fermentation and pasteurization processes. Joseph Lister (1867) Used carbolic acid in OR to prevent surgical infections. Robert Koch (1876) Identified Bacillus anthracis as causative agent of anthrax + developed Koch postulates. Hans Christian Gram (1884) Developed Gram staining technique to make bacteria more visible in stained tissue. Paul Ehrlich (1910) Discovered cure (Salvarsan) for syphilis: was first specific agent for infection. Alexander Fleming (1928) Discovered antimicrobial properties of penicillin. Jonas Salk (1955) Developed first successful polio vaccine. Louis Pasteur French scientist, developed germ theory of disease. Helped understand causes of infectious disease. Research led to pasteurization process: process that kills harmful bacteria in food + drink. Pasteur’s work in vaccination helped lead to creation of first rabies vaccine. Pasteur’s experiment to disprove spontaneous generation of infection: 1. Had 2 flasks of soup broth: boiled both of them to kill microorganisms. a. Then left both flasks at room temp.  2. 1 diff b/w flasks: one had straight neck, one was curved. 3. Both flasks exposed to air. a. After some time: straight neck was discoloured/cloudy, curved neck didn’t change. 4. So: germs in air could fall into straight neck + contaminate it. a. Disproved spontaneous generation bec if it was true, both flasks would be contaminated equally. 5. Then: tipped curve neck flask on side and sat it back straight again. a. Broth became contaminated: bacteria entered flask and entered broth. Joseph Lister Introduced surgery septic techniques: used carbolic acid to disinfect skin, instruments, wound, hands. Wanted to know why so many post-op deaths were from infection. ○ From Pasteur’s work: realized he needed to kill germs before they enter wound. Diluted carbolic acid + nebulized it so it fell onto wound, surgeon hands, instruments, etc. Decreased post-op wound infections from 50% to 15%. Was also first person to use penicillin: used it to heal a nurse’s abscess. Paul Ehrlich Found cure for syphilis: it contained arsenic, but is still considered the first antibiotic. Described toxin-antitoxin interaxn. Bacteria Erhlichia named after him. 1908: won Nobel Prize for immunology contributions. Other important events: 1. 1887: Petri dish made by Robert Petri. 2. 1901: blood groups (Landsteiner). 3. 1976: Legionnaires disease, waterborne pathogens could be nebulized in air + inhaled to cause infection. 4. 1979: smallpox eradicated in CAN. 5. 1983: HIV identification. Major branches of medical microbio: 1. Bacteriology: study of pathogenic bacteria + role in health/disease. 2. Virology: study of viruses, structure, replication, impact on health. 3. Mycology: study of fungi, identification, classification, role in infection. 4. Parasitology: study of parasite,s life cycle, interaxns w/human host. 5. Immunology: study of immune system + response to pathogens. Microbiomes Normal flora: 1. Resident/commensal flora: always founds in/on us in a particular site. a. E.g. S. epidermidis on skin. b. Usually has beneficial or neutral relationship w/host. c. Beneficial: e.g. prevents growth of dangerous microorganisms. 2. Transient flora: found briefly/intermittently at site. a. Is usually elim by competition w/resident flora or host’s immune system. b. E.g. group A streptococci in oral pharynx. 3. Carrier state: potential pathogen found in healthy host, may or may not result in disease. a. E.g. MRSA in nares. b. May pass these pathogens onto somebody else. Types of microbiomes: 1. Gut: microbial communities in human gut for digestion, immune fcn, health. 2. Skin: bacteria, fungi, etc. on skin that protects from pathogens + helps skin health. 3. Oral: microbial ecosystem in oral cavity, helps w/dental health + overall wellbeing. 4. Vaginal: in fem repro tract, maintains vaginal health + prevent infection. Skin 10 -10 organisms/cm2 of skin (higher hair follicles). 4 6 [Higher] in moist areas: e.g. skin folds, pits. Primarily Gram (+) organisms: S. epidermidis, S. aureus, cutibacterium acnes. ○ Some yeast, few Gram (-) organisms. Oral Cavity + Upper Respiratory Tract Mouth: organisms vary w/age + dental state. Young: gram (+) cocci + anaerobes. Elderly: gram (-) bacilli. Upper resp tract: nose contains S. epidermidis, S. aureus. GI Tract 1. Esophagus: transient oral flora. 2. Stomach: very few bacteria (103), killed by stomach acid. a. Lactobacillus, enterococcus. 3. SI: similar acidic env as the stomach at the start of intestine. a. Move thru duodenum - jejunum: increasing quantity + diversity. b. Further down: anaerobes, clostridial species. 4. Large intestine/colon: 1011 organisms/g of feces. a. > 90% organisms are gram (-) anaerobes, enterococci, streptococcal spp.  Urogenital Tract 1. Urethra: sterile once past the first distal cm, colonization more likely closer to outside world. a. Mostly same gram (+) organisms as skin. 2. Vagina: a. Premenopause: lactobacilli predominate to keep pH 4-5. b. Postmenopause: increase in gram (-) organisms, causes increased pH. Infection How can normal flora go rogue + cause infection: 1. Opportunistic infection: a. If host is immunocompromised: organisms usually held back by immune system. b. Antimicrobial therapy: antimicrobial can decrease competition for resident bacteria. c. Penetrating trauma: e.g. gunshot. 2. Tissue invasion: microorg crosses tissue, moves from site of neutrality to harmful. a. E.g. S. aureus from skin moves to blood + can invade heart tissue. 3. Translocation: movement from usual site (GIT?) thru mucosal/other mems to a non-normal site. a. E.g. bacteria from inside gut lumen translocates to blood. Host Defenses Non-Specific: Local Non-Specific: Systemic Specific 1. Skin 1. Fever 1. Ab-mediated or humoral immunity: 2. Secretions: tears, saliva, sweat 2. Interferons a. IgM, IgG, IgA 3. Acid: low pH in stomach 3. Phagocytosis b. Mediated by B lymphocytes 4. Mucus secretions 4. NK cells 2. Cell mediated immunity: 5. Gut motility a. Cytotoxic T cells: elims virus infected cells 6. Flushings: tears, urine b. Macrophages: kill intracellular bacteria 7. Normal flora competition c. Mediated by T lymphocytes 8. Cilia (lungs) When Host Defenses Fail Definitions: 1. Pathogen: any microorganisms capable of causing disease. 2. Pathogenesis: mech/route where microbe causes disease. 3. Infection: persistence on/within another living organisms. 4. Disease: end product/damage from infectious process. 5. Incubation: time from infection to onset of symptoms/disease. Routes of microorganism transmission: Horizontal Vertical 1. Direct contact: secretions, blood, penetrating trauma Mother to fetus 2. Respiratory: aerosol, droplet 3. Contaminated inanimate objects: fomites on bedrails 4. Zoonotic vector: insect bite  Infectious dose required to cause disease: 1. Shigella spp.: 10-103 via oral route. a. Incubation: 1-2 days. 2. Salmonella typhi: 105 via oral route. a. Incubation: 14 days. 3. Mycobacterium tuberculosis: 1-10 via inhalation. a. Incubation: variable. Virulence factors: structures/systems that allow pathogen to infect host. 4 types: Capsules Adhesions External structures to cell wall: allows org to avoid phagocytosis. Allow bacteria to stick to mucosal cells + prevent elim. Adhesions = bacterial surface structures: ○ Pilli, proteins, lipopolysaccharides. Enzymes + Toxins Invasiveness 1. Enzymes: break down host cells + antimicrobials. Organism’s ability to invade host cell. 2. Toxins: 2 broad categories (exotoxin, endotoxin). Includes multiple factors: e.g. adhesion, enzyme, a. Exotoxin: protein made + released by organism. toxin. b. Endotoxin: part of org, signal to host immune system presence of pathogen. Antimicrobial resistance: Resistance Type Impact on Medical Treatment Antibiotic Resistance Reduces antibiotic effectiveness. Causes longer + more severe infections, increased hospitalization + mortality rate. Antiviral Resistance Compromises ability to treat viral infection. More difficult to manage + control outbreaks (esp for vulnerable pops). Bacteria Basics Prokaryotes Eukaryotes 1. < 2 um, no nucleus 1. Defined nucleus 2. Circular DNA strand, divided by binary fission 2. DNA in paired chromosomes, divide by mitosis 3. Has ribosomes, but lack other organelles 3. Cytoplasm contains ribosomes, mito, etc. 4. Bacteria 4. Fungi, protozoa, worms Bacteria structures: flagellum (movement), pilli. Also: capsule, cell wall, plasma mem, cytoplasm, ribosomes, plasmid, nucleoid (circular DNA).  Bacterial Structures 1. Endospore: formed by some gram (+) bacteria (clostridia spp., bacillus spp.). a. Resists heat + drying, contains nuclear material + protein. b. Germinates into dividing bacteria under optimal conditions. 2. Nucleoid: in nuclear region, contains single circular DNA strand w/o a nuclear mem. a. Plasmids occasionally present. 3. Ribosomes: transcribes mRNA into essential proteins. 4. Cell mem: encloses bacteria cell, has selectively permeability + transports solutes. a. Contains energy producing enzymes. b. Produces compartmentalization needed for e- transport + oxidative phosphorylation. 5. Cell wall: rigid enclosure, surrounds cell mem + gives shape to bacteria cell. a. Protects against osmotic lysis. b. Structure: mixed polymers + peptidoglycan. c. Cell wall structure + thickness determines gram stain characteristics. Bacterial Classifications 1. Gram stain: a. Positive = purple: thick cell wall retains crystal violet dye. b. Negative = pink-red: thin cell wall decolourizes + loses purple colour. i. Then: wall takes up counter stain (Safranin). 2. Morphology: cocci, bacilli. a. Cocci (round): streptococci, diplococci, tetrad, staphylococci, sarcina. b. Bacilli (rod): chain of bacilli, flagellate rods, spore former. c. Others: vibrios, spirilla (H. pylori), spirochaetes. 3. Ideal growth conditions: a. Aerobic: must have O2 b. Obligate anaerobic: reqs no or near absence of O2. c. Facultative anaerobic: can use O2, but also grows w/o O2. Gram stain importance: 1. Provides direction for further testing + direction for therapy. 2. It may stain other microorganisms: fungi, protozoa, worms. 3. But: doesn’t stain acid fast bugs (mycobacteria). a. May poorly or not stain bacteria that don’t have cell wall (chlamydia, mycoplasma, etc.). Gram stain steps: 1. Fixation of organism. 2. Crystal violet dye: turns purple. 3. Iodine treatment. 4. Decolourization: removes purple dye in gram (-). 5. Counter stain w/safranin: stains gram (-) bacteria pink. Taxonomy: Domain < Kingdom < Phylum < Class < Order < Family < Genus < Species Dear King Phillip Came Over For Good Soup  Microbiology Laboratory Diff phases of microbiology process: 1. Pre-analytic phase: a. Ordering: how to order diff lab tests. b. Specimen collection, transportation, receipt. c. Specimen processing. 2. Analytic phase: testing + interpreting results. 3. Post-analytic phase: reporting results. Infection Control Measures Hand Hygiene Hand washing w/soap + water, or using hand sanitizers. PPE Gloves, gown, mask, eye protection: creates barrier b/w HCP + sources of infection. Env Cleaning + Disinfection Cleaning + disinfection of equipment, surfaces, env to elim presence of pathogens. Proper Waste Disposal Safe + regulated disposal of waste (sharps, contaminated materials, etc.) to prevent infection spread. Sterilization of Medical Kill + remove all microorganisms from instruments + equipment to ensure they’re safe to use on pts. Instruments Future trends in medical microbio: 1. Advancements in genomic sequencing: for fast ID + characterization of microorganisms. 2. Personalized microbial therapeutics: tailors probiotics/therapies to treat microbial disease based on pt’s microbiome. 3. Antimicrobial resistance monitoring: surveillance + early detection to track spread of antibiotic resistant pathogens. 4. Microbiome based diagnostics: human microbiome as diagnostic to detect/monitor condition. 5. Nanotech in microbio: use nanomaterials/devices for better antimicrobials, drug delivery, etc.  Lec 2: Staphylococci (S. aureus) Staphylococcus aureus Introduction Etymology: staphyle = bunch of grapes, kokkus = grain/seed, aureus = golden. Morphology: gram (+) coccus in grape-like clusters. S. aureus: major pathogen within Staphylococcus genera. ○ Periodically colonizes skin + mucous mems in healthy adults (≥ 60%). Opportunistic pathogen: impetigo, carbuncles, abscess, invasive disease in right conditions. ○ Can also cause pneumonia, bloodstream infections, etc. Epidemiology S. aureus colonization in general pop vs nasal carriers: General Population Nasal Carriers of S. aureus Nose: 27% Nose: 100% Neck: 10% Pharynx: 25-50% Pharynx: 10-20% Axilla: 19% Axilla: 8% Skin abdomen: 40%. Chest: 45% Skin abdomen: 15%. Chest: 15% Forearm: 45% Forearm: 20% Hand: 90% Hand: 27% Perineum: 60%. Perineum: 22%. Vaginal: 5% Ankle: 10% Ankle: 10% Colonizes skin + mucosa in multiple body sites. Primarily: primary anterior nares, secondary skin, perineum, pharynx. If nasal carrier: there’s increased colonization in other areas (increased extra-nasal carriage). 3 types of carriage patterns: 1. Non (20%): never been colonized by S. aureus. 2. Intermittent (60%): colonized at some points, then decolonized, then colonized, etc. 3. Persistent (20%): cannot be decolonized. S. aureus easily spread to hands, clothing/inanimate objects, env. ○ Can remain dormant for months in specimens + env. S. aureus killed by: heat, light, disinfectants. Pathogenesis Has many virulence factors that allow for infection. Virulence factors can be: niche adaptions or response to stress. Niche Adaptations Stress Response Surface binding proteins: Quorum sensing Immunoglobulin, fibrinogen, collagen Toxin production Fibronectin, Hb, epithelial cells Cassettes: genetic elements w/resistance genes Biofilms ○ Protects against env + innate immunity How S. aureus establishes infection: Entering body: breach epithelial layer of skin w/alpha toxin production. Alpha toxin: forms pores in target cells + breaches junctions. Allows bacteria to enter cell. Once inside body: 1. Biofilm production on prosthetic/plastic devices/products (e.g. tampons). a. Can cause staphylococcal toxic shock syndrome. b. Once bacteria enters blood: disseminates throughout body. 2. In blood: can destroy immune cells directly using diff methods. a. a-toxin: major cytolysin. b. Leucocidins: panton-valentine leucocidin (PVL), γ-toxin, LukDE, LukAB, etc. c. Phenol soluble modulins (PSMs). 3. MSCRAMMs: microbial surface components recognizing adhesive matrix molecules. a. MSCRAMM: surface protein on bacteria, helps adhere to cell + invade. b. Facilitates attachment + invasion to other tissues. c. E.g. protein A, fibronectin/fibrinogen/collagen/elastin binding proteins. Protein A: surface protein A (SpA). ○ Binds to Fc receptors on Ig + polymorphonuclear cells. ○ Camouflages itself w/nonspecific IgGs on surface: prevents immune response. ○ Also interferes w/opsonization + phagocytosis. Fibronectin binding protein: adheres to fibronectin + allows bacteria internalization. Fibrinogen binding protein: adheres to fibrin clots + forms aggregates. ○ Covers + camouflages bacteria w/fibrinogen: inhibits immune recognition. ○ Also adheres to implanted biomaterials. ○ Can use card lab test (not done often): place bacteria on card + mix w/reagent. Detects fibrinogen binding protein by forming aggregates/clumping. Confirms sample is S. aureus. 4. Further abscess formation w/exoenzymes, toxins, surface proteins. a. IsdA/B: acquires Fe for initial abscess formation. b. Coa + vWbp: produces fibrin clots + inhibits leucocyte infiltration. c. Protein A: pro-inflammatory (recruits WBCs). d. Exoenzymes: digests released nutrient macromolecules from cell lysis via cytolysins.  Toxins in S. aureus: Cytotoxins: ⍺, β, γ toxins, PVL, ETs. ○ ⍺-toxin: pore forming, lyses RBCs, platelets, WBCs, endothelial/epithelial cells. ○ β-toxin: hemolytic activity. ○ γ-toxin: hemolysin, lyses WBCs, leukocytes, etc. ○ PVL: causes severe disease. ET: exfoliative toxin. ○ ET: can cause staphylococcal scalded skin syndrome (SSSS), bullous impetigo. Enterotoxins: non-specific T-cell stimulators, called superantigens. ○ Causes cytokine overload: only small amt needed to be toxic. ○ Bioterrorism potential;: responsible for food poisoning + TSS (macular papular rash). Lab Diagnosis Specimen collection, microscopy, culture, supplementary tests, confirmation. Specimen Collection What specimens to collect depends on clinical manifestation. E.g. superficial infection: swabs, aspirate pus in lesion (better option). ○ Pneumonia: resp samples (sputum), blood. ○ Sepsis: blood sample. Sent to microbiology lab ASAP: time is correlated w/morbidity + mortality. Microscopy Depending on specimen: either use a film or smear. Microscopy = first piece of diagnostic info we get. Provides info on probable staphylococci or another gram (+) cocci in clusters. ○ Only indicates that specimen might be S. aureus. ○ But: could be non-pathogenic or commensal. Blood Culture Specimen Sputum Culture Specimen Pure S. aureus Culture Outside of RBCs Culture Culture specimen on many types of media, distinct colonies appear after 24h. Diff media allows for diff growth reqs. S. aureus grows on typical lab media: blood agar, chocolate agar, etc. Appearance: round, opaque, +/- golden, +/- B-hemolytic colonies. ○ Still only indicates that it may be S. aureus. ○ Outside colony: has dark ring (indicates B-hemolysis).  Supplementary Tests Less commonly used now: but is a rapid test. Most S. aureus has unique property: can produce coagulase (free + bound). ○ (+) coagulase test: highly likely that it’s S. aureus complex. Coagulase test: coagulase (clumping factor) reacts w/fibrinogen (virulence factor). ○ Causes precipitation + cell clumping when bacteria mixed w/plasma. ○ Can do test on a card (less accurate) or in tube. Tube coagulase test: plasma coagulase reacting w/CLP causes free coagulase activation. ○ This forms coagulase CLP complex: complex reacts w/fibrinogen to form clot. ○ Takes 4-24 hrs for results. ○ LHS tube: clot (positive test). RHS: no clot (negative test). Card coagulase test: (+) = clumps, (-) = no clumps. Confirmation MALDI-TOF MS: matrix assisted laser desorption ionization time-of-flight mass spectrometry. A proteomic method: detects diffs in proteins (mostly ribosomal proteins). ○ Then produces fingerprint that’s unique to species. Obtained within min from a colony or directly from specimen. Antibiotic Resistance Can have resistance to: 1. B-lactams: methicillin/cloxacillin (MRSA). 2. Glycopeptides: vancomycin (VRSA, VISA). 3. Oxazolidinones: linezolid. Resistance can occur to multiple antimicrobial classes. B-lactams, glycopeptides, lincosamides, macrolide, quinolone, rifampicin, tetracycline, co-trimoxazole, linezolid. Resistance occurs thru: chromosomal de novo mutations or horizontal gene transfer. B-lactams: penicillin, aminopenicillin, cephalosporin, carbapenem, B-lactamase inhibitor combos. B-Lactam Resistance Structure: all have B-lactam ring, thiazolidine ring, differing side chains. Penicillin Resistance Penicillin resistance is common (80%+): rarely tested, we assume it’s resistant. Resistance occurs thru blaZ gene (B-lactamase). ○ Hydrolyzes B-lactam ring.  Methicillin/Oxacillin Resistance MRSA MRSA: methicillin resistant Staphylococcus aureus. Caused by horizontal acquisition of mecA (or mecC) gene that encodes PBP2a. ○ PBP: penicillin binding protein. ○ S. aureus modifies structure to avoid binding. mecA: is a genetic element called SCCmec. ○ Derived from animal adapted lineages of S. aureus. MRSA has low affinity for most B-lactams. MRSA = 1st line agents are ineffective (cefazolin, cloxacillin) + most B-lactams ineffective. 2021: 17% of S. aureus infections were MRSA. Canadian antimicrobial resistance surveillance system (CARSS): ○ Looks at epidemiology of antimicrobial resistance across Canada. 2 classifications: HA-MRSA, CA-MRSA. HA-MRSA CA-MRSA Healthcare associated MRSA: Community acquired MRSA: absence of HC exposure. 48h after hospital exposure. Resistant to ≤ 2 classes. ○ Or: outside hospital, within 12 months of HC exposure Caused by SCCmec type IV/V. Resistant to ≥ 3 antibiotic classes. ○ Higher virulence, PVL more common. Caused by SCCmec type I/II/III. ○ Has lower virulence, PVL less common. Graphs of rates: 1. Incidence rates of MRSA (2017-2021): a. CA-MRSA increased 80%, HA-MRSA decreased 2.3% b/w 2017-2021. b. Proportion of MRSA from BSI (bloodstream infection): increased 15.8% to 19.9%. 2. All cause mortality from MRSA BSI (2017-21): 18.6% died ≤ 30 days of diagnosis. 3. Antimicrobial resistance patterns from all MRSA BSI (2017-2021): a. MRSA has multiple antimicrobial classes that are resistant. b. High resistance: ciprofloxacin, erythromycin. Detecting MRSA in lab: many methods available. 1. Oxacillin agar screen: grow bacteria on petri dish, impregnate w/antibiotic. a. See if bacteria still grows or if it’s killed.  2. Disc diffusion: place discs w/antibiotic in dish of bacteria. 3. Chromogenic media: media contains things that detects if MRSA has grown. a. Look for navy blue colonies. 4. Latex agglutination: looks at specific virulence factors. a. Agglutination = S. aureus. 5. Nucleic acid amplification test: look at genome. What antimicrobials we’re looking for when looking at resistance: 1. B-lactam combo agents: amoxi-clav, pip-tazo. 2. Oral cephems: cephalexin, cefuroxime, cefpodoxime. 3. Parenteral cephems: cefazolin, cefepime, cefotaxime, ceftriaxone, cefuroxime, ceftaroline. 4. Carbapenems: ertapenem, imipenem, meropenem. PIDAC: provincial infectious diseases advisory committee (within Canada). Gives advice/guidelines on measures to prevent spread of specific infections (e.g. MRSA). Risk factors for MRSA: Definite risk factors: screen upon arrival into HC system/facility. Possible risk factors: screening can be done, but not necessary. Definite Risk Factors Possible Risk Factors Previous colonization/infection w/MRSA Home health care, household contact of pt w/MRSA > 12 hrs in any HCF (HC facility) within last 12 months Indwelling device Recent exposure to unit/area of HCF w/MRSA outbreak ICU, burn unit, transplant unit HC in another country Communal setting Injection drug use Immunocompromised CA-MRSA risk (e.g. sports teams) Screen for MRSA: Take specimens from anterior nares (tip of nose): has highest MRSA yield. ○ But: in some cases, may only be isolated from perianal/perineal area. ○ So: do 2 swabs (1 in anterior nares, 1 in perianal/perineal). CA-MRSA: do additional culture of any lesions/abscesses. Children: do throat swab instead of nasal. Newborns: take umbilicus sample. Screening lab tests for MRSA: Screen using chromogenic agar: looking for dark blue/denim colour colonies. ○ Also do confirmation tests. Then: report as MRSA or no MRSA isolated. MODSA Modified penicillin binding protein S. aureus: methicillin/oxacillin resistance. Caused by PBP mutations that encode for PBP2 + PBP4: causes oxacillin resistance. Are mecA negative: won’t pick it up if using methods that detect mecA. ○ Normally, may use mecA tests to determine if you have B-lactam resistance. ○ So: use alternative methods. MODSA very rare. BORSA Borderline oxacillin resistance S. aureus. Hyper-produces B-lactamase: causes oxacillin resistance. ○ If using oxacillin: will be resistant. ○ If using cefoxitin: will be susceptible (so not MRSA). Has mutations in PBP2 + PBP4 (like MODSA) + mecA negative. Grows on MRSA chromogenic media: so may think it’s MRSA, but it’s not. Glycopeptide Resistance Vancomycin Treatment of choice for serious infections + empiric Rx before knowing if it’s MRSA. Inhibits peptidoglycan synthesis: prevents new building blocks. ○ Stops bacteria from growing. Gram (+) structure: thick peptidoglycan layer. VRSA: vancomycin resistant staphylococcus aureus. Occurs from VanA operon derived from enterococcus spp. ○ Operon synthesizes an altered precursor of cell wall. ○ Result: can’t get binding of vancomycin. Very rare (52 isolates worldwide). ○ Likely due to: reduced fitness + MRSA incompatibility. ○ MRSA incompatibility: bec PBP2a cannot cross-link the modified wall precursor. ○ Can’t be vancomycin resistant and be an MRSA. Lab: grow bacteria in presence of antibacterials. ○ Can see if bacteria is vancomycin resistant or not. ○ LHS: vancomycin resistant. Middle: vanco susceptible. RHS: oxacillin susceptible. VISA: vancomycin intermediate staphylococcus aureus (intermediate phenotype). Causes prolonged hospitalization/treatment, treatment failure. Causes of VISA: 1. Increased cell wall thickness.  2. Changes in cell wall + cell surface charge. 3. Decreased autolysis + cross-linking. 4. Increased cell wall synthesis. Unique mech of resistance. Uncommon: but difficult to detect (esp bec there’s several types). ○ E.g. hVISA, sVISA. Oxazolidinone Linezolid Linezolid: used for nosocomial pneumonia + SSTIs by MRSA/MSSA. Targets 23S rRNA: blocks protein synthesis. Resistance occurs thru mutations in 23S rRNA gene. ○ AND/OR acquiring ribosomal methyltransferase cfr gene. ○ Cfr may be transferred from S. epidermidis. Rare (2%): but more common (11%) in CF pts. Clinical Case D1: 45 y/o male (previously healthy), pain in left calf, expressed pus from ingrown hair. D2: cellulitis extended from knee to ankle. Red, warm cellulitis, no lymphadenopathy (given CRO + CEX) D4: had area of fluctuance in centre of cellulitis, low-grade fever. Sent 1 mL pus for gram stain + culture. Excised + drained lesion. Antimicrobial susceptibility testing results: Resistant against: erythromycin, penicillin, cefoxitin. ○ I.e. MRSA. Questions: 1. Where did S. aureus come from + why/how did it cause infection? a. Pt likely colonized (intermittent or persistent). b. Manipulated original lesion: bacteria entered dermis. 2. Why incision + drainage req?: antimicrobials alone cannot penetrate centre zone of abscess. a. Drainage removes large bioburden of bacteria, allows drugs to penetrate. 3. What is significance of the lab results? a. Resistant against cefoxitin = MRSA. b. Cephalosporins originally given would have no activity. c. Susceptible against vancomycin: reqs vanco. d. Other options: clindamycin, doxycycline. Coagulase Negative Staphylococci (CoNS) Etymology: staphyl = bunch of grapes, kokkus = grain/seed. Gram (+) cocci in grape like clusters. ○ Morphologically similar to S. aureus.  ○ But: doesn’t coagulate plasma + most don’t have clumping factor (caveat). Rarely causes infection. ○ Opportunistic pathogen: may cause infection in immunocompromised pts. ○ Does this by colonizing medical devices (e.g. prosthetic valves, IV lines). Epidemiology Colonizes skin + mucosa: diff species have diff preferred sites. S. epidermidis: axillae, head, arms, legs, nasopharynx. 1. Hair: can go from hair shaft down to follicle. 2. Face: sebaceous/oily lipids, sebum, anaerobic follicles. 3. Antecubital crease: moist, low pH, sweat glands, site of atopic dermatitis. 4. Volar forearm: dry, nutrient poor. 5. Foot: toe webs. S. capitis: scalp, arms, other regions. S. lugdunensis: abdomen + extremities. S. saprophyticus: rectum + GU tract (esp in sexually active females). NICU: can have nosocomial outbreaks of single clones of MDR + biofilm producing CoNS. Pathogenesis CoNS has diff factors that facilitate: 1. Survival on skin 2. Protects against other pathogens (e.g. S. aureus). 3. Adhere to prosthetics. 4. Evade immune system. 5. Produce biofilms: icaADBC operon encodes genes promoting biofilm production. a. Protects from immune system, antimicrobials, shear stress, desiccation. 6. MSCRAMMs: has less than S. aureus. a. Produces surface anchor proteins that allows bacteria binding to tissue. b. SdrF (collagen), SdrG (fibrinogen), SdrH, Embp (fibronectin), 7. Accumulation associated protein (Aap): anchored to cell, mediates attachment to host. a. Helps colonization. b. Also helps biofilm attachment + accum (not thru polysaccharide based biofilms). Lab Diagnosis Microscopy, culture, supplementary tests, confirmation. Microscopy Depending on specimen, do work-up on film or smear. First piece of diagnostic info. Blood culture specimen: indistinguishable from S. aureus. Culture Similar to S. aureus. White grey, smooth, opaque. Non-hemolytic: no ring around colony. Supplementary Tests Coagulase negative: some exceptions for bound (clumping factor) + free coagulase. Due to exceptions: we don’t do this test anymore. Confirmation MALDI-TOF MS Pathogen vs Flora Microbiology lab must determine if the tested bacteria is normal or is causing disease. Assessing clinical significance of CoNS can be difficult. Blood cultures: may flag “positive” w/CoNS due to contamination upon specimen collection. ○ So: looks like CoNS is causing BSI, but it isn’t. So: do multiple sets of blood cultures drawn from diff venipuncture sites @ diff times. ○ If multiple sets (+) from same species/morphotype/AST: then likely the true pathogen. Resistance Can be resistant to multiple classes. In some cases: is the origin of SCCmec that leads to MRSA. S. epidermidis + S. haemolyticus: high rates of MR. Some have reduced susceptibility to glycopeptides (like S. aureus). S. saprophyticus: has intrinsic resistance to novobiocin, fosfomycin, fusidic acid (always R). Usually: can’t use B-lactams.  Lec 3: Streptococci Structure + Classification Etymology Strepto = easily twisted, coccus = seed/berry. Morphology Round/ovoid bacteria, joined in pairs or chains (short/long). Round bacteria that are bound in linear fashion. Staining Gram stain (+) (no outer mem, so cell wall exposed to env). Biochem characteristics Coagulase + catalase negative Mobility Non-motile Oxygen tolerance Facultative or obligate anaerobe Streptococcus: large group of bacteria, has own genetic + microbiologic niches. Classification: 55+ species, 8 subspecies, at least 5 groups. Categorization: originally, was categorized bec they looked the same under microscope. 1. Hemolysis: destruction of blood cells (sheep’s blood agar), diff types. a. Alpha hemolysis: partially hemolyzes (turns plate green). b. Beta hemolysis: complete hemolysis of sheep’s blood agar. c. Gamma hemolysis: doesn’t hemolyze sheep’s blood agar (not many exist). d. But: some species have variable hemolysis based on culture media/conditions. 2. Serologic/antigen grouping: sugar types that organism has on surface. 3. Genetic markers: e.g. 16S RNA sequencing. Alpha Hemolytic Streptococci (AHS) 2 ‘groups’: 1. Streptococcus pneumoniae: technically part of VGS, but behaves differently. a. Most predominant pathogen for pneumonia (?). 2. Viridans group streptococci (VGS): large umbrella term, ‘grab bag’ (turns plate green). S. pneumoniae Most important of all pathogens: bec it has most diversity for possible disease. S. pneumoniae + pneumococcus = same thing. Morphology Gram (+), diplococci (pairs) Classification Serotype/antigen classification (> 97 diff serotypes). Adults may develop serotype immune response against S. pneumoniae. Natural habitat Human resp tract carriage: common in children (65%), less common in adults. Virulence factors Factors that help organism cause disease: 1. Polysaccharide capsule (not a universal thing for bacteria): protects from immune system. a. Cap can also help bacteria enter areas other bacteria can’t go.  2. Pneumolysin: hydrolysis of cell connections. 3. Autolysin. 4. Pneumococcal surface protein A/C. 5. Pneumococcal surface adhesion A. 6. Pili, neuraminidase. Overall: allows bacteria to adhere to host, prevent macrophage digestion, defend against immune system Serotype (surface antigens) distribution: some serotypes cause lots of disease, some cause less. Childhood vaccination helps w/disease morbidity + mortality. Epidemiology: world-wide distribution. Likelihood of carrier + disease based on: 1. Age 2. Immune status + vaccination status, type of infection 3. Year Associated infections: S. pneumoniae can cause many infections, many in resp tract. 1. Non-invasive infections (affects surfaces): S. pneumoniae is predominant cause for these. a. Acute otitis media (AOM). b. Acute bacterial rhinosinusitis, pneumonia. 2. Invasive infection: bacteremia, meningitis, infective endocarditis. Pneumococcal disease rates + cases: U shaped curve. Prevalence increases at age extremes (young or old). ○ Children (< 2): higher prevalence rate than middle aged. ○ Seniors (> 65-75): higher risk. Microbiological Identification Sample Collection Identification Grows well on standard agarose Biochemical methods: Find in: blood, CSF, resp tract, urine (urine antigen test) 1. Optochin susceptible ○ Urine tests not sensitive, not routinely used 2. Bile acid soluble GPC in chains Rapid identification methods (MALDI-TOF), often used. ○ Lasers lyse + aerosolize bacteria ○ Mass spec: see components of bacteria ○ Compare components to library of bacteria PCR methods: multiplex PCR Antigen testing: not routinely used  Viridans-Group Streptococci Morphology Gram (+) cocci in short/long chains Classification Grouped tgt due to hemolysis + phenotypic similarity. All VGS means is that organism will turn plate green. Lots of diffs in clinical presentation + antimicrobial susceptibility. Natural habitat Variable, mostly GI tract (mouth, etc.) Virulence factors Dextran + exopolysaccharide formation: forms microfilm deposits, protects organism. Typically more indolent infection, except S. pneumoniae + S. angionosis. Classification + Associated Infections 5 major groups of VGS: 1. S. angionosis: S. angionosis, S. constellatus, S. intermedius. a. Abscess forming: lungs, GI tract, liver, brain, etc. b. Unlikely to cause endocarditis, but more virulent than other VGS groups. c. Butterscotch smell, S. constellatus may be beta-hemolytic too. 2. S. mitis: S. mitis, S. infantis, S. pneumoniae, S. parapneumoniae. a. Unlikely to cause human disease in immunocompetent ppl (besides S. pneumoniae). b. Can cause odontogenic infections, bacteremia, infective endocarditis. i. Mostly in immunocompromised ppl. 3. S. bovis: S. bovis, S. gallolyticus, S. infantarius. a. Found in GI tract, benign (but can propagate + cause issues). b. If in blood: assoc w/bacteremia, colon malignancy (cancer), infective endocarditis. 4. S. mutans: S. mutans, S. sobrinus. a. Local infections, rarely causes significant infections in immunocompetent. b. Assoc w/odontogenic infection, but rare for it to be main cause. 5. S. salivarius: S. salivarius, S. thermophilus, S. vestibularis. a. Local infection, rarely causes significant infections in immunocompetent. b. Assoc w/odontogenic infections or infection in immunocompromised. Microbiological Identification Sample Collection Identification Blood Morphologic/hemolysis Tissue Rapid identification: MALDI-TOF Abscess, biliary tract Summary S. pneumoniae: leading cause of infection (e.g. AOM, sinusitis, pneumonia, bacteremia, meningitis). VGS: more indolent, assoc w/abscess formation, endocarditis, local oral/GI diseases.  Beta Hemolytic Streptococci (BHS) Pyogenic streptococci: focus on Lancefield group A, B, C, G. Pyogenic: fully hemolyzes sheep blood agar. Can cause mild - life threatening disease in many hosts. Can cause acute + post-acute/post-infectious sequelae (post infection complications). Each streptococci has unique surface antigens. ○ Group A, B, C, G: refers to the surface antigens. S. pyogenes Group A streptococci (GAS). Morphology Gram (+) cocci in chains Classification emm gene/M-protein (> 220 genotypes) Emm gene: expresses surface markers for virulence factors Classified based on antigen presentation Natural habitat Human skin, upper GI tract Virulence factors 1. M-protein (encoded by emm gene): coiled protein on cell surface a. Helps bacteria adhesion to cells, brings in host factors (e.g. fibrin) to conceal itself. b. Helps resist detection/neutralization. 2. Hyaluronic acid capsule: camouflage, protects against immune invasion. a. Also helps bind to epithelial cells. 3. S protein: crosslinks RBCs to bacteria, avoids detection/ 4. Secreted factors: streptokinase, SpeB, streptolysin, DNAase. a. Breaks down cell components to allow bacterial invasion. 5. Superantigens: T-cell cross link proteins. a. Crosslinks inactivated T-cells + activates them (causes toxic shock). Epidemiology: world-wide, increasing invasive disease trends after COVID. Associated infections: 1. Non-invasive: acute bacterial pharyngitis (strep throat). 2. Impetigo/erysipelas/cellulitis. 3. Scarlet fever: scarlet, rough rash. 4. Invasive infection: bacteremia, necrotizing fasciitis, toxic shock syndrome. Post-infectious complications: usually from group A strep. 1. Glomerulonephritis: deposits on kidney basement mem, causes immunoactivation + failure. a. Can cause temporary or permanent renal dysfcn. 2. Rheumatic heart disease: deposits on heart valves, destroys them (e.g. sticky valves). Invasive rates/cases for invasive group A strep: had downward trend in COVID.  Post-COVID iGAS in Ontario: increased a lot last yr, now back down to typical #s. S. agalactiae Group B streptococci (GBS). Diff b/w GAS + GBS: GBS has capsule. Morphology Gram (+) cocci in chains Classification Serotyping Natural habitat GI/GU tract, upper resp tract. Particularly in pregnant or child-bearing aged females. Virulence factors Less well characterized: assoc w/more invasive + problematic disease. 1. Adherence: Ssr1/Ssr2, HygA, FbsA/B/C, Lmb, pilli. 2. Immune evasion: C5a peptidase, BibA. 3. Tissue invasion: PbsP, SfbA, polysaccharide capsule. Epidemiology: world-wide. Many ppl have colonization in lower GI tract + GU microbiome. ○ 10-30% of pregnant females: not tested for GBS unless pregnant. Associated infection: many ppl asymptomatic. 1. Non-invasive: asymptomatic carrier, UTI, cellulitis. 2. Invasive: bacteremia, infective endocarditis, neonatal sepsis/meningitis. a. Neonatal: high [bacteria] in birth canal, can ingest it. Microbiological Identification of BHS Sample Collection Identification 1. Blood 1. Hemolysis on blood agar 2. Wound/pus 2. Rapid identification: MALDI-TOF 3. Sputum, throat swab, urine 3. Rapid antigen testing (GAS) Summary Group Genus/Species Natural Habitat Associated Disease A S. pyogenes Human skin Skin/soft tissue infection, pneumonia, bone/joint infection, bacteremia, toxic shock B S. agalactiae Human GI, GU tracts Neonatal sepsis, meningitis, infective endocarditis, skin/soft tissue infection C/G S. equi, S. dysgalactiae, others Skin, GI tract, zoonotic sources Skin/soft tissue infection, bacteremia, toxic shock (GAS-like) Rapid Antigen Performance in Pediatrics Case: 8 y/o has sore throat, fever, swollen lymph nodes. Children more likely to have group A strep than adults. Avg sensitivity of rapid antigen: 85.6%. Avg specificity of rapid antigen: 95.4%. Some children misdiagnosed as not having Group A strep if relying on rapid antigen testing. How to approach: depends on age + chances of developing complications. Child: suggest rapid antigen test. 1. If (+): highly suggestive of group A streptococcal pharyngitis. 2. If (-): refer for gold standard testing (throat culture). a. Just bec rapid antigen is (-), doesn’t mean they don’t have group A. Lec 4: Enterococcus Structure + Classification Etymology Entero (intestine), coccus (seed/berry) Morphology Round + ovoid bacteria, in pairs or chains. Non-spore forming Staining Gram (+) (purple/blue) Biochemical characteristics Catalase negative, coagulase negative Mobility Non-motile (most species, a couple are motile) Oxygen tolerance Facultative anaerobe Enterococcus are very versatile: can survive extreme temps, pH, O2 conditions, etc. Classification Until 1984: was part of streptococci genus (was group D streptococci). Made of 40+ diverse species: very ubiquitous organism, so exact # is debatable. 90% of infections are caused by 2 species: both part of normal feces. 1. E. faecalis (80-90%). 2. E. faecium (5-15%). E. gallinarium + E. casseliflavus: mobile. Categorization Most strains are non/low virulent. Most gamma hemolytic (non-hemolytic). ○ Common virulent factor = hemolysis. ○ So: presence of hemolysis shows virulence potential. Virulent enterococci strains show ⍺ or ꞵ hemolysis.  Epidemiology Most common GPC in lower GI tract: 105 x 107 cfu/gram of feces. Also found on skin + oral cavity: [higher] in perineum areas. Nature: animals, soil, water, food, sewage, plants. Easily transmissible to: hands, clothing, env, inanimate objects. ○ Very resilient + can survive against antiseptics + disinfectants. GI Tract Nature (Soil, Water, Food, Sewage, etc.) Is commensal organism. Food: helps meat + cheese fermentation, food preservation. Stims immune system + maintains intestine homeostasis. Also used as probiotic. But: can also cause food contamination. How: enterococci belong to Firmicutes phylum. Firmicutes roles: 1. Helps metabolize nutrients (carbs, lipid, protein). a. Maintains pH of env. 2. Helps make vitamins + metabolites for normal fcn 3. Prevents binding/spreading of putrefactive bacteria a. Prevents other bacteria from becoming dominant 4. Immune system: stims humoral + cellular immunity. a. Activates CD4, CD8 (CD-cluster differentiation) cells + B cells Enterococci part of normal flora: but can go rogue + turn pathogenic. Pathogenesis Doesn’t produce significant toxins: has 2 main virulence factors. 1. Cell-surface proteins: a. Extracellular surface protein (Esp) b. Aggregation (Agg) substances: Asa1, Asp1, Asc10. c. MSCRAMS (Acm/Ace/Scm) adhesions. d. Pili: Ebp, Bee. 2. Externally secreted factors: a. Cytolysin/hemolysin (Cyl). b. Gelatinase (GeIE). c. Serine protease (SprE). d. Secreted antigen A (SagA). e. Hyaluronidase (Hyl): only E. faecium belonging to CC17. Virulence Factors 1. Surface adhesins aggregation substance: hair-like protein (pilli) in cytoplasmic mem. a. Helps binding to host cell + cell-to-cell contact b/w donor + recipients for conjugation. b. Helps entero bind to host or other entero/bacteria to transfer genetic info. 2. Enterococcal surface protein (Esp): discovered in E. faecalis for adherence to bladder. a. Produces biofilm + causes nosocomial infections related to medical devices. b. Faecalis: a main UTI cause, but immobile.  i. Can stick to foreign objects (e.g. medical devices) + form biofilm. ii. Biofilm protects it + allows it to grow. 3. Cytolysin: hemolytic protein in virulent strains. a. Breaks down blood cells, inhibits growth of other gram (+) bacteria. b. Lyses macrophage + neutrophils: protects against immune system. i. Also gains nutrients from these cells. 4. Gelatinase: hydrolyzes gelatin, collagen, Hb, etc. a. Damages tissue: allows bacteria to grow. b. Assoc w/enhanced virulence for endocarditis. 5. Extracellular superoxide: purpose unknown, potentially helps lyse RBCs. 6. Antibiotic resistance (multiple plasmid + chromosome genes). a. Resistance factors in entero are intrinsic, but can also be transferred. b. Transferred via: conjugation, transposons, bacteriophages. c. Makes them resistant to aminoglycoside, B-lactams, vancomycin. Normally: enterococci not very virulent. Properties of enterococci: 1. Can survive in hostile envs (bile, antimicrobial rich milieu, etc.). 2. Able to evade immune system. 3. Can attach to human host cells. 4. Has ECM, can form biofilms. 5. MSCRAMMS (microbial surface components recognizing adhesive matrix molecules). a. Surface elements that help entero adhere to host tissues. b. Similar to staphylococcal MSCRAMMS. Opportunistic Pathogen Enterococci are opportunistic pathogens: normally in check, but turns opportunistic in right env. Can spread from normal flora to other body sites. ○ GIT has defense mechs to keep entero in place: 1. Intestinal associated lymphoid tissue: immune organ. a. Responsible for sIgA (secreting IgA). b. IgA prevents microorganism adhesion to epithelium. 2. Enterocytes: forms strong cell junctions, hard for organism to pass thru. If these defenses weakened: bacteria can pass thru intestinal barrier + into blood/other sites. ○ E.g. heart valves. Enterococci not normally in urinary tract: how does it get from perineum to urethra? Usually caused by presence/prior presence of catheter: entero can attach to catheter. ○ Causes translocation to urinary tract + activation of virulence factors (e.g. biofilm). Can also translocate from GIT. Kidney/bladder stones: can adhere to stones + translocate. Men: prostate colonization can cause enterococcal urinary infections. ○ Esp if they have prostate stones.  Enterococci can spread within diff envs: 1. Hospital/health care env: can adhere to inanimate objects (e.g. rails, tables). 2. Fecal matter from other species: e.g. dogs, can cause fecal-oral transfer to human. 3. Contaminated plant/food: also a key transfer site for resistant strains from env to human. One health: antimicrobial isn’t just a human issue, it’s a one health problem. Affects agriculture, animals, etc. Antimicrobial use in food can create resistant strains that infect humans. Enterococci Infection Sites UTI Dysuria + pyuria: usually nosocomial, in pts w/catheters Bacteremia Caused by entero entering blood from UTI/abdominal abscess, increases endocarditis risk Endocarditis Heart endothelium/valve infection, occurs from infection sources (e.g. GIT, GUT, wounds) Meningitis Rare, mostly in neonates ([high] in perineum, neonate can ingest during delivery) Wound Infections Largely caused by surgery, decubitus ulcers, burns Peritonitis Intra-abdominal/pelvic infections, abd swelling, etc. Enterococcus Resistance Enterococcus resistance: can be intrinsic or acquired. Intrinsic Resistance Bacteria is inherently able to survive against antimicrobials: 1. Low-affinity penicillin binding proteins (PBPs): called PBP4 or PBP5. a. PBP: enzymes in cell wall that creates integrity of cell wall structure. b. PBP is B-lactams’ target: some gram (+) bacteria’s PBP has high affinity for B-lactam. c. Enterococcus: can have low-affinity PBP in E. faecalis (PBP4), E. faecium (PBP5). d. This feature removes one class of antibiotics as a potential treatment. 