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Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim SPORE 2. Exotoxins Oxygen Requirement Catalase Test...

Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim SPORE 2. Exotoxins Oxygen Requirement Catalase Test Protective factor- Binding molecules to edema factor with 15% H2O2 and lethal factor Bacillus Strict aerobe + Edema factor- Adenylate cyclase enzyme causes Clostridium Strict anaerobe - edema Lethal factor- Protease enzyme that kills the host cell GENUS: BACILLUS Characteristics: Bacillus anthracis: CLINICAL INFECTIONS Large gram (+) rods in singles, pairs, or serpentine 1. Cutaneous anthrax changes Malignant pustule/black eschar (most common & least Catalase (+) severe) Capable of producing endospores in the presence of Occurs at the site of spore penetration oxygen (aerobically) Manifested by: erythematous papule → causing Species differentiation within the genera is based on: ulceration → black eschar ○ The size of vegetative cell MOT: contact with infected spore while handling wool ○ Sporulation resulting in swelling of vegetative cells from sheep on open wounds ○ Biochemical analysis of bacillus Occurs at site of spore penetration usually 2-5 days after exposure Cultivation Condition May progress to malignancy/toxemia and death Blood Agar Media ○ Colonies are non-hemolytic (gamma hemolytic) 2. Inhalation/Pulmonary anthrax ○ Narrow zone of hemolysis Aka Woolsorter’s/ Ragpicker’s disease/ Hide porter’s ○ Chocolate Agar, routine blood culture media and Follows inhalation of spore and progresses from mild nutrient broths can also be used form to respiratory distress to severe inhalation of ○ Will NOT grow on Mac Conkey Agar spores MOT: Occupational hazard (sheeps, wools) Incubation Conditions and Duration Patient manifestations: Will produce detectable growth within 24 hours ○ Malaise with mild fever Needs an oxygen at 35 °C (ambient air) or in 5% carbon ○ Non-productive cough dioxide- Bicarbonate agar ○ Massive chest edema ○ cyanotic/cyanosis Bacillus anthracis ○ Death A.K.A. Anthrax bacillus Biosafety level 3 (inhalation) Most significant because it is an agent of bioterrorism causing disease (Anthrax) 3. Gastrointestinal anthrax Non-hemolytic, non-motile on BAP and with centrally Least common but most severe located endospores Follows ingestion of live spore from improperly Microscopically- Forms disjointed bamboo rod cooked meat (sheep) appearance/fishing rod Causes overwhelming sepsis (bacterial invasion in On gram-stained smears it forms box car- due to its square blood stream) ends Affects the GI area, oropharynx (oropharyngeal), Pathogenic due to capsule (Polypeptide D-Glutamate abdominal area capsule) (encapsulated) Irregular, round raised dull opaque grayish white colonies 4. Injection anthrax with a frosted ground glass appearance Associated with the use of illicit drug thru injection TOP VIEW- Edge of the colony is composed of long, interlacing chains of bacilli, resembling locks of matted Bacillus anthracis: LABORATORY DIAGNOSIS hair (medusa head-like appearance) 1. Microscopy Pine tree/Inverted fir tree appearance with slow Largest bacteria, gram +, square ends, singly or in liquefaction starting from top on gelatin stab chains Bamboo rod / box car appearance Bacillus anthracis: VIRULENCE FACTORS Spores are oval and located centrally or subterminal 1. Glutamic acid capsule - Heat-resistant and Resistant to host proteolytic enzymes 2. Cultural Characteristics Stain: M’ Fdyean Stain (Polychrome Methylene blue) Non-hemolytic, large, gray and flat with irregular margin (not perfect circle) Medusa head appearance Large and mucoid capsule in bicarbonate