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STUDENT NOTES: MICRO 1  

SPORE FORMING GRAM-POSITIVE BACILLI  

parenchyma

AEROBIC SPORE FORMING GRAM-POSITIVE BACILLI  Gastrointestinal anthrax  

Bacillus - ​Via ingestion of the spores
- ​>100 species within the genus - ​Symptoms: abdominal pain, nausea, anorexia, and
- ​metabolically diverse; some species grow best at 55 deg C or vomiting, bloody diarrhea (can also occur)
higher
- ​Aerobic/ facultative anaerobes Injectional Anthrax  
- ​Spores = aid in survival - ​Characterized by soft tissue infection associated with “skin
- ​Motile popping”
- ​Catalase (+), ferments glucose, hydrolyzes starch ​- - ​Lack of eschar, severity of disease, and increased mortality
Most grow well on SBA rate make this form clinically distinct from the cutaneous
form
Bacillus anthracis
Laboratory Diagnosis:
General Characteristics: Microscopy  
• ​Anthrax bacillus - ​large (1.0 to 1.5 µm × 3.0 to 5.0 µm)
• ​Causative agent of anthrax - ​square-ended (bamboo rod/ bamboo fishing rod
• ​Grows aerobically/ anaerobically appearance)
• ​Non-motile - ​gram-positive or gram-variable rod found singly or in chains ​-
• ​Halophilic (7% NaCl) unstained central spore
• ​Catalase (+) Cultural Characteristics  
• ​Ferments glucose - ​SBA: non-hemolytic
• ​Produces lecithinase - ​Large (2-5 mm)
• ​Growth factor: thiamine (vit. B1) - ​Gray
- ​Flat w/ irregular margin
Virulence Factors: - ​Medusa head colonies
• ​D-glutamic Acid Capsule = prevents organism from - ​beaten egg white appearance
phagocytosis - ​String of pearl appearance on agar w/ penicillin (0.05-0.5
• ​Anthrax toxin/ Protein Exotoxins (EA, PA, LF) = nontoxic U/mL)
but act collectively to produce damaging effects Diagnostic Tests  
Anthrax toxin consists of three proteins:   - ​Ascoli test (precipitin test): skin test
• ​Protective antigen (PA) - serves as a necessary binding - ​Detects thermostable anthrax antigen
molecule for EF and LF - ​Uses rabbit antiserum to observe precipitin
• ​Edema factor (EF) - an adenylate cyclase that increases the formation
concentration of cAMP in host cells; PA + EF = Edema ​• - ​Sample soln: 2g of sample in 5 ml saline placed in
Lethal factor (LF) – protease; PA + LF = Death 1/100 final concentration of acetic acid
- ​(+) result: precipitin band formation (<15 minutes) ​-
Clinical Infections: Catalase test: ​Bacillus vs Clostridium
Cutaneous Anthrax   - ​Direct fluorescent antibody test: (+) cell wall polysaccharide &
- ​a small pimple (2 to 3 days after exposure) ​→ ​ring of capsule antigen
vesicles develops → ​ ​merge → ​ ​small dark area appears ​→
ulcerates and dries →
​ ​eschar or black eschar   Treatment:
- ​Penicillin
- ​CDC recommendation: ciprofloxacin or doxycycline for initial
Inhalation Anthrax  
IV therapy until antimicrobial susceptibility results are
- ​Woolsorter’s disease/ Ragpicker’s disease
known
- ​acquired when spores are inhaled into the pulmonary
 - ​Clindamycin: to inhibit exotoxin production
- ​Metronidazole: injectional anthrax Bacillus cereus
- ​Oral ciprofloxacin or doxycycline: PEP for pulmonary General Characteristics:
anthrax - ​Fried Rice bacillus

DDC Medical Laboratory Science Program | 2020-2021 Prepared by: Rachel Ann O. Eromon, RMT
- ​Causes food poisoning due to ingestion of contaminated Spore-bearing; Catalase (-)
rice/ other food products • ​Saprophytes; frequently encountered in exogenous
- ​Most commonly encountered sp. in opportunistic infections ​- anaerobic infections/ intoxications
Can be part of the normal fecal biota • ​MOT: ingestion/ open wounds contaminated w/ soil ​•
- ​Motile Histotoxic clostridia: ​C. perfringens, C. novyi, C. septicum, C.
- ​Penicillin (R) histolyticum, C. bifermentans
- ​2 forms of food poisoning: diarrheal and emetic Clostridium perfringens
General Characteristics:
Virulence factors: ​enterotoxins (heat stable & heat labile), cerelysin, • ​Gas gangrene bacillus
phospholipase C. pyogenic toxin • ​Most commonly isolated member of the genus in blood
cultures
2 Types of Food Poisoning: • ​Causes outbreak after ingestion of contaminated meat &
- ​Diarrheal type = associated w/ ingestion of contaminated gravy
meat/ poultry & vegetables • ​Capsulated
- ​Emetic type = associated w/ ingestion of improperly stored • ​Non-motile
fried rice/ reheated rice • ​Causative agent of gas gangrene
• ​Grown quickly on selective media