2. Cell envelope: causes poor penetration of drugs/other foreign material. a. Does anaerobic metabolism, prevents molecule transport across cytoplasmic mem. 3. Isa(A) gene: encodes proteins that moves some molecules out of cell once it enters cell. a. If antibiotic crosses mem + enters cytoplasm: proteins will push it out of cell. 4. Enterococcus can absorb folate from env: doesn’t synthesize folate itself. a. So can bypass antibiotics whose MOA affects folate synthesis. These features are seen in E. faecalis + E. faecium. Acquired Resistance Has genome plasticity: acquires foreign DNA from conjugation + phage-mediated transduction. Transduction occurs less than conjugation. Enterococcus has 4 ways to share genetic material: e.g. conjugation. Conjugation: genetic material transfer from donor to recipient, e.g. conjugated plasmids. Conjugated plasmids: encodes proteins needed for plasmid transfer during conjugation. ○ Allows bits of DNA to be shared b/w bacteria. ○ Has broad host range: can transfer genetic material b/w many bacterial species. ○ E.g. enterococci, staphylococci, streptococci. Vancomycin Resistance Enterococci (VRE) Can have glycopeptide (vancomycin) resistance. Caused by acquisition of operons that affect nature of peptidoglycan precursors. ○ Crosslinking peptidoglycan chains = final steps of creating cell wall infrastructure. ○ B-lactams interfere w/crosslinking by binding to proteins. Binds to D-Ala-D-Ala terminal peptide chain. Operon substitutes terminal D-alanine for D-lactate. ○ So: drug doesn’t bind as effectively anymore. Most common operons: vanA or vanB. Operon: replaces pentapeptide terminus D-Ala-D-Ala w/D-Ala-D-Lac. Operon is carried on a transposon. ○ Transposon located on chromosome or transferable plasmids. vanA + vanB found in E. faecalis + E. faecium. ○ More prominent in E. faecium: bec entero overgrowth causes faecium overgrowth,. vanC operons: intrinsic to E. gallinarium + E. casseliflavus. ○ Produces peptidoglycan precursors that end in D-Ala-D-Ser. ○ These operons causes lower glycopeptide resistance than vanA/B. Due to genome plasticity: operons can be transferred to other species + cause VRSA. VRE in Canada: 30% of healthcare associated enterococci infections are VRE. Most infections are E. faecium. Lab Diagnosis Specimen collection, microscopy, culture, supplementary tests, confirmation. Specimen Collection What specimens you collect depends on the clinical scenario. Specimens you can collect: 1. Urine 2. Blood cultures 3. Intra-abdominal/abscess/intra-OR 4. CSF Microscopy First piece of info you get is Gram stain. Other info you can get from microscopy: 1. Morphology: entero = coccus, chains.  2. Quantity: few, many, heavy. a. Quantity of organisms in specimen can be a marker of bacterial burden. 3. Quality of specimen: good or bad quality. a. Quality = how many epithelial/contaminated cells you have in specimen. b. E.g. are there many contaminant cells in specimen? Culture Specimen is cultured to get growth: allows for further identification. Usually on blood agar: distinct colonies appear ~24 hrs later. Entero appearance: wet-looking colonies. ○ But: can’t say for sure if it’s entero + what species it is, so MALDI-TOF to confirm. Confirmation MALDI-TOF MS: done using culture or from smear of the specimen. Provides result within min: a game changer for clinical care. Doesn’t provide susceptibility results. ○ I.e. doesn’t tell us what drug to use for treatment. Susceptibility Testing Diff methods to test bacteria’s susceptibility to diff drugs. Method Description Examples Phenotypic Method Add drug to bacteria: see if susceptible or not. Broth dilution test: 48h Reqs bacterial growth, takes at least 48h for results. Disk diffusion: 48h Constrained by bacterial growth time. Gradient test: 48h Can be automated w/predetermined bug-drug combos. Chromogenic media: 24h Automated devices: > 20h Molecular Based Directly detects specific resistance genes + mutations. PCR, qPCR: several hours Is an alternative or complementary method to phenotypic. Cycle sequencing: 24-48h Faster: 1 to few hrs. NGS: several days More $$$: reserved for finding specific resistance genes. ○ Can also be used for difficult to grow organisms. Mass Spectrometry MALDI-TOF MS: takes several hours If bacteria is confirmed enterococcus: must rule out VRE. Screen for VRE w/agar screening plates: vancomycin incorporated into agar. Other alternatives: e-test methods, immunoassays, PCR, disk diffusion. Detection of low-level resistance has improved. ○ Used to be problematic for some automated systems.  Case 83 y/o fem, has fatigue, weakness, anorexia, falls, feverish. Hx: prosthetic mitral valve. Cultures sent: blood + urine. ○ Blood: must rule out bacteremia (elder w/prosthetic + fever). Has higher risk of infection at prosthetic site (prosthetic valve endocarditis). ○ Urine: elderly fem, can have atypical features of UTI. Blood culture results: GPC in chains in 2 of 2 blood cultures. Blood cultures are sent in sets: 1 set = 2 bottles of blood. ○ 1 bottle: anaerobic medium. 1 bottle: aerobic medium. ○ 2 out of 2 = 2 out of 2 sets. ○ Send 2 sets bec there’s chance of blood contamination w/bacteria from skin. Also shows a constant state of bacteremia. Conclusion: has streptococci or enterococci in blood (bacteremia). Did MALDI-TOF MS: determined to be E. faecalis. Must do reporting: determine bacteria’s susceptibility to diff drugs. Report this in 2 ways: MIC or susceptible vs resistant. MIC: minimum inhibitory concentration. Lowest [ ] of antibacterial (ug/mL) that completely prevents visible growth of organism. ○ Done under strictly controlled in vitro conditions. Final step in micro lab analysis: determine which drugs the organism is susceptible to. ○ Define this w/MIC: use standard bug-drug combos + concentrations. Breakpoint: a value (zone of inhibition, MIC) that differentiates b/w resistant + susceptible. ○ Is a surrogate measure: reps whether you can use drug to treat infection. Possible methods to determine susceptibility: 1. Disk diffusion: a. Prepare disks w/standard [antibiotics] + prepare bacteria culture. b. Place disk on bacterium culture: then measure width of zone of inhibition. 2. E strips: strip contains gradient of [antibiotic] (top = [highest], bottom = [lowest]). a. Put strip in bacteria dish: measure zone of inhibition. b. See where zone crosses strip (this is MIC). 3. Standardized card: contains dilutions of diff [antibiotic]. a. Add bacteria: based on colour, tells you if bacteria is susceptible or not. Where did E. faecalis come from in pt: likely translocated from GIT to blood. Likely has endocarditis due to prosthetic valve: do further workup to rule this in/out. What if it was E. faecium: prob still came from GIT. ○ But: if it was a hospital-acquired infection, could have come from env. Lec 5: Fungi Introduction Fungi = eukaryotic: closer to animals than plants (e.g. crabs, lobsters). Cell wall: made of chitin (chitin also found in crabs/lobsters). Fungi = major env decomposers. Many things are made of/derived from fungi: 1. Plastic car parts, synthetic rubber, lego: made from itaconic acid. a. Itaconic acid: derived from Aspergillus terreus fungus. 2. 15% of vaccines + therapeutic proteins are made in yeast (e.g. Hep B vaccine). 3. Penicillin, statins. Largest living organism on Earth = fungus: honey mushroom (Oregon). ○ Fungi can infect ants (Ophiocordyceps unilateralis) + controls their brain. Morphology Cell mem: bilayer mem made of ergosterol. Cell wall: made of chitin + glucans (+ other proteins). Can have basic fungal cells or multicellular fungus. 1. Fungal cell: cell wall, cell mem, mitochondria, ribosomes, nucleus, cytoplasm, etc. 2. Multicellular fungus: a. Hyphae: long branching filaments, individual cells may be divided by septations. b. Mycelium/mycelia: collection of hyphae filaments. c. Pseudohyphae: long branching structures, in b/w budding cells + true hyphae. i. Have cytoplasmic connections b/w individual cells. d. Conidia: reproductive spore, at tip of hyphae. e. Spores: fungal seeds, produced from hyphae. Pathogenesis Most fungi are endogenous to humans: present in normal microbiome. E.g. GIT, UT, resp tract. ○ Immune system usually capable of protecting us from fungi. Most fungal infections are opportunistic: fungus overcomes host immune defenses. Ways that fungi overcome host immune defenses: 1. Fungal overgrowth (e.g. antibiotic use + candida): antibiotics can elim healthy bacteria. a. This bacteria may be used to prevent overgrowth of fungi. b. So: fungal overgrowth + infection. 2. Compromise in immune system: either first, second, third line of defense. a. First line: breach in mucosal barriers + skin (e.g. Blastomyces, candida). i. Blastomyces: can enter open wounds/skin pricks + cause skin infection. ii. Candida: antibiotics/chemo destroy oral mucosal barriers = oral candidiasis. b. Second line: affected neutrophils (e.g. mold) = neutropenia. i. Neutropenia causes: meds, hematological deficiency (leukemia), autoimmunity. ii. Neutrophils: contains fungi + prevents infections, so can cause mold infections.  c. Third line: cell mediated immunity (T + B cells). i. Abnormality in this line = fungal infections (e.g. cryptococcus). 3. High inoculum load that overcomes immune defenses: e.g. inhaling spores, droppings. 