agar Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim Beaten egg white consistency 3. Culture Media Produces string of pearl appearance in Mueller BCM - Bacillus Cereus Medium Hinton Agar containing 0.05-0.5 u/mL penicillin MEYP/ MYP - Mannitol Egg Yolk Polymyxin B PEMBA - Polymyxin B Egg Mannitol Bromothymol Blue Culture Media CHARACTERISTICS B. anthracis B. cereus PLET (Polymyxin Lysozyme EDTA Thallous Acetate Hemolysis Gamma Beta Agar) String of Pearls + - ○ Selective media used for isolation of Gelatin Hydrolysis - + Bacillus anthracis from contaminated spx ○ Media: 5% sheeps BA Motility Non-motile motile ○ Detects: Medusa head colonies 3. Other Tests Bacillus subtilis Lecithinase Test Known as blood bank contaminant B. anthracis can produce the lecithinase Hay bacillus Media: Egg yolk media in plate Motile, beta hemolytic on BAP Positive: Opaque zone around colonies BSL-1: Minimal risk Ascoli test Causes: opportunistic infection, eye infection among heroin Serological test for detection of Anthrax addicts Source of bacitracin Bacillus cereus Penicillin: Resistant Fried Rice bacillus Selective media: MEYP agar Motile, beta hemolytic on BAP Used as biological indicator in oven (160-180°C) Ideal spx for testing: Suspected food ○ Bacillus subtilis var niger- Quality control organism Ideal media: Sheep’s Blood Agar B. subtilis var globijii – Ethylene oxide Most encountered species of Bacillus in opportunistic infections: Bacillus stearothermophilus ○ Post-traumatic eye infection Kilit ampoule ○ Endocarditis Used as biological indicator in autoclave (121°C 15 psig ○ Bacteremia 15-30 mins) Infections of other sites are rare and usually involve: Causes flat sour spoilage ○ Intravenous drug users ○ Immunocompromised patient Bacillus circulans, Bacillus licheniformis, and other Bacillus spp. CHARACTERISTICS DIARRHEAL EMETIC Less virulence Incubation period 8-16 hours after 1-5 hours after Unknown virulence factors ingestion ingestion Food poisoning but not common Predominant Diarrhea Vomiting May be involved in opportunistic infections like those symptoms described for B. cereus Duration of illness 12-24 hours 6-24 hours (9 hours is the average) Bacillus mycoides Food implicated Meat producers Fried and Boiled rice Less significant but almost same with B. cereus 5% SBA- Produce rhizoid colonies appearing fungus Stability to heat Negative Positive Toxins HBL (Hemolysin BL) Cereulide Cyt K (Cytotoxin K Bacillus thuringiensis hemolysin IV) Larvicidal- Commercial ingredient for pesticides/insecticides Nhe (Nonhemolytic Presence of organism in feces without gastrointestinal enterotoxin) symptoms Rare cases: Bacillus cereus: LABORATORY DIAGNOSIS ○ Wounds, burns, pulmonary, eye infection 1. Microscopy Large, gram + bacilli Spores are oval and located centrally or subterminally 2. Colon appearance Large, feathery and spreading Wide zone of beta-hemolysis Frosted glass appearance Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim Bacillus thuringiensis: LABORATORY DIAGNOSIS GENUS: CLOSTRIDIUM 1. Lecithinase test Characteristics Determines the ability of the organism to produce Strict anaerobe but some are aerotolerant lecithinase All are single hemolytic except Clostridium perfringens- Positive: target hemolysis (double zone of hemolysis/target ○ Colonies surrounded with opaque zone of hemolysis; Outer- Alpha, Inner- Beta) precipitation Virulence factor: Toxin forming ○ B. anthracis, B. cereus, B. thuringiensis, B. TOXINS mycoides ENTERIC Clostridium difficile (GIT infection) Negative: ○ No wide opaque zone HISTOTOXIC Clostridium perfringens (tissue infections) ○ Other bacillus NEUROTOXIN Clostridium tetani & Clostridium botulinum 2. Motility test (semisolid