GRAM-POSITIVE BACILLUS

Overview

Gram Stain Other Features Organism

Aerobic, non motile, box-car-
shaped, medusa head Bacillus anthracis
Spore-forming morphology
G (+) rods Aerobic, motile, reheated fired Bacillus cereus
rice, Fried Rice Bacillus
Anaerobic, tennis racket-like, Clostridium tetani
lollipop shaped
Anaerobic, bulging cans, Canned Clostridium botulinum
good bacillus
Anaerobic, lecithinase, gas- Clostridium perfringens
forming, gas-gangrene
Anaerobic, causes Clostridium difficile
pseudomembranous colitis,
antibiotic related diarrhea
Aerobic,non motile, curved, Corynebacterium diptheriae
Non-sporeforming Chinese characters appearance
G (+) rods on GS
Aerobic, curved, tumbling Listeria monocytogenes
motility

 Bacillus sp. - aerobes

 Clostridium sp. - anaerobes

Endospores
 Formed when cells are unable to grow (when environmental conditions change or when nutrients are exhausted)
 Made up of a complex multilayered coat surrounding a new bacterial cell
 Presence of dipicolinic acid & high calcium content (confer extreme resistance to heat & chemicals)
 Can remain viable in a dormant state for many years
 SPORULATION: triggered by near depletion of nutrients (carbon, nitrogen, phosphorous)
 Involves the production of many new structures, enzymes & metabolites along with the disappearance of many
vegetative cell components
 Axial filament formation  forespore septum formation  engulfment of forespore cortex synthesis  coat
deposition  maturation  lysis of mother cell
 Properties of endospore:
1. Core
2. Spore Wall
3. Cortex
4. Coat
5. Exosporium
 GERMINATION
1. ACTIVATION
2. INITIATION
3. OUTGROWTH

Important Diseases:
 Anthrax (Bacillus anthracis)
  Food poisoning (Bacillus cereus)
 Tetanus (Clostridium tetani)
 Gas gangrene (Clostridium perfringens)
 Botulism (Clostridium botulinum)

Bacillus
 Large, aerobic, Gram-positive rods occurring in chains
 Most members are saprophytic organisms prevalent in soil, water & air and on vegetation ( Bacillus cereus, Bacillus subtilis)
 Insect pathogens
 Bacillus cereus grow in foods, produce enterotoxin or an emetic toxin  food poisoning; produce disease in
immunocompromised humans (e.g. meningitis, endocarditis, endophthalmitis, conjunctivitis, acute gastroenteritis)
 Bacillus anthracis causes anthrax

 MORPHOLOGY & IDENTIFICATION
Typical cells: (1 x 3-4 mm) have SQUARE ENDS, arranged in long chains, spores located at the CENTER of a
nonmotile bacteria
Culture: round, “cut glass” /“ground-glass” appearance in transmitted light
Hemolysis Uncommon with B. anthracis
Gelatin is liquefied
Growth in gelatin stabs resembles an INVERTED FIR TREE
Growth Characteristics:
saprophytic bacilli utilize simple sources of nitrogen & carbon for energy & growth
spores are resistant to environmental changes, withstand dry heat, & certain chemical
disinfectants for moderate periods; persist for years in dry earth

 Bacillus anthracis (Anthrax bacillus)

 Large, Gram-positive, nonmotile, spore-forming rods (bacilli) found in chains

 Virulent strains are pathogenic for animals, encapsulated, with a capsule composed of poly-D-
glutamic acid
 Aerobic anthrax bacteria grow well on blood agar media after overnight incubation at 35oC
without CO2
 Characteristic colonies (2-5 mm) have a “ground-glass” appearance; nonhemolytic,
nonpigmented, edge-irregular with comma projections (“Medusa head”colonies)
 On gelatin medium, inverted pine tree/ fir tree growth is seen
 In PLET (Polymyxin Lysozome Ethylenediamine Tetraacetate, colonies stand-up like beaten
egg whites when pushed and lifted with an inoculating needle

ANTHRAX- Primarily a disease of hervivores

Humans become infected incidentally by contact with infected animals or their products
In animals: portal of entry – mouth & the GIT
In humans:
 entry of spores through injured skin (cutaneous anthrax) : entry of bacilli trough skin abrasions
  or rarely, the mucous membranes (gastrointestinal anthrax): improperly cooked meat

 or by inhalation of spores into the lung (inhalational anthrax) handling wools or animal hides

The vegetative bacteria have three virulence factors:

Antiphagocytic capsule (poly-D-glutamic acid)-plasmid-coded
2 toxins - EDEMA TOXIN
LETHAL TOXIN