4. Endemic fungi: fungi that isn’t endogenous to humans. a. Get thru respiratory acquisition: e.g. inhaling contaminated soil. Antifungal Targets 1. Ergosterol: in fungal cell mem. a. Azoles: inhibit fungal cytochrome P450 3A-dependent C 14-demethylase. i. This enzyme converts lanosterol to ergosterol. b. Amphotericin: forms complex w/ergosterol, pokes holes in cell mem. c. Result: rapidly deteriorates fungal cel. 2. 1,3 D-glucan: in fungal cell walls. a. Echinocandins: inhibit glucan synthesis, helps destroy fungal cell integrity. b. 1,3 D-glucan not present in mucor spp: so won’t be effective. i. Some fungi resistance to antifungals if fungi doesn’t have antifungal’s target. Fungi Classification 3 morphological types: yeast, dimorphic fungi, mold. Yeast Unicellular: some (candida) form pseudohyphae. But: yeast usually refers to single fungal cell. Candida, cryptococcus, saccharomyces, trichosporon Dimorphic Fungi Exists as yeast + mold: depends on env factors. Sporothrix, histoplasma, blastomyces, coccidioides, paracoccidioides, penicillium Mold Multicellular. Aspergillus, rhizomucor, fusarium, microsporium, trichophyton, scedosporium. Yeast Yeast = candida spp + cryptococcus spp. Case: 31 y/o fem, has signs of UTI. Given 5 days of TMP-SMX. 2 days later: urinary symptoms gone, but has oral burning + white lesions on buccal mucosa + tongue. This is a case of candida mucositis. Candida spp. Epidemiology Unicellular fungi: endosymbionts in human (GI + GU tracts). Most common cause of fungal infections: often caused by antibiotics. ○ Antibiotics promote overgrowth by inhibiting advantageous bacteria. ○ This bacteria usually keeps candida growth in check. 200 species of candida: only some cause infections. ○ Candida albicans: dimorphic fungus.  Notable species: 1. C. albicans: most common infection. 2. C. krusei + glabrata: azole resistant, but dose dependent. a. High enough azole dose may overcome resistance. 3. C. parapsilosis, guilliermondii: echinocandin resistant. 4. C. lusitaniae: amphotericin resistant. 5. C. auris: can be pan resistant (causes outbreaks in HC settings, LTC, etc.). a. Not susceptible to any agents. Disease Usually opportunistic infections (e.g. antibiotic use). Manifestations: oral thrush, esophageal candidiasis, vulvovaginitis, rarely UTIs. ○ Esophageal candidiasis: esophageal burning, dysphagia, etc. ○ UTIs: usually caused by indwelling catheter. Can cause invasive infections in immunocompromised pts + stem cell transplant recipients: 1. Bloodstream infections, endocarditis. 2. Hepatosplenic candidiasis: often seen in ppl undergoing leukemia chemo. a. These pts usually have catheters + candidiasis colonization. 3. Catheter associated infections: e.g. catheters for dialysis, bladder, etc. a. Risk of candida infection bec candida forms biofilms on foreign objects. Diagnosis Gram stain: gram (+) oval budding yeast cells w/pseudohyphae. Silver stains, KOH stains: helps visualize fungi under microscope. Specimen + blood cultures: gold standard. Immunodiagnosis: Ab/AB detection against cell-wall mannan. ○ Usually not req since you’re performing the other tests. Management Management depends on fungal infection type. Azoles: fluconazole first line if fungus is susceptible to it. ○ If DDR: higher doses of other azoles used. Echinocandins: first choice for invasive infections until susceptibility returns. Dual therapy w/azoles + echinocandins: for endocarditis. Amphotericin: reserved for invasive infections unresponsive to first-line therapy. Remove catheter/hardware: bec candida forms biofilms. Cryptococcus spp. Epidemiology 2 major pathogenic species in cryptococcus: cryptococcus neoformans, cryptococcus gattii. C. neoformans: found in pigeon guano (droppings) + rotting trees. C. gattii: found in eucalyptus + coniferous trees (western CAN). Can colonize humans w/o causing disease: causes more disease in immunodeficient. 1 million cases of cryptococcosis/yr: 600k+ deaths annually.  ○ Reqs a low threshold for diagnosis + treatment. Disease Disease usually caused by deficiency in third line of immune defense. Risk factors: AIDS, corticosteroid Tx, transplant, cancer, sarcoidosis, lymphopenia, cirrhosis. ○ Transplantation: most meds that prevent organ rejection suppress T cells. ○ Cancer: any cancer that affects T + B cells. ○ Sarcoidosis: can be caused by steroid use. Major sites: CNS (meningoencephalitis, sometimes cryptococcoma), lungs. ○ Cryptococcoma: ringed lesions, caused by concentrated fungal infection in CNS. ○ Lungs: cavitary, nodular, interstitial pneumonia. Other sites: skin, prostate, peritoneum, eye. ○ Disseminated cryptococcus assoc w/flesh coloured papular lesions. People w/underlying liver disease/cirrhosis may have invasive cryptococcus. Immune cells in liver contain cryptococcus. ○ If liver non-functioning: causes invasive infections. Diagnosis Serum cryptococcus Ag (blood test): sensitivity = 99%, specificity = 94%. ○ Higher specificity if detecting thru CSF sample. CSF cryptococcus Ag: sensitivity = 99%, specificity = 99%. ○ Used for things like cryptococcus meningoencephalitis. Culture of body site: CSF, blood, pulmonary lavage. Direct microscopy: use India ink + histopathological stains (mucicarmine). ○ Characteristic feature of cryptococcus: thick capsule/wall. Management 1. Amphotericin + 5FC. 2. Then fluconazole. 3. Usually need lifelong suppression if pt has ongoing immunosuppression. Dimorphic Fungi Exists in 2 forms: yeast or mold. Yeast in heat (inside host), mold in cold (in outside env). ○ Mold: multicellular form better for colonizing surfaces + survival. Use of spores also helps propagation of fungi. ○ Yeast: adapts to host defenses (unicellular, small enough to evade defenses). Also small enough to reproduce rapidly. Transition b/w mold + fungi: mediated thru signals. Candida albicans: considered a dimorphic fungus as well. ○ Has pseudohyphae in heat (inside hosts/animals). Dimorphic fungi = endemic mycoses: not endogenous in humans (except C. albicans). ○ Usually acquired thru spore inhalation.  ○ Can cause asymptomatic disease: more severe if immunodeficient. Sporothrix spp (schenckii) Epidemiology Worldwide distribution: acquisition assoc w/exposure to soil, plants, plant products (hay, straw). May be transmitted w/cat scratches or bites. Disease Causes sporotrichosis (rose handler’s disease). 1. Inoculation occurs thru puncture wound/cut → 2. Formation of ulcerated nodule at site of injury → 3. Fungi enters wound + spread thru lymphatics. a. Causes appearance of similar nodules along lymphatics. May be disseminated in immunocompromised hosts. Diagnosis 1. Direct visualization: swab, culture it in Sabouraud agar or brain heart infusion culture. 2. Molecular techniques: rRNA amplification is being studied. a. But: usually only need low threshold + direct visualization under microscope. Management Easy to manage: Oral saturated potassium iodide (used historically). Itraconazole or terbinafine: for local infections. Amphotericin: for disseminated infection. Histoplasma spp (capsulatum) Epidemiology Distributed worldwide except Antarctica. Most prevalent: Ohio, Mississippi river valleys, St. Lawrence Valley, Mt. Royal, St. Thomas, London, Manitoba, Quebec, Nova Scotia. Present in: bird droppings, bat droppings, soil. ○ Common ways to get infected: spelunking (cave exploring), cleaning bird coops. ○ Clean bird coops = inhaling bird droppings. Exists as mold (outside env) + yeast (inside hosts). Mold Yeast Individual short stalks. Narrow-budding yeast, formed in infected tissue. Readily airborne/spores released when colony is disturbed. Small (2-4 um), in clusters within phagocytic cells. ○ E.g. histiocytes, macrophages, monocytes.  Disease Can cause many types of disease: 1. Localized pulmonary infection: asymptomatic/mild pneumonia, symptomatic pneumonia. a. Asymptomatic infection/mild pneumonia: immunocompetent hots. b. Symptomatic pneumonia: lesions calcify + form granulomas (mimics TB). i. Found in spleen + lungs. ii. In immunodeficient hosts or high inoculum. 2. Disseminated infection: immunocompromised host, can infect any organ (liver, spleen, lungs). a. Subacute fever, pancytopenia, hypoadrenalism, mucosal lesion, lung lesions. 3. Oral ulcers. 4. Granulomatous mediastinitis: rare, but affects lymph nodes. a. Causes granulomas + chronic infection. Diagnosis 1. Direct visualization w/GMS stain: narrow budding yeast. a. Features: no capsule, clusters inside phagocytic cells (e.g. histiocytes). 2. Histoplasma antigen assay: cross-reacts w/blastomyces Ag (antigen). 3. Antibody (Ab) test: non-specific, doesn’t show if pt has current infection. a. Or if Abs are from previous infection. 4. Specimen culture: blood, BAL, tissue, etc. Management Itraconazole, amphotericin (for systemic, disseminated infection). Blastomyces spp (dermatitidis) Epidemiology Soil, decaying wood. Locations: Mississippi river Valley (extends North around Great Lakes). ○ North Ontario, SE Manitoba, southern Quebec, Asia, Africa. Mold Yeast Network of mycelium: penetrates substratum where it grows on. Larger budding yeast: in infected tissue/bodily fluids. After 3-5 days: asexually reproduce w/small (2-10 um) conidia. 