Anaerobic Bacteria PDF

Anaerobic Bacteria Definitions Anaerobic bacteria organisms do not need O2 to grow vary in ability to tolerate oxygen Obligate (strict) anaerobes require anaerobic conditions for growth Oxygen is toxic Killed in presence of oxygen Definitions Aerotolerant anaerobe...

Anaerobic Bacteria Definitions Anaerobic bacteria organisms do not need O2 to grow vary in ability to tolerate oxygen Obligate (strict) anaerobes require anaerobic conditions for growth Oxygen is toxic Killed in presence of oxygen Definitions Aerotolerant anaerobes Can grow in atmosphere with O2, but grow best in anaerobic environment Facultative anaerobes Do not require O2, but will use it if available Oxygen Reduction - O2 + e- ➔ O2 - + O2 + e ++2H ➔ H2O2 Superoxide anion and hydrogen peroxide are toxic Superoxide dismutase O 2- + H+ H 2O 2 + O 2 catalase H 2O 2 H2O + O2 Strict aerobes and facultative anaerobes have superoxide dismutase &/or catalase Oxidation-Reduction (Redox) Potential Anaerobic bacteria require low redox potential high redox environment contains oxygen Normal human tissue and aerobic culture media have high redox potential Reducing agents (thioglycollate, cysteine, dithiothreitol) included in anaerobic media Normal Habitat Soil, water, animals Human NF oral cavity, URT, intestinal tract, genitourinary tract, skin facultative organisms use up oxygen in protected areas reduce redox potential inactivate harmful oxygen molecules Diseases Exogenous infection organisms from outside body organisms, spores, or toxins enter body through ingestion or trauma examples: tetanus, botulism Endogenous infection host’s NF infections near mucosal surfaces bacteremia, abscesses, gas gangrene, pneumonia  Clues to Anaerobic Diseases Trauma (deep or puncture wounds) Animal or human bite Foul-smelling, gaseous discharge Necrotic tissue, vascular stasis Contains sulfur granules (actinomycosis) Black or fluorescent red color Previous therapy with aminoglycosides aminoglycosides are ineffective against anaerobes Failure to grow organism seen on gram stain Specimen Collection Appropriate specimens – best to aspirate with a needle and syringe Blood and sterile body fluids (CSF, bone marrow, cavity fluid) Urine when collected as a suprapubic aspirate Abscesses, ulcers, draining wounds Biopsy material Specimen Collection Inappropriate specimens – site containing a resident flora (oral, GI, GU) Superficial skin sites Voided or catheterized urine Expectorated sputum, throat or nasopharyngeal swabs, bronchial washings Vaginal, cervical and urethral swabs Stool or rectal swabs Transport and Processing Inoculate media and immediately place in anaerobic environment Limit exposure to room air Do not allow to dry out Do not refrigerate Minimize time at room temperature Transport and Processing PRAS media - anaerobic transport system Prereduced, anaerobically sterilized transport medium Agar transport medium (modified Cary-Blair or Amies) Rezasurin (oxygen indicator) Reducing substances (protect against oxygen and inhibit NF) Sterile container Culture Specimens for anaerobic culture should be cultured aerobically on BAP, CHOC, MAC and on anaerobic media to correlate aerotolerance Anaerobes require Vitamin K, hemin, and yeast extract Nonselective Media CDC or Anaerobic Blood Agar Plates (anaBAP) support growth of obligate and facultative anaerobes Anaerobic Broth, Thioglycollate or chopped (cooked) meat support all obligate and facultative anaerobes anaerobes grow toward bottom (facultative grow throughout) Anaerobic-BAP Thioglycollate Broth Selective Media Phenylethyl alcohol blood agar (anaPEA) Contains phenylethyl alcohol Supports GN and GP obligate anaerobes Supports GP facultative anaerobic Inhibits enteric GNR Colistin naladixic acid blood agar (anaCNA) Supports GN and GP obligate anaerobes Supports GP facultative anaerobic Inhibits enteric GNR Selective Media Bacteroides Bile-Esculin (BBE) agar Selective gentamicin and bile Differential esculin hydrolysis Supports growth of bile tolerant organisms like B. fragilis Brucella blood agar Supports facultative and obligate anaerobes Best for GN Other Selective Media Kanamycin-vancomycin laked blood (KVLB) agar selects for Bacteroides and Prevotella kanamycin inhibits most facultative GNR vancomycin inhibits most GP laked blood (hemolyzed) encourages Prevotella to produce brown-black pigments Other Selective Media Cycloserine-cefoxitin-fructose agar (CCFA) selective and differential for Clostridium difficile cycloserine & cefoxitin: antibiotics frucose: source of CHO pH indicator: neutral red (red in acid and yellow in alkaline) C. difficile metabolize proteins ==> alkaline (yellow) Anaerobic Incubation 35 to 37°C for 48 hours Nitrogen gas (80-90%) Hydrogen gas (5-10%) removes oxygen ==> water Carbon dioxide (5-10%) some anaerobes are capnophilic Anaerobic Systems Anaerobic jars Anaerobic bags Anaerobic chambers Anaerobic Jars Jar technique (gas pak jar) Classic principle of anaerobic culture Catalyst - palladium pellets Envelope generates H2 and CO2 when water is added sealed, incubated @ 35°C Methylene blue or resazurin indicator is blue when oxidized, white when reduced (anaerobic) Anaerobic Bags commercially available hold 1-3 plates contains oxygen removal system indicator gas-impermeable plastic bag Anaerobic Chamber Optimal anaerobic incubation system Provides O2 free environment for inoculating, incubating and examining media Contains palladium catalyst desiccant indicator anaerobic gas mixture Anaerobic Chamber Culture Examination examined in chamber at any time bags and jars should be kept sealed for 48 hours oxygen exposure minimized after 48 hour incubation examined and processed as quickly as possible and returned to anaerobic atmosphere Anaerobic Identification Tests Presumptive ID Gram-stain is key identification test Use colony morphology Rapid tests  Anaerobes Gram Stain RXN Gram Stain Gram Negative Rod Gram Positive Cocci Bacteroides spp. Gram Positive rods Peptococcus Fusobacterium spp. Peptostreptococcus Prevotella spp. Porphyromonas spp. Non spore forming Spore forming Actinomyces Clostridium spp. Propionbacterium spp Bifidobacterium Rapid Identification Tests Catalase Aerotolerance Motility test Fluorescence Urease Disk tests Indole Lecithinase Esculin hydrolysis Naglar test Lipase Reverse CAMP Aerotolerance Determines if isolate is a strict anaerobe or a facultative anaerobe Incubate the suspected isolate in both aerobic and anaerobic environments anaBAP anaerobically CHOC aerobically in CO2 The plates are examined @ 48 hours for growth  Aerotolerance Growth only on anaBAP Strict anaerobe Growth on anaBAP > growth on CHOC aerotolerant anaerobes Growth on anaBAP and CHOC facultative anaerobes  Aerotolerance Anaerobic Aerobic Se, Staphylococcus epidermidis; Af, Alcaligenes faecalis; Cb, Clostridium butyricum; Lp, Lactobacillus plantarum; Ea, Enterococcus aerogenes; Pf, Pseudomonas fluorescens Fluorescence Fluoresce under ultraviolet light (366 nm) Red, orange, pink, chartreuse Antibiotic Disk Test Confirm gram stain Special potency disks Vancomycin, colistin, kanamycin Most GN are R to vancomycin Most GP are S to vancomycin and R to colistin Van S, Col R = Clostridia, Porphyromonas (Kan R) Van R = GN, not Porphyromonas Disk Tests Sodium polyanethol sulfonate (SPS) disk Identify ana GPC Peptostreptococcus are sensitive Nitrate disk Reduction of nitrate Bile disk Ability to grow in 20% bile Bile tolerant anaGNR = Bacteroides fragilis group  Lecithinase Egg Yolk Agar (EYA) Detects lecithinase and lipase activity Clostridium spp. ID Lecithinase cleaves lecithin in EYA Releases insoluble fat makes an opaque zone Positive reaction white opaque zone in agar surrounding growth Negative reaction no change in the agar surrounding growth Clostridium spp. Lecithinase positive negative  Nagler Test The Nagler reaction is used to detect the alpha toxin of C. perfringens on EYA Alpha toxin is a specific form of lecithinase produced by C. perfringens Antiserum to alpha toxin neutralizes the activity of the alpha toxin type of lecithinase by C. perfringens, but not that produced by other clostridia Nagler Test Lipase Lipase hydrolyzes triglycerides in EYA Produces glycerol + free fatty acids Positive reaction oil-on-water surface or multicolored sheen in colony Negative reaction no sheen observed normal colony morphology Clostridium spp. Lipase POS Reverse CAMP alpha toxin of C. perfringens works synergistically with beta hemolytic group of S. agalactiae → area of hemolysis test organism on anaBAP GBS perpendicular to test organism streak (close but not touching) arrowhead-shaped zone = positive Reverse CAMP S. agalactiae Clostridium perfringens Definitive Identification Tests Biochemical tests Commercial systems Gas-liquid chromatography Cellular fatty acid analysis 16S rRNA gene sequencing Biochemical and Commercial Systems PRAS or non-PRAS biochemical test media Commercial biochemical and preexisting bacterial enzymes minisystems Gas Liquid Chromatography Gas-liquid chromatography Analysis of cellular fatty acids or metabolic end products (volatile acids) Produce characteristic pattern Gene Sequencing 16S rRNA gene sequencing Ribosomal DNA extracted, amplified via PCR, sequenced Clostridium Species Catalase negative Motile (except C. perfringens) Anaerobic GPR Some gram variable or GN Susceptible to vancomycin Spore-forming appear as unstained refractile structures in gram stain oval to round terminal to subterminal Clostridium Species Some species are aerotolerant Clostridia cause exogenous infections Gain access to body via ingestion and wounds Tetanus, gas gangrene, botulism, food poisoning Produce potent toxins Genera Differences Test Clostridium Bacillus Lactobacillus Optimal Anaerobic Aerobic Varies Growth Conditions Sporulation Anaerobic Aerobic No spores conditions Catalase Negative Positive Negative Clostridium perfringens Most common Clostridium spp. Found in undercooked meat, soil, water GP boxcar-shaped rods Double zone of beta hemolysis on anaBAP Positive reverse CAMP, lecithnase, Nagler Spore seldom observed C. perfringens C. perfringens Clinical Significance Isolated from tissue infections and bacteremia Produces gas in infected tissue Myonecrosis (gas gangrene) Deep, penetrating wound or surgery Diabetic foot ulcers Myonecrosis C. perfringens Clinical Significance Food poisoning from meat or meat products (gravy) Ingested spores, germinate in intestines and produce an enterotoxin nausea vomiting, diarrhea, abdominal pain Clostridium difficile May be NF of stool Antibiotic-associated diarrhea and pseudomembranous colitis antibiotics upset intestinal ecosystem by killing indigenous NF resistant C. difficile increase in number All toxigenic strains produce enterotoxin (toxin A) and cytotoxin (toxin B) C. difficile C. difficile Characteristics Yellow ground glass colonies on cycloserine- cefoxitin-fructose agar (CCFA) Smells like horse manure Spore forming GPR Chartreuse fluorescence Can be NF, must test for toxin production  C. difficile Toxin Tests Cytotoxin test toxic effects of stool on human cells grown in culture more sensitive method to detect toxin, but it requires 2 – 3 days to get the result Detects toxin B C. difficile Toxin Tests Kit testing Determine if toxin A, toxin B or glutamate dehydrogenase (not a virulence factor) is present in stool Enzyme immunoassay Molecular testing Under development Clostridium botulinum Botulism Ingestion of botulinum toxin Home canned veggies, home cured meat Infant botulism associated with honey Neuormuscular toxins cause paralysis or death Foodborne botulism (ingested), wound botulism (inoculated), infant botulism (intestinal - infants lacking NF) C. botulinum C. botulinum Diagnosis Diagnosed clinically (patient hx and symptoms) Isolating organism or detecting toxin (reference lab) Clostridium tetani Soil and intestinal tract of animals Enters body through wound site Produces potent neurotoxin (tetanospasmin) Severe muscle spasm in unimmunized individuals Tetanus (lockjaw) C. tetani Diagnosed clinically Terminal spores Tennis Racquet Shaped C. septicum Aerotolerant Swarming colonies Sub-terminal spores Myonecrosis and bacteremia Associated with leukemia, lymphoma, large bowel carcinoma Other Species Cause bacteremia, intra-abdominal infections, wound infections, myonecrosis Non-Spore-Forming Anaerobic GPR Actinomyces spp. Bifidobacterium spp. Propionibacterium spp. Eggerthella and Eubacterium spp. All can cause actinomycosis Actinomyces Actinomyces spp. includes aerobic and anaerobic bacteria Nocardia is an aerobic actinomyces Causes mycetomas and nocardiosis A. israelii is the most common anaerobe Causes actinomycosis Inhabit human and animal mucosal surfaces Anaerobic actinomyces are not weakly acid fast like Nocardia spp. Actinomyces Gram Stain Aerobic and anaerobic Actinomyces spp. GPR irregularly stained (beaded appearance) coccoid to filamentous filaments branch  A. israelii Actinomycosis Actinomycosis Mainly A. israelii Usually in jaw Chronic, granulomatous infection Develop fistulae – drain pus with sulfur granules (colonies of bacteria) A. israelii Cultures Associated with “lumpy Jaw” Pus and sulfur granules grown on anaBAP Grow slowly, 7-9 days Molar-tooth colonies Anaerobic branching GPR Molar Tooth Colonies Bifidobacterium Actinomyces-like morphology Gram stain branched, or bifurcated GPR NF intestines and oral cavity Rarely cause diseases Can be the cause of actinomycosis Propionibacterium Anaerobic diphtheroid like GPR resembles corynebacteria NF skin Most common anaerobe isolated in lab contaminant in blood cultures and specimens collected by penetrating the skin P. acnes linked to acne Catalase and indole positive May cause SBE and bacteremia Eggerthella and Eubacterium spp. Eggerthella previously Eubacterium Anaerobic diphtheroid like GPR No branching Anaerobic GPC Peptostreptococcus GPC in chains P. anaerobius is susceptible to sodium polyanethol sulfonate (SPS) disk all other anaerobic cocci are resistant Peptococcus GPC in clusters Anaerobic GNR NF mucous membrane Bacteroides Prevotella Prophyromonas Fusobacterium Veillonella Bacteroides fragilis Group Anaerobic GNR NF of GI tract B. fragilis most common Intra abdominal infections, bacteremia, soft tissue infections B. thetaiotaomicron is second most common Bacteroides fragilis Group Bile resistant Resistant to kanamycin, vancomycin, and colistin disks Growth on KVLB agar Growth on Bacteroides Bile-Esculin (BBE) agar (growth stimulated by bile) Growth = bile tolerance Brown to black colonies = esculin hydrolysis B. fragilis on BBE Bacteroides ureolyticus Group Anaerobic GNR Bile sensitive and bile tolerant nonpigmented organisms Some pit the agar Growth in formate and fumerate Resistant to vancomycin Sensitive to kanamycin, colistin Prevotella Anaerobic GNR Bile susceptible Resistant to kanamycin & vancomycin Growth on KVLB agar but not on BBE Some produce protoporphyrin dark pigmented colonies Colonies fluoresce a “brick red” under UV light Prevotella spp. Prophyromonas Anaeroic GNR Require hemin and vitamin K Resistant to kanamycin & colistin Sensitive to bile & vancomycin Do not grow on KVLB Produce pigmented colonies and fluoresce brick red Fusobacterium Susceptible to kanamycin & colistin Vancomycin resistant Fluoresce chartreuse F. nucleatum – thin fusiform rods bread-crumb, speckled colonies F. necrophorum lipase positive Mobiluncus Associated with BV, PID, and abdominal infections Curved bacilli gram-variable Motile, catalase and indole negative Inhibited by vancomycin  Selective Anaerobic GNR ID Kanamycin/Colistin sensitive resistant Fusobacterium Vancomycin B. ureolyticus (pits agar) Sensitive resistant Pigmented Porphryomonas bile sensitive resistant Red pigmented Prevotella B. fragilis  Selective Anaerobic GNR ID Kanamycin S R bile vancomycin S R S