Anthrax toxin – made up of 3 proteins:

 PROTECTIVE ANTIGEN (PA)
binds to specific cell receptors; forms a membrane channel that mediates the entry of the EF & LF into the cell
 EDEMA FACTOR (EF)
Calmodulin dependent adenylyl cyclase; part of edema toxin (with PA) ,cause cellular sweling
 LETHAL FACTOR (LF)
with PA, forms the lethal toxin , zinc metalloprotease

 LETHAL TOXIN
 Specific endopeptidase that cleaves the kinase family of proteins and interferes with intracellular
signaling
 Targets the macrophages, causing hemorrhagic necrosis in the lymph nodes, resulting in the release of
large numbers of B anthracis

 Inhalational Anthrax (“Woolsorters’ disease)

Incubation period: 1-6 days
Spores from the dust of wool, hair or hides are inhaled  phagocytosed in the lungs  lymphatic drainage to the
mediastinal LN  GERMINATION  TOXIN PRODUCTION
 95% - cutaneous anthrax, 5% inhalation
 CUTANEOUS ANTHRAX
 Occurs on exposed surfaces of the arms or hands, face & neck
 Starts as pruritic papule 1-7 days after entry of organisms or spores through the scratch
 Papule  vesicle or small ring of vesicles that coalesce  necrotic ulcer
 Lesions are 1-3 cm in diameter
 Central black eschar
 Marked edema
 Lymphangitis, lymphadenopathy
  Nonspecific flu-like illness - fever, malaise, headache, nonproductive cough
 Healing by granulation, leaves a scar
 Can lead to sepsis, meningitis, death
 Incubation period as long as 6 weeks
 Marked hemorrhagic necrosis & edema of the mediastinum
 Substernal pain
 Hemorrhagic pleural effusions (involvement of the pleura)
 Cough (effects to the trachea)
 Sepsis
 Hematogenous spread to the GIT  bowel ulceration, meninges  hemorrhagic meningitis
 High fatality rate

 Diagnostic Laboratory Tests

Specimens: fluid or pus from a local lesion, blood & sputum
 Stained smears show chains of large gram-positive rods
 Anthrax can be identified by immunofluorescence staining techniques
 On blood agar plates: nonhemolytic gray to white colonies with a rough texture & a ground-glass appearance
 On blood agar plates:
- comma-shaped outgrowths (Medusa head) may project from the colony
 Gram stain: large gram-positive rods
 Carbohydrate fermentation not useful
 On semisolid medium: B anthracis nonmotile, B cereus exhibit motility by “swarming”
 Demonstration of capsule: growth on bicarbonate-containing medium in 5-7% CO2
 ELISA- measure antibodies against edema and lethal toxins (positive result: 4-fold change or a single titer of greater
than 1:32)
 String of Pearl Test: penicillin sensitivity test (pearl-like colonies)
 Ascoli Test: diagnostic precipitation of B anthracis

 Treatment
 Must be started early
 Ciprofloxacin recommended
 Two or more antimicrobial agents predicted to be effective recommended
 Treatment of systemic B anthracis infection with penicillins, cephalosporins or TMP-SMX is not recommended
because bioterrorism-related strains may be resistant to these drugs
 Supportive care includes controlling pleural effusions
  Prophylaxis
 Ciprofloxacin or Doxycycline
 Given for 4 weeks while 3 doses of vaccine is being given
 Given for 8 weeks if no vaccine
 Prevention
 Clinical suspicion should be high for bioterrorism-related inhalational anthrax
 In the event of a presumptive bioterrorism event, medical personnel must be alert to coordinate testing, packaging &
transporting with the public health laboratory (obtain appropriate specimens)
 Suspected or confirmed anthrax cases must be reported immediately to local or state departments of health
 Direct contact with wound or wound drainage should be avoided
 Disposal of animal carcasses by burning or by deep burial in lime pits
 Decontamination (autoclaving) of animal products
 Protective clothing & gloves for handling potentially infected materials
 Active immunization of domestic animals with live attenuated vaccines
 Immunization of persons with high occupational risk