8-15 um, buds thru a broad base/neck. ○ Conidia: asexual spores, main infectious particles. ○ Highly recognizable. ○ Can inhale conidia. Disease 1. Blastomycosis: Chicago Disease. 2. Localized cutaneous infections: at site of inoculation/lesion. 3. Pneumonia (asymptomatic or symptomatic): can mimic cancer.  4. Disseminated infection: in immunocompromised pt, affects any body site. a. E.g. prostatitis, arthritis. 5. CNS infection. Diagnosis 1. Direct visualization w/GMS or PMS stains: shows broad based budding yeast. 2. Blastomyces Ag assay: cross-reacts w/histoplasma. 3. Ab testing: non-specific (doesn’t say if it’s active or previous infection). 4. Specimen culture. Management Itraconazole, amphotericin B (disseminated infection), voriconazole or fluconazole (CNS infection). Coccidioides spp (immitis) Epidemiology Dry, hot, desert areas: SW US + Mexico (moving north). In sandy soils: most prevalent in Arizona. Infection usually occurs in dry seasons. Mold Yeast Mycelial cells (arthroconidia): 2-5 um. Spherules divide internally by developing internal septae. Can reach terminal bronchioles when inhaled. Septae divide spherule into compartments. Inside lung: arthroconidia remodels to spherical forms (spherule). ○ Changes from rectangular form. Disease 1. Coccidioidomycosis: Valley Fever. 2. Pneumonia: most asymptomatic, can be symptomatic. 3. Disseminated infection: immunocompromised host. a. Affects any organ system: e.g. bones, CNS. 4. Erythema nodosum: fleshly nodular lesions on skin, can accompany infection. a. Is immune response to infection, not infection itself. Diagnosis 1. Direct visualization w/GMS stain: shows spherules within tissues. 2. Serological tests: immunodiffusion, EIA to IgG + IgM (may be falsely positive). 3. Specimen cultures: gold standard. Management Fluconazole, itraconazole, amphotericin (invasive infections).  Mold Aspergillus spp, rhizomucor spp, fusarium spp. 55 y/o male, 10 days after chemo, developed fever, no resolution after antibiotics. CT thorax: nodular opacities w/halo signs. BAL cultures obtained + microscopy done. Common properties: Contains hyphae, reproduces thru spores. Few molds grow below 4℃ (fridge temp). Penicillium discovered in 1928 (Fleming): produced Penicillin antibiotic. Most molds are harmless colonizers: can cause disseminated infection in immunocompromised. ○ Disseminating infection: necrotizing, angioinvasive. Aspergillus spp Epidemiology Name derived from aspergillum: ubiquitous in nature. Asymptomatic colonization in GI + resp tract. Has septate hyphae. Disease 1. Usually respiratory infection: ABPA + aspergilloma local, can be asymptomatic. a. Allergic bronchopulmonary aspergillosis (ABPA): i. Asthmatics, elevated IgE + IgE aspergillus Ab. ii. Determination: eosinophilia, skin test. b. Aspergilloma: fungal ball, in asthmatics + diabetics. c. Angio-invasive aspergillosis: disseminated necrotizing pulmonary infection. i. Immune deficiency. 2. Aflatoxin consumption: can cause liver damage + cancer if consume A. flavus. Neutrophils: most important line of defense for containing aspergillus. Neutropenic (esp longterm) ppl will have more invasive infections. Diagnosis 1. Direct visualization w/GMS: branching hyphae @ acute angles. 2. Biomarkers: galactomannan (taken from BAL, common test for immunocompromised). 3. Aspergillus PCR: not readily available. 4. Specimen culture (tissue biopsy, BAL): BAL cultures doesn’t always indicate disease. a. Can indicate colonization, but not acc infection. 5. Blood cultures: usually contamination, aspergillus doesn’t grow in blood (throw away).  Management Voriconazole: first line Isavuconazole/posaconazole Amphotericin: disseminated infection High dose echinocandins: if unresponsive to first line Rhizomucor spp Rhizopus, rhizomucor, mucor. Epidemiology “Black fungus”: all 3 types cause similar syndrome + ubiquitous in env. Inhaled: can cause asymptomatic colonization in resp + GIT. Contains aseptate hyphae: larger, wider hyphae than other species. Disease Mucormycosis in immunocompromised, DKA, iron overload, steroid use. Usually present in normal microbiome. Characteristics: 1. Sinusitis, ophthalmitis, periorbital mucormycosis 2. Cavitary pneumonia 3. Disseminated disease, > 50% mortality Diagnosis Requires high index of suspicion. 1. Direct visualization w/PAS + GMS: broad based, ribbon like hyphae w/right angle branching. 2. Cultures: have poor sensitivity, but can grow. 3. Histopathology: gold standard, see direct tissue invasions from samples. Management Amphotericin B (first line), oral posaconazole or isavuconazole (when pt stabilized). Surgical management important to decrease fungal burden. Fusarium spp Ubiquitous in nature, can be biological weapon (1930s, alimentary toxic aleukia, > 60% mortality). Disease: causes opportunistic infection in immunocompromised. ○ Necrotizing pneumonia, sinusitis, skin lesions (necrotic centre, erythema halo). ○ Localized infection: keratitis (contaminated lens solution), local skin nodule. ○ Has characteristic skin rash. Diagnosis: specimen culture, blood cultures may be positive. ○ If blood culture isolates mold + matches fusarium presentation: likely fusarium. Treatment: amphotericin + voriconazole combo (severe disease). ○ Can step down to voriconazole monotherapy.  Superficial Fungal Infections Malassezia furfur: chronic, superficial infection. Involves stratum corneum (outer part of epidermis). Causes tinea versicolor. Characteristics: painless, hypo or hyperpigmented patches. Dermatophytes > 30 species of dermatophytes: causes chronic stratum corneum infection. Body Inguinal folds Feet Scalp Nails Ringworms Jock itch Athlete’s foot Tinea capitis Onychomycosis (tinea unguium) Treatment: ketoconazole, systemic antifungals are rare (for severe, refractory infections). Summary: Notable Yeast Notable Dimorphic Fungi Notable Mold Candida: commensal organisms. Endemic mycoses. Aspergillus, rhizomucor, fusarium. Localized infections > disseminated Histoplasma, blastomyces, Angioinvasive infection. coccidioides. Cryptococcus: not endogenous, opportunistic ○ Resp acquisition. infection. Asymptomatic > disseminated Meningitis (AIDS, cirrhosis) Treatment: highest to lowest severity. 1. Molds: a. Rhizomucor: posaconazole, isavuconazole. b. Aspergillus: voriconazole. 2. Dimorphic fungi: itraconazole. 3. Yeast: fluconazole. Superficial Mycoses Dermatophytes: derma = skin, phyton = plant. 3 genera: trichophyton, epidermophyton, microsporum. Other superficial mycoses: malassezia furfur, candida spp. Dermatophytes: free living in env. Made of: septate, hyaline, filamentous molds. ○ Mainly made of mycelium (hyphae cluster). Mycelium: site of nutrient absorption + spore creation. ○ Conidia (spores): single nucleus. Hyphae: multinucleated cells. 3 types of asexual spores: macro, micro, arthroconidia. ○ Arthroconidia: infectious fragment of hyphae.  Pathogenesis 1. Spores contact unhealthy tissue. a. Unhealthy: young, nutritional deficiency, immunosuppression, trauma, ↑ temp/humidity. 2. Arthroconidia adhere to keratinized tissue: skin, palm, foot sole, masticatory mucosa, hair. 3. After adherence: germination occurs. a. Germ tubes appear + penetrate first layer of epidermis + stratum corneum. 4. Organism degrades keratin: uses keratin for nutrients. 5. Fungal hyphae proliferate: produces more arthroconidia. Transmission: direct transmission from infected animal or contact w/viable conidia in env. Dermatophytes are not part of normal skin flora. Env: animal shelter, swimming pool/change room, nail salon, wrestling mats. Arthroconidia: viable in env for long time (4.5 yrs). Diff types of dermatophytes: some prefer humans, animals, etc. ○ Anthropophilic dermatophyte: preferentially infects humans, 30+ species. Anthrophiles: some are zoophiles. Anthrophiles adapted to human host, developed preference for specific locations on body. ○ E.g. tinea pedis: foot infection (Trichophyton rubrum = most common dermatophyte). Common species: T. rubrum, T. interdigitale, T. tonsurans, microsporum canis (z), epidermophyton floccosum, T. tinea. ○ (z): zoophilic. T. barbae T. capitis T. faciei T. corporis T. cruris T. manuum T. unguium T. pedis Beard Scalp/hair Face Body Groin Hands Nail Foot Diagnosis 1. Specimen collection: appropriate sample collection is important. a. Well-defined skin lesion: take sample from near the edge. i. This is the most active viable fungal cells. b. Poorly defined edges/lesion: scrape sample that covers representative area of infection. 2. Direct examination: hand-held magnifying tool (dermoscopy). 3. Microscopy + histopathology: a. KOH: doesn’t speciate + differentiate b/w dead or alive cells. b. Hair samples: look if it’s ectothrix or endothrix. i. Ectothrix: hyphae + conidia doesn’t invade hair shaft. ii. Endothrix: hyphae + conidia invades hair shaft. iii. Hyphae structure can help differentiate species. c. Histopathology: rare for superficial infections, done for deeper tissue infection. i. Periodic acid Schiff (PAS), Gomori’s modification of methenamine silver (GMS) ii. Calcofluor white stain. 4. Fungal culture: gold standard. a. Dermatophyte test medium (DTM): phenol red, changes colour when pH increases. i