R formate-fumarate Catalase pigmented bile nitrate nitrate Porphyromonas + negative + - S R B. ureolyticus Bilophila F. mortiferum lipase pigmented B. fragilis (red) + - + - F. necrophorum F. nucleatum pigmented nonpigmented Prevotella Prevotella Veillonella Only commonly encountered anaerobic GNC Sensitive to kanamycin & colistin Resistant to vancomycin Smallest gram-negative cocci  Gram Positive Rods Non-Spore-Forming GPR Non-Spore-Forming Branching GPR Spore-Forming Non-Branching GPR Non-Spore-Forming GPR Corynebacterium and Coryneforms Listeria Erysipelothrix Acranobacterium Lactobacillus Gardnerella Corynebacterium C. diphtheriae C. jeikeium C. urealyticum C. pseudodiphtheriticum C. striatum C. xerosis Corynebacterium Normal skin and mucous membrane flora Called diphtheroids or coryneforms “club shaped” GPR, pleomorphic, non-spore forming Gram stain looks like “Chinese letters” (V, L, Y formation) or palisades (line up side by side) Corynebacterium Facultative anaerobic Small gamma colonies on SBA “Chinese letters” Catalase positive Nonmotile Frequent contaminants  C. diphtheriae Significant pathogen Virulence factor is Diphtheria toxin In strains infected with bacteriophage carrying tox gene Exotoxin blocks protein synthesis Destroys host cells May be absorbed heart, nervous system damage Lethal at 130 ng/kg of body weight 150 lb person = 0.3 ounces C. diphtheriae Two forms of disease Cutaneous Non-healing ulcer Respiratory Diphtheria Diphtheria URT infection Tonsils, pharynx Bacteria multiply, release toxin Tissue necrosis Exudate Inflammation → Pseudomembrane Necrotic epthithelial cells, WBCs, fibrin, bacteria Suffocation C. diphtheriae Treated with antitoxin Prevented by immunization (DIP/TET) C. diphtheriae Lab Diagnosis Pleomorphic GPR Darker staining area of cell Metachromatic granules Methylene blue stain pockets of inorganic phosphates or nutrient reserves C. diphtheriae Media SBA – small zone of beta hemolysis C. diphtheriae Media Serum or blood containing media Loeffler medium enhances development of metachromatic granules Pai agar egg-based agar C. diphtheriae Media Tinsdale agar (cystine-tellurite blood agar) Selective Potassium tellurite inhibits non-coryneform bacteria Differential Tellurite reduction produces brown or black colonies Cystinase activity = halo around colony C. diphtheriae, C. ulcerans, C. pseudotuberculosis WARNING: Staphylococcus can produce brown colonies too! C. diphtheriae Media  Elek Test Immunodiffusion test for toxin production Strip of filter paper saturated with diphtheria anti- toxin imbedded in agar medium Isolate, positive, and negative control streaked on agar parallel to each other and perpendicular to filter paper Incubate (1-2 days) Line of precipitate forms in agar when toxin interacts with anti- toxin (arc of identity) Corynebacterium Spp. Normal skin flora most isolates are considered contaminants C. jeikeium – catheters or prosthetic devices, diptheropid prosthetic valve endocarditis C. urealyticum Urinary pathogen Other Non-spore-forming GPR Rothia dentocariosa oral NF endocarditis and wound infections filamentous or coryneform Undesignated CDC Coryneform Groups Opportunistic or nosocomial infections Listeria monocytogenes Vaginal and intestinal NF in humans Clinical infections due to ingestion of contaminated food Virulence factors Hemolysin Listeriolysin O Others Listeria monocytogenes Listeriosis (sepsis) Neonates Fatality rate ~ 50% Pregnant women Can cause spontaneous abortion and still birth Immunocompromised Predilection for CNS L. monocytogenes Cultures Specimens CSF, blood, amniotic fluid Facultative anaerobe Grow on routine media (BAP, CHOC) Prefers increased CO2 Colonies resemble S. agalactiae Cold enrichment Growth at 4 °C L. monocytogenes ID L. monocytogenes on SBA GPR Non-spore forming Small beta colonies on BAP sometimes hemolysis is hidden under colonies L. monocytogenes ID Catalase positive Differentiates it from streptococci Bile-esculin positive Motile at 25 °C  L. monocytogenes Motility Tumbling motility at 25°C, not at 35°C L. monocytogenes Motility One tube incubated at 35°C, other at RT Tumbling motility on broth wet mount in broth incubated at 25°C Umbrella pattern at 25°C, not at 35°C Semisolid Agar Motility umbrella-like growth pattern Positive CAMP test distinguishes L. monocytogenes from other Listeria spp. Block vs. arrowhead http://www.youtube.com/watch?v=fjD_ruKmSfA&NR=1 L. monocytogenes CAMP Differentiation of Listeria Esculin β- 6.5% Catalase hydrolysis Motility hemolysis NaCL L. monocytogenes + + tumbling + + Corynebacterium + N V V V spp. S. agalactiae N N N + V Enterococcus N + N V + spp. Erysipelothrix rhusiopathiae domestic swine major reservoir occupational hazard butchers, vets, fisherman causes erysiploid (red skin lesion) in animals and humans can disseminate into bacteremia and endocarditis Erysipelothrix rhusiopathiae GPR catalase negative alpha or gamma hemolysis nonmotile H2S positive in TSI Cultures specimens skin biopsies, blood grow on routine media (BAP, CHOC, CNA, PEA) very small colonies @ 48 hours Arcanobacterium haemolyticum Formerly Corynebacterium significant: A. haemolyticum, A. pyogenes, and A. bernardiae. Causes pharyngitis Facultative anaerobe Beta hemolysis Catalase negative Reverse CAMP positive inhibits S. aureus hemolysis Reverse CAMP Lactobacillus Normal vaginal flora Tiny alpha colonies on BAP Produce lactic acid Reduce vaginal pH Inhibits grow of other organisms Rarely causes disease L. acidophilus probiotic Lactobacillus Non-spore forming GPR Medium to long rods Aerotolerant anaerobes Catalase negative Nonmotile Gardnerella vaginalis Pleomorphic coccobacilli Cell wall with GP characteristics, stains Gram variable Nonmotile Facultative anaerobe NF in 50-70% women Linked to bacterial vaginosis 41 Bacterial Vaginosis (BV) Clue cells Change in NF (lactobacillus) increase in pH allows growth of G. vaginalis Foul-smelling discharge – amine odor Untreated can lead to PID, UTIs Culture not recommended Perform wet prep or gram stain Clue cells squamous cells with bacteria clustered at edges 42 Clue Cells 45 Cultures Appropriate for extravaginal sites Grow on CHOC, BAP, not MAC Presumptive Identification Small pleomorphic gram variable coccobacilli Catalase and oxidase negative Hippurate hydrolysis positive 46 Non-Spore-Forming Branching GPR (Aerobic Actinomycetes) Nocardia spp Tropheryma whipplei Streptomyces Actinomadura Gordonia Tsukamurella Rhodococcus equi Nocardia spp. Found in soil Usually infect immunocompromised Pulmonary Cutaneous Nocardia spp. Pulmonary N. asteroides complex Pneumonia → abscess → necrosis May disseminate Cutaneous (mycetomas) N. brasiliensis Abscess → invasive Draining pus with sulfur granules Nocardia spp. Aerobic GPR morphologically similar to fungus Form beaded, branching filaments on gram stain Weakly acid fast Nocardia spp. Microscopic Nocardia spp. Grow slowly (1 Nocardia spp. Colony Morphology on SBA week or more) on nonselective media Waxy, chalky, crumbly colonies Nocardia ID Filamentous, branching organism Weakly acid fast Acid Fast Stain acid-fast stains (Ziehl Neelsen uses heat or Kinyoun Stain uses detergent) Primary stain carbolfuchsin (minutes) Rinsed and decolorized with acid Rinsed and counterstained with methylene blue Reddish purple filaments (partially acid fast), blue: negative Nocardia is positive with Modified acid fast stain Other Actinomycetes Tropheryma whipplei Whipple Disease Intracellular pathogen Uniformly fatal if untreated Diarrhea, weight loss, malabsorption Identify by PCR or 16S rRNA Other Actinomycetes Streptomyces Actinomadura Gordonia Tsukamurella Rhodococcus equi Spore-Forming Non- Branching GPR Bacillus Spore-forming, non-branching bacilli Spores Protective, metabolically inactive Increase survival in nature Resistant to biocides Thick outer wall Bacillus Found in nature Metabolically diverse Lab contaminants B. anthracis and B. cereus are two most important pathogens Important to rule out Bacillus Large aerobic GPR Spore-formers Spores, not stained by gram stain, appear as holes Beta hemolytic except for B. anthracis Catalase positive Bacillus Catalase positive and aerobic spore formation distinguishes them from the Clostridia Growth on SBA and Phenylethyl alcohol (PEA) Does not grow on enteric agars, it is GPR B. anthracis Causes anthrax, usually in herbivors Bioterrorism agent Virulence factors Glutamic acid capsule Exotoxin Protective antigen Edema factor Lethal factor  B. anthracis Clear zone is capsule Clinical Significance Three forms cutaneous, pulmonary, or gastrointestinal Cutaneous anthrax Pimple → vesicles → erythematous ring Produces necrotic lesion (black eschar) 1 to 2 weeks to heal Clinical Significance Inhalation anthrax Pulmonary Woolsorter’s disease Flu-like phase followed by respiratory distress, coma, death GI anthrax Ingestion Pain, nausea, vomiting B. anthracis Gram stain very long chains, “bamboo shoots” Square ended  Non-motile and non-hemolytic Large, gray, flat colony with irregular edges Medusa head colonies B. anthracis B. anthracis Identification Aerobic/anaerobic Spore-forming GPR Nonhemolytic on SBA Nonmotile Catalase positive In biological safety hood If cannot R/O, send to reference lab B. cereus B. cereus is beta hemolytic and motile Distinguishes it from B. anthracis Produces enterotoxin Causes food poisoning Colony Morphology Self-limiting Frosted Glass Colonies  Identification of GPR Spores positive negative Bacillus Catalase positive negative branching H2S positive negative positive negative aerobic actinomycetes motility Erysiplothrix Lactobacillus bile-esculin positive negative Listeria Corynebacterium  1 Spirochetes  2 Introduction Borrelia, Leptospira, Treponema Long, slender, spiral-shaped Motile Differ in number and tightness of spirals Not seen in gram stain Require silver stains Seen in wet preparations by darkfield or phase contrast microscopy  3 Borrelia Borrelia contain several species of spirochetes Transmitted by arthropods (ticks & lice) May be seen with bright-field microscopy 3 to 10 spirals/organism Most cause relapsing fever B. burgdorferi causes Lyme disease  4 Borrelia Under Darkfield Microscopy  5 Borrelia recurrentis Causes relapsing fever repeated febrile episodes of spirochetemia fever, headache, muscle pain Cyclic relapses caused by antigenic variation evades immune system Episode ends when immune system responds to new antigen makeup Epidemic relapsing fever (louse-borne) Endemic relapsing fever (tick-borne)  6 Relapsing Fever Lab Tests Microscopic examination of blood during febrile periods Giemsa or Wright’s stain preferred method only spirochete disease where organisms are seen in blood with bright field microscope Can be cultured in Kelly medium rare Serology antigenic variation makes this impractical  7 Relapsing Fever  8 Borrelia burgdorferi Lyme borreliosis Lyme disease Originally in northeast (spreading) Transmitted by Ixodes tick deer or mouse ticks  9 Ixodes  10 3 Stages of Lyme Disease Stage 1 Erythema chronicum migrans target lesion, bull’s eye lesion Lymphadenopathy Flu-like symptoms  11 3 Stages of Lyme Disease Stage 2 Dissemination Fever, bone and joint pain Splenomegaly Malaise Stage 3 Late or persistent infection Months to years later Chronic arthritis, neurologic defects, meningioencephalitis, cardiac problems  12 Lyme Disease Lab Tests Serologic tests – Most common and fastest Antibody detection tests with a western blot confirmation Direct microscopic examination of skin or blood Usually negative Cultures Labor intensive Kelly medium  13 Leptospira L. biflexa Nonpathogenic L. interrogans Animal pathogen passed to humans via water contaminated with animal urine Occupational hazard for vets, farmers, sewer workers  14 Leptospira interrogans Spirals with hooked ends Tightly coiled Scanning electron micrograph  15 Leptospirosis Zoonotic infection Dogs, rats, cattle Spirochete infects kidneys of animals ➔ urine ➔ water indirect Leptospires enter humans via breaks in skin or intact mucosa  16 Leptospirosis  17 Leptospirosis Clinical manifestations Asymptomatic to severe Incubation period 3-30 days Onset of symptoms abrupt Nonspecific influenza like  18 Leptospirosis Severe systemic disease Weil’s disease – CSF positive Renal failure Hepatic failure icteric leptospirosis Intravascular disease  19 Leptospirosis Lab Tests Blood & CSF in first week, urine after that Direct examination Darkfield microscopy or DFA Cultures Fletcher’s semi sold media Incubate in the dark @ 30°C for 6 weeks Darkfield microscopy from media Serology ELISA assay  20 Treponema 4 to 14 spirals/organism Four pathogenic organisms T. palladum subsp. pallidum T. palladum subsp. pertenue T. palladum subsp. endemicum T. palladum subsp. carateum  21 Treponema pallidum subspecies pallidum Venereal syphilis Great imitator – before serological tests Variety of clinical presentations Transmitted sexually or by active nongenital lesions (mouth or skin) Can cross the placenta (congenital syphilis) Three stages primary, secondary, tertiary  22 Primary Syphilis Few days to months after organism acquired Firm chancre forms at inoculation site Lesion contains many spirochetes and is highly infectious Painless  23 Secondary Syphilis 2 to 12 weeks after primary lesion Organisms disseminate throughout body Fever, lymphoadenopathy, headache Secondary infectious lesions of skin and mucous membrane Widespread unusual skin rashes May include palms and soles  24 Secondary Syphilis Secondary lesions Syphilis rash  25 Tertiary or Late Syphilis 1/3 of untreated develop tertiary syphilis years after initial infection Not infectious Many body sites affected gummans (granulomatous lesions) in skin, liver, bones CNS: neurosyphilis deafness, blindness, partial paralysis, shuffling walking gait, mental disturbances Cardiovascular lesions: syphilitic aortitis  26 Tertiary Syphilis  27 Early Onset Congenital Syphilis Mother has early syphilis Treponemes cross placenta Severe infected fetus often dies Affects many body systems Manifestations include skin and mucous membrane lesions, anemia, hepatosplenomegaly, meningitis, bone lesions  28 Late Onset Congenital Syphilis Mother has chronic, untreated syphilis Symptoms > 2 years old blind, deaf, mentally retarded bone deformities tooth malformation Prevented by screening pregnant women and treatment if necessary  29 Serological Laboratory Diagnosis Nontreponemal tests - screening Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR) Screening, easy, inexpensive Treponemal tests - confirmation Enzyme Immunoassay (EIA) Treponema pallidum particle agglutination assay (TPPA) Fluorescent treponemal antibody absorption (FTA- ABS)  30 Nontreponemal Tests Detects antibodies reagin or reaginic antibodies formed against lipids in response to treponemal lipids released during infection antigen is cardiolipin-lecithin Sensitive but not specific false positive can occur in Lyme disease, certain viral infections, autoimmune disease, pregnancy Screen, monitor therapy (titers), detect reinfection  31 VDRL and RPR  32 Treponemal Tests Detects treponemal specific antibodies Confirmation Remain positive after treatment Enzyme immunoassay Treponema pallidum particle agglutination assay (TPPA) Previously FTA-ABS  33 TPPA Positive control Negative control  34 Direct microscopic exam Darkfield microscopy requires patience and experience fluid from lesions looking for corkscrew motility oral lesions not appropriate, nonpathogenic treponemes may be present  35 Other Pathogenic Treponemes Spread through direct contact or through contact with contaminated drinking and eating utensils May exhibit primary, secondary, tertiary disease is rare Not usually sexually transmitted No congenital infections  36 Other Pathogenic Treponemes T. endemicum Endemic syphilis (non-venereal) Middle East/hot, arid areas