Laboratory Diagnosis: Virulence Factors:

- ​>10​5 ​cells per gram of food = food poisoning by this - α toxin (phospholipase C, lecithinase)
organism is confirmed • ​the most important toxin
- ​At least 10​5 ​cells per gram of stool = organism as the • ​Lyses RBCs, platelets, leukocytes and endothelial cells ​•
cause of the disease is confirmed Increased vascular permeability with massive hemolysis and
- ​BAP: ​large, feathery, frosted glass colonies, beta hemolytic ​- bleeding tissue destruction
Biochemical tests: ​ferments salicin; lecithinase (+) • ​Hepatic toxicity and myocardial dysfunction
- ​β​-toxin
Treatment: • ​responsible for necrotic lesions in necrotizing enterocolitis -
- ​Resistant to penicillin and all of the other β-lactam Enterotoxin
antibiotics except for the carbapenems • ​heat labile toxin produced in colon → food poisoning -
- ​Vancomycin or Clindamycin w/ or w/o an aminoglycoside DNAse
• ​lowers viscosity of exudates
Bacillus subtilis
General Characteristics: Clinical Infections:
- ​Hay bacillus/ Grass bacillus Gas gangrene/ Myonecrosis  
- ​Common laboratory contaminant • ​“eating sore”
- ​Halophilic (7% NaCl) • ​Caused by α toxin
- ​Source of bacitracin antibiotic • ​Organisms contaminate wounds via trauma, frostbite, or
- ​Culture: large, flat, dull, ground glass appearance, beta surgery
hemolytic, pigmented • ​Accompanied by bullae, pain, swelling, serious discharge,
- ​Biochemical test: ferments mannitol, xylose, & arabinose discoloration, & tissue necrosis
• ​Tx: hyperbaric oxygen therapy
Bacillus pumilus
- ​Culture: large, moist, blister colony, beta-hemolytic Food poisoning/ Enteritis necroticans  
• ​“​pig-bel”
Bacillus thurigiensis • ​Via ingestion of enterotoxin in contaminated food ​•
- ​Insect pathogen Symptoms: diarrhea (foul-smelling stool), crampy
- ​Produces parasporal crystals abdominal feeling
2 types:
Type A food poisoning: mild & self-limiting; w/ diarrhea & cramping
ANAEROBIC SPORE FORMING GRAM-POSITIVE 
abdominal pain
BACILLI Clostridia  
Type C food poisoning: serious; w/ diarrhea & vomiting
• ​Obligate anaerobe
• ​Gram-positive
Laboratory Diagnosis:
• ​Straight or slightly curved rods with slightly rounded ends ​•
 Specimen​: ​Histological specimen ​or ​wound exudate​ ​➢ • ​Boxcar-shaped bacilli
Histological specimen: transferred aseptically into a sterile • ​capsulated & sporulated
screw-capped bottle • ​oval, sub-terminal & non bulging spores
➢ ​Specimens of exudates: should be taken from the • ​Spores are rarely observed
deeper areas of the wound Culture: ​Anaerobically at 37 deg C
Microscopy   • ​BAP: dome-shaped, gray to white colonies; beta-hemolytic
• ​Gram-positive colonies; double zone hemolysis
DDC Medical Laboratory Science Program | 2020-2021 Prepared by: Rachel Ann O. Eromon, RMT
• ​On Robertson's cooked meat medium → blackening of
meat will be observed w/ the production of H2S and NH3 Prevention & Control:
Biochemical tests   1. Tetanus toxoid vaccine
• ​Ferments many carbohydrates with acid & gas -DTaP = <7y/o; safer; lesser side effects than DTP
• ​Acidifies litmus milk with stormy clot production -Tdap = ≥11y/o; lower concentration of diphtheria and pertussis
• ​Nagler reaction (+) toxoids than DTaP.
• ​Reverse CAMP test (+) -DTP = no longer used in USA
Treatment:
​Nagler’s Reaction: ​done to detect lecithinase activity ​– 1. Aggressive wound care
Inoculated on the medium containing human serum or egg yolk 2. Antibiotics
(contains lecithin) 3. Tetanus immune globulin (TIG)
– ​The plate is incubated anaerobically at 37 deg Celsius for 4. Tetanus toxoid booster
24 hrs 5. Agents to control muscle spasm (e.g. Benzodiazepine)
​Reaction on Litmus Milk   6. Supportive care

Clostridium botulinum
General Characteristics:
• ​Canned Good bacillus
• ​Obligate anaerobes
• ​Found in soil & aquatic sediments
• ​Potential agent for bioterrorism
• ​Catalase (-)
• ​Gram-positive rods, Spore-forming
• ​Produces toxin that causes botulism
(+)
• ​First recognized and isolated in 1896 by Emile Pierre Marie
Stormy Fermentation of Litmus Milk
van Ermengem
Clostridium tetani
General Characteristics:
Clinical Infections:
• ​Tack head bacillus
Botulism  
• ​Gram positive, straight, slender rod with rounded ends ​•
• ​neuroparalytic disease
Forms endospores (drumstick with a large round end) ​•
• ​Virulence factor: botulinum toxin
Motile
• ​7 types (A-G)
• ​Soil and environmental inhabitant ✓ ​A, B, E = associated w/ human infxns
• ​Endospores: found in hospital environments, soil, dust,
✓ ​A = used to treat strabismus & as a beauty enhancer
feces of many farm animals
- Foodborne Botulism  
• ​Results from ingestion of ​preformed toxin
Clinical Infection:
​- Infant Botulism  
Tetanus  
• ​“Floppy baby syndrome”
- ​Virulence factor: Tetanospasmin
• ​Caused by ingestion of ​organism ​from honey/ via
– ​causes increasing excitability of spinal cord neurons
breastfeeding
and muscle spasm
​- Wound Botulism  
– ​Characterized by “trismus” and “risus sardonicus” • ​Seen in people who inject illegal drugs
Laboratory Diagnosis: Laboratory Diagnosis:
• ​Specimen: ​Wound exudates using capillary tube ​• • ​Microscopy: ​gram (+) rods w/ oval subterminal spore ​•
Culture:   Culture:  
– ​BAP: heavy, “smooth swarming”, matte colonies; ​- BAP: large (3mm), irregularly circular, smooth, grayish,
beta-hemolysis, grow slowly translucent with a fibrillar edge that may spread, β
• ​Microscopy: ​gram (+) rods w/ terminal spore hemolytic
• ​Biochemical tests: ​motile, gelatinase & indole (+), • ​Biochemical test: ​lipase (+), catalase (-)
lecithinase & lipase (-)
 – ​High enemas
Treatment: 4. Antibiotics
1. Antitoxin administration (trivalent or heptavalent) 5. Botulinum Immunoglobulin (BIG)
2. Supportive care
– ​Mechanical ventilation Clostridium difficile
– ​Body positioning General Characteristics:
– ​Parenteral nutrition • ​Obligate anaerobe
3. Elimination • ​Most common cause of antibiotic-associated diarrhea &
– ​Induced vomiting pseudomembranous colitis

DDC Medical Laboratory Science Program | 2020-2021 Prepared by: Rachel Ann O. Eromon, RMT
• ​Part of the GI flora in: • ​For clinical use: two-step testing uses initially EIA detection
◦ ​1-3% of healthy adult screening followed by cytotoxicity assay or toxigenic culture
◦ ​70% of children < 12 months for confirmation
• ​Toxin-producing strains cause ​C. difficile ​Infection (CDI) | • ​Gold standard: stool culture followed by toxigenic culture
Toxin A and B assay

Diagnostic Laboratory Test: References:

Culture   Delost, Maria Dannessa. ​Introduction to Diagnostic Microbiology for
• ​BAP: non-hemolytic, “barnyard”/ “horse stable” odor ​• the Laboratory Sciences​. 1​st ​Edition, 2014
CCFA: yellow, ground glass colonies Mahon, Connie & Lehman, Donald. ​Textbook of Diagnostic
Microscopy: ​gram (+) bacilli, oval subterminal Microbiology, ​6th​ ​Edition, 2018
spores ​Biochemical test: ​glutamate dehydrogenase Tille Patricia M. ​Bailey and Scott’s Diagnostic Microbiology​, 14th
(+) Edition, 2018

Best Strategy for ​C. difficile ​Testing:

DDC Medical Laboratory Science Program | 2020-2021 Prepared by: Rachel Ann O. Eromon, RMT