agar deep) Determine if the organism is motile Clostridium perfringens Positive: Also known as C. welchii/Frankel’s Bacillus / Gas Gangrene ○ Spread of organism out from site of inoculation Bacillus ○ B. cereus, B. subtilis, B. mycoides Negative: Clostridium perfringens: VIRULENCE FACTOR ○ Organism remained at site of inoculation B & A-toxin ○ B. anthracis, B. thuringiensis ○ Type A- Milder form ○ Type C- Food poisoning type causing enteritis 3. Penicillin necrotans (vomiting, bloody diarrhea, abdominal To test for susceptibility to penicillin pain) Positive: Enterotoxin ○ Zone of inhibition (+) / susceptible ○ GI irritations ○ B. anthracis (string of pearl) Negative: Clostridium perfringens: ASSOCIATED DISEASE ○ No ZOI (-)/ Resistant Bacteremia ○ B. cereus, B. mycoides, B. thuringiensis Gas Gangrene (putrefaction)- Myonecrosis (necrosis of tissue) 4. Parasporal body/crystals ○ Common for diabetic px Tightly packaged insect pro-toxin molecules Food poisoning- Pig-bel disease/Enteric necroticans Crystalline protein that forms around spore in some Heat kills vegetative, spore formers survive & germinate bacteria that acts as a toxin precursor when digested ○ Leads to necrotic damage Lethal (pathogenic) to Lepidopteran insects (moth) ○ Gas → blackens Positive: B. thuringiensis Negative: B. anthracis, B. cereus, B. mycoides Clostridium perfringens: CELLULAR MORPHOLOGY Gram variable straight rods with blunt ends Spores: oval, sub terminally located Large box car morphology Clostridium perfringens: COLONIAL APPEARANCE Large, irregular shape, double/target hemolysis Alpha toxin- Outer zone partially hemolyzed ○ Due to lecithinase Beta toxin- Inner zone complete hemolyzed ○ Due to beta & theta toxin Other Characteristics and Presumptive Test 1. Lecithinase positive in Egg-yolk Agar (EYA) C. perfringens Lecithinase (+) (opaque zone around colonies) Lipase (-) (no sheen) Bacteroides fragilis Lecithinase (-), Lipase (-) F. necrophorum Lipase (+) (iridescent, multicolored sheen) Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim 2. Nagler Reaction (+) Clostridium tetani Positive: presence of zone of precipitation Drumstick/Tennis Racket/Tackhead/Tetanus Bacillus C. perfringens Terminal swollen spores One-half of the surfaced is streaked Type A antitoxin Causative agent: Tetanus (wound infection) 3. Reverse CAMP test (+) Asaccharolytic (CHO Utilization test (-) Y axis: GBS (S. agalactiae) “Teinien” - To stretch X axis: C. perfringens Loves iron (rust) and spores is present on infected rust Positive: Arrowhead formation and enhanced beta hemolysis Clostridium tetani: VIRULENCE FACTOR 4. Stormy fermentation in Milk (+) Exotoxin- Blocks the neurotransmitter C. perfringens - Has this characteristic Tetanospasmin- Neurotoxin that causes a spastic Litmus Milk Test type of paralysis with continuous muscle spasms Performed to find out the ability of a bacteria to ○ Patients can experience locked jaw differentiate milk components which is manifested in color or litmus, production of gas, curd Clostridium tetani: ASSOCIATED DISEASE formation, etc. Tetanus- Paralysis with continuous muscular spasms Qualitative test to determine action of bacteria on Trismus- Lockjaw milk Risus sardonicus- Distorted grin / sardonic smile Lactose- Milk sugar Breathing difficulty Litmus- pH indicator Opisthotonos- Spasms of all muscles into backward Casein- Milk protein arching on back muscles Contained within the medium can all be metabolized by different types of bacteria. Clostridium tetani: CELLULAR MORPHOLOGY The test differentiates microorganisms based on Gram variable straight rods with blunt ends various metabolic reactions in litmus milk, including Swollen terminal spore reduction, fermentation, clot formation, digestion, Looks like drumsticks and the formation of gas Use Schaeffer and Fulton stain for spores ○ Primary: Malachite green ○ Counterstain: Safranin Clostridium tetani: COLONY MORPHOLOGY Smoothly swarming but slow growing Narrow zone of beta hemolysis Anaerobic blood agar Clostridium botulinum Von emergen’s/Canned good bacillus Medium: Anaerobic environment NOT cultured Alpha hemolytic on BAP Lipase reaction (+) RESULT INTERPRETATION Clostridium botulinum: VIRULENCE FACTOR Pink color Acid Botulinum toxin (neurotoxins) Pink and solid Acid clot ○ Release of acetylcholine Fissures or spaces Gas Result in flaccid type if paralysis ○ Botulism- Fatal type of food poisoning Clot broken apart Stormy fermentation ○ Small amount- Paralysis White color (lower portion) Reduction of litmus Treats Strabismus “wandering eye”, “frown lines” Semisolid, not pink; clear Curd formation gray fluid at the top Clostridium botulinum: ASSOCIATED DISEASE Clarification of medium, Peptonization loss of “body” Foodborne- Canned goods Infant Botulism- Ingestion of spore via breastfeeding Blue medium or blue band Alkalinity or basic at the top (food / blood) No change No change ○ Floppy baby syndrome- Hypotonia leads to SID (crib death) Wound Botulism Weakness, paralysis, blurred vision, impaired speech Clinical Bacteriology LECTURE|Module 3- Part 2|Gram positive bacilli (spore formers)|1st semester |Prelim Clostridium botulinum: CELLULAR MORPHOLOGY Clostridium difficile: OTHER INFORMATION Gram (+) straight rods with blunt ends Confirmed by demonstration of: Main difference: Toxins clinical manifestations ○ Toxin B in feces by tissue culture ○ Organism in feces (liquid or unformed) Clostridium botulinum: COLONY MORPHOLOGY Beta hemolytic Clostridium septicum Clostridium septicum: CELLULAR MORPHOLOGY Other Information Thin rods with subterminal spores Confirmation ○ Check for neurotoxin (blood, stool, vomit, gastric) Clostridium septicum: ASSOCIATED DISEASE Malignancies Clostridium difficile Colorectal cancer Enteric clostridium: Normal flora of the colon but can cause Neutropenic enterocolitis pseudomembranous colitis Myonecrosis ○ Due to an antibiotic- Clindamycin Clostridium septicum: COLONY MORPHOLOGY Clostridium difficile: VIRULENCE FACTOR Resembles medusa head-like colony of B. anthracis Toxin A (enterotoxin)- Toxic to cell on intestine because of its irregular edges in culture media Toxin B (cytotoxin)- Necrosis of colonic mucosa Smoothly swarming Beta hemolytic Clostridium difficile: ASSOCIATED DISEASE Pseudomembranous colitis BIOCHEMICAL TESTS FOR MAJOR CLOSTRIDIUM SPP. Antibiotic associated diarrhea (Clindamycin) MOTILITY LECITHINASE LIPASE LACTOSE GLUCOSE Clostridium difficile: DIAGNOSIS C. perfringens - + - + + Cytotoxin Detection (Cytotoxicity Assay) C. tetani + - - - - C. botulinum + - + - + Organism can be cultivated but seldom done in the C. difficile + - - - + laboratory The identification of C. difficile is through detection of toxin B Enzyme Immunoassay (EIA); serotest Confirmatory test: ELISA (Enzyme-linked immunosorbent assay ○ To detect glutamate beta dehydrogenase (sensitive to toxin B) Toxin assay: Stool or rectal swab to check for carrier state of infection Polymerase Chain Reaction (PCR) Gold standard Detects genes of toxin via amplification to identity TED A and TED B genes) Clostridium difficile: CULTURE MEDIA Cycloserine Cefoxitine Fructose Agar (CCFA) ○ Selective ○ pH indicator: Neutral red ○ On CCFA: Colonies of C. difficile → horse stable/barn yard odor Blood Agar Plate ○ Beta hemolytic organism Clostridium difficile: CELLULAR MORPHOLOGY Chartreuse ground glass on CCFA Odor: Horse stable barn yard