T. pertenue Yaws (skin and bones) Humid, tropical areas  37 Other Pathogenic Treponemes T. carateum Pinta (skin) Central and South America Can not be distinguished by laboratory tests Clinical manifestations and patient history Mycobacteria Mycobacteria Introduction Aerobic - require increased levels of CO2 Non-spore forming Non-motile Cultures held for 6 weeks before negative Slim gram variable rods High lipid content (mycolic acid) in cell wall Don’t gram stain well Mycobacteria Gram Stain gram stain poorly (if at all) cell wall lipids interfere with penetration of crystal violet and safranin gram stain: no organisms beaded GPR ghost cells Mycobacteria Safety Incidence of positive tuberculosis skin test (PPD) test is 3X higher in mycobacteriology MT Mycobacterium lab safety Lab is separate Nonrecirculating ventilation Negative air pressure Work done in biosafety cabinet Mycobacteria Safety Airborne hazard BSL2 for processing specimens (wear gloves and gowns) in biological safety cabinet BSL3 when working with cultured organisms specimens double sealed during centrifugation  Mycobacteria Species Major pathogens M. tuberculosis complex Nontuberculous mycobacteria (NTM) M. leprae Hansen’s disease (leprosy) Specimen Collection Sputum, bronchial washing, gastric contents, urine, stool, and tissue Sputum first morning; on 3 consecutive mornings 5-10 ml sputum minimum volume refrigerate overnight If sputum can’t be produced, bronchoscopy performed collect bronchial washings (BAL) or biopsy Specimen Collection Gastric Aspirates and Washings young patients radioactive CO2 instrument samples gas in vial, reported as growth index BACTEC 9000MB fluorescent sensor in bottom of vial check oxygen concentration fluorescence increases as oxygen consumed Instrumentation - Liquid Media MB/BacT system sensor changes color from green to yellow as CO2 generated color detected by colorimetric sensor of instrument ESP Myco System manometric system (pressure-measuring) measures pressure as gases consumed or produced  Approach to ID AFB Positive growth on TB liquid or solid media other organisms grow on TB media First test is acid fast stain to confirm mycobacteria ID of AFB – slow, cumbersome, ? Growth parameters Biochemical tests Analysis of cell wall lipids Growth parameters Growth rate rapid growers: visible colonies within 7 days slow growers: longer incubation period > 7 days place NMT into subgroups Colony morphology described as smooth and soft, or MTB like: ruff and buff, cording “cauliflower” colonies on LJ Photoreactivity Some produce carotenoid pigments Some require light for pigment production Evaluate ability to produce pigments +/- light Isolate inoculated into LJ slants Tube +/- aluminum foil wrap Incubated  Identification Testing Biochemical tests Miscellaneous Tests Catalase NAP Niacin Growth on MAC Nitrate Growth temperature Tween 80 hydrolysis Tellurite reduction Catalase Heat -stable (68 °C) catalase Suspension of organism in 2 tubes, one heated in 68 °C water bath for 20 minutes, other at RT Tween 80-hydrogen peroxide added Bubbles = positive No bubbles = negative  Niacin Most mycobacteria possess enzyme to convert free niacin to niacin ribonucleotide M. tuberculosis lacks enzyme Niacin accumulates in medium Nicotinic acid + cyanogen bromide → yellow color Niacin Positive Negative Nitrate Reduction Nitrate reduced to nitrite/nitrogen gas MTB, M. kansasii, M. szulgai, and M. fortuitum Organism inoculated into nitrate substrate Reagents added sulfanilamide and N-naphthylenediamine dihydrochloride Positive - red color If red color does not appear zinc added to see if nitrate reduced all the way to nitrogen Positive – if no color change after zinc added Negative – red color nitrate still present Tween 80 Hydrolysis Common nonpathogenic mycobacteria can hydrolyze Tween 80 Destruction of Tween 80 turns media from amber to red Red = positive for Tween hydrolysis Amber/no color change = negative  Tellurite Reduction Tellurite reduction to tellurium Black = positive Reduction rate varies among MB Few species reduce tellurite in 3 days M. avium complex tellurite positive distinguish from other nonchromogenic species All rapid growers reduce tellurite in 3 days Growth Inhibition NAP P-nitroacetylamino-β-hydroxypropiophenone Inhibits MTC growth T2H Thiophene-2-carboxylic acid hydrazide Distinguishes MTB and M. bovis (inhibited by lower levels) Growth on Special MAC Determines if organism capable of growing on MAC without crystal violet Rapid growers M. fortuitum-chelonae complex can grow, other AFB can’t Growth Temperatures Isolate inoculated onto culture media Incubated at 24, 30, 37, 42°C Examined for growth Classification of Mycobacteria Mycobacterium tuberculosis complex (MTC) Tubercle bacilli Cause human tuberculosis Atypical mycobacteria Nontuberculosis mycobacteria (NTM) or Mycobacterium other than tubercule (MOTT) May produce pulmonary disease No person-to-person transmission Acquired from environment M. tuberculosis Complex Members of MTB complex causing (TB) M. tuberculosis (most common) M. bovis M. africanum, M. canettii, M. microti King of the pathogens > 1 billion infected worldwide M. tuberculosis Complex Spread by respiratory droplets Person-to-person Slow growers Colonies are non pigmented M. tuberculosis Complex Mycobacterium bovis atypical tuberculosis transmitted by drinking unpasteurized milk was a major cause of TB in children rarely causes TB now since most milk is pasteurized (undergoes a heating process that kills the bacteria) M. tuberculosis Complex Colony morphology MTB: “ruff” and buff (color), cording “ cauliflower” colonies on LJ Requires long term treatment-MDR variants Use skin test screening in U.S. (PPD) Primary TB Exposure to respiratory droplets Many people form lung granulomas Tumor-like inflammatory lesion → tubercle Formed as host walls off infecting organisms forming scar tissue (CMI) Necrotic centers with soft “cheesy” appearance Described as caseous Tubercles Granuloma in lung apex Center of tubercle dissentegrates Cheese-like mass = caseation  Primary TB Risk for TB reactivation in the future Occurs after suppression of the immune system 15-20% of infected develop disease Extrapulmonary TB Occurs in small percentage of MTB infections Can cause disease in any body site lungs (pulmonary TB - most common) meninges kidneys bones (Pott’s disease) genital tract Miliary TB Disseminated TB Small tubercles scatter throughout the body resembling millet seeds Lung – lesion erode blood vessels PPD Tuberculin Skin Test Mantoux test MTB infected individuals become hypersensitive to MTB protein antigens Detected by injecting purified protein derivative (PPD) MTB antigen PPD Tuberculin Skin Test Positive PPD Delayed hypersensitivity Red hard area at injection site within 48 to 72 hours does not distinguish between active and latent infections Negative PPD skin test No response to PPD MTB Symptoms Coughing Hemoptysis Weight loss Low-grade fever  M. tuberculosis ID Slow grower Rough, buff colored colonies Cells arranged in serpentine cords due to cord factor Positive niacin and nitrate Negative 68°C catalase NAP susceptible Reportable to CDC Colonies on LJ Acid Fast Stain – Cord Factor  M. bovis ID Colonies resembles MTB Negative nitrate and niacin* Negative 68°C catalase Susceptible to NAP and T2H  Runyon NTM Classification I Photochromogens Slow growers, > 7days Nonpigmented in dark Yellow/orange pigment in light II Scotochromogens Slow growers, > 7days Yellow/orange pigment in dark Orange/red pigment in light  Runyon NTM Classification III Nonphotochromogens Slow growers, > 7days Lightly (tan, buff) pigment in light or dark IV Rapid growers Visible growth in < 7days  I Slow Growing Photochromogens M. kansasii Pulmonary disease Can infect other body sites joints, BM, skin, lymph nodes Hydrolyze Tween 80 Nitrate positive I Slow Growing Photochromogens M. marinum Swimming-pool granuloma Acquired when traumatized skin comes in contact with fresh or salt water Optimal temperature: 30°C, grow poorly at 37°C Hydrolyzes Tween 80  II Slow Growing Scotochromogens M. scrofulaceum Mycobacterial cervical (neck) lymphadenitis Nitrate and Tween 80 negative M. gordonae Tap water bacillus III Slow Growing Nonphotochromogens M. avium Complex (MAC) M. avium and M. intracellulare Found in environment (coastal marshes) Increase in infections due to AIDS Most common NMT causing tuberculosis 68 °C catalase positive Distinguished by GLC, HPLC, or nucleic acid probes III Slow Growing Nonphotochromogens M. ulcerans Skin ulcers Third most common AFB isolated worldwide 32°C optimum temperature Does not grow at 37°C difficult to isolate IV Rapid-Growers M. fortuitum, M. chelonae, M. smegmatis Environmental organisms Can cause wound infections, abscesses, osteomyelitis, pulmonary infections Growth on special MAC Nitrate negative M. chelonae Nitrate positive M. fortuitum and S. smegmatis Uncultivable M. leprae causes leprosy (Hansen’s disease) Disease of skin, mucous membrane, and peripheral nerves Grows best at 30°C Two forms of disease Tuberculoid leprosy Skin and nerves CMI ➔ recovery Lepromatous leprosy No CMI ➔ slowly progressive, fatal Uncultivable Mycobacterium Rare in US and is treatable Does not grow in vitro Use armadillos footpads or lab mice Diagnosis based on Presence of non-culturable AFB in skin biopsies Patient clinical manifestations Other Pathogenic Species M. genavense M. simiae M. xenopi M. szulgai Others… Newer Identification Tests Chromotography Gas-liquid chromatography (GLC) or high- performance liquid chromatography (HPLC) Cell walls contain mycolic acids (long chains fatty acids) Different species have different mycolic acids Identified by lipid profile  Newer Identification Tests Molecular ID of AFB Hydridization and nucleic acid amplification Rapid identification of clinically significant AFB by nucleic acid probe Decrease turn around time (TAT) Increased accuracy and reproducibility Antimicrobial Susceptibility Tests M. tuberculosis tested for susceptibility to Isoniazid Rifampin Ethambutol Streptomycin Susceptibility Testing Methods Absolute concentration Determines minimum inhibitory concentration (MIC) Resistance ratio Compares test organism MIC to lab strain MIC Proportional Predicts resistance by testing resistance of 1% of bacterial population Liquid media, commercial methods Antimicrobial Susceptibility Tests To prevent MDR variants two to three drugs given Isoniazid (INH) and Rifampin for 9 months INH prophylaxis given for positive PPD Susceptibility testing not done on NTM except rapid growers