Bacillus cereus
 FOOD POISONING
 Emetic type – associated with fried rice
 Begins 1-5 (mean 2) hours after ingestion of rice, pasta dishes
 Nausea, vomiting, abdominal cramps, occasional diarrhea
 Self-limiting; recovery within 24 hours
 Soil organism that commonly contaminates rice
 When large amount of rice is cooked and allowed to cool slowly, spores germinate & vegetative cells
produce the toxin (heat stable) during log-phase growth or during sporulation
 Diarrheal type – associated with meat dishes &sauces
 Incubation period: 1-24 hours (mean 9)
 Profuse diarrhea with abdominal pain and cramps
 Fever & vomiting uncommon
 Enterotoxin (heat labile) may be preformed in the food or produced in the intestine
 Produces toxins that cause disease intoxication > food-borne infection
 Diagnostic: concentration of 105 bacteria or more/ gram of food
 EYE INFECTIONS
 Severe keratitis
 Endophthalmitis Associated with trauma
 Panophthalmitis
 SYSTEMIC INFECTIONS
 Endocarditis
 Meningitis Predisposed by presence
 Osteomyelitis of medical device or IV drug use
 Pneumonia

Bacilus subtilis (Hay bacillus)

 Source of bacitracin
 Cause eye infections in herion addicts
 BAP: colonies usially large, flat and dull with ground glass appearnce

BIOCHEMICAL TEST SUMMARY

PARAMETERS B.anthracis B. cereus

MOTILTIY - +
CAPSULES + -
HEMOLYSIS GAMMA BETA
SALICIN FERMENTATION - +
GROWTH @ 45 C - +
PENICILLIN SENSITIVITY SENSITIVE RESISTANT
GAMMAPHAGE SENSITIVE RESISTANT
LECITHINASE + +
OXIDASE + +
CATALASE + +
CLOSTRIDIUM
 Anaerobic
 Gram-positive
 Motile
 Rods
 Decompose proteins, form toxins or both
 Natural habitat: soil or intestinal tract of animals and humans
 MORPHOLOGY & IDENTIFICATION
 Typical Organisms
 Spores wider than the diameter of the rods in which they are formed
 In various species, spores are placed centrally, subterminally, or terminally
 Most are motile and possess peritrichous flagella
 Culture - Anaerobes, few aerotolerant, Grow well on the blood-enriched media used to grow anaerobes

 Colony Forms
 Some clostridia produce large raised colonies (C. perfringens)
 Some produce smaller colonies (C. tetani)
 Some form colonies that spread on the agar surface
 C perfringens produces multiple zones of hemolysis around colonies

Double zone
-hemolysis

 Growth Characteristics
 Can ferment a variety of sugars; many can digest proteins
 Milk is turned acid by some, digested by others & undergoes “stormy fermentation” (clot torn by gas)
 Various enzymes produced by different species
 With peritrichous flagella except: C. perfirngens, C. ramosum, C.innoculum
 All have swollen sporangia except: C. perfringens, C.bifermentans
 All are non-encapsulated except: C. perfringens
 All are single hemolytic except: C. perfringens (double zone)

Classification
 Histotoxic group/Gas Gangrene
o Tissue death due to ischemia (lack of blood flow)
o Cause sever infection of the muscle termed as clostridial myonecrosis
 Clostridium perfringens
 Toxigenic group
o Clostridium tetani
o Clostridium botulinum

Clostridium botulinum (Canned Good Bacillus)

 Worldwide distribution
 Found in soil, occasionally in animal feces
 Types are distinguished by the antigenic type of toxic they produce
 Spores are highly resistant to heat (withstand 100oC for several hours)
 Heat resistance diminished at acid pH or high salt concentration
 Botox is a commercial preparation of exotoxin A uses: wrinkle removal
 TOXIN
- Liberated during growth and autolysis into the environment
- 7 antigenic types known (A-G)
- Types A, B & E (occasionally F) are the principal causes of human illness
- Types A & B associated with varied foods
- Type E associated with fish products
- Types C & D are not human pathogens
- MW 150,000
 - Protein cleaved into 100,000-MW & 50,000-MW proteins linked by disulfide bond
- destroyed by heating for 20 min at 100 0C
- Lethal dose = 1-2 μg enough to eradicate the population of the world/ humankind

Toxin absorbed from the gut

Binds to receptors of presynaptic membranes of motor

neurons of the PNS & CNs

Inhibition of the release of acetylcholine at the synapse

(proteolysis of the target SNARE proteins in the
neurons)

Lack of muscle contraction and paralysis (flaccid)

 SNARE proteins
 Synaptobrevin
 Cleaved by toxin B
 SNAP 25
 Cleaved by toxins A and E
 Syntaxin
 C. botulinum toxins are among the most toxic substances known
 3 kinds of botulism:
 Infant botulism (72%) Floppy Baby Syndrome
 is caused by swallowing the spores, which then grow inside of the infant's intestines and release toxin
 Causes SIDS by feeding contaminated “honey”, corn syrup
 Foodborne botulism (25%)
 is caused by eating foods that contain the botulinum toxin
 spiced, smoked, vacuum-packed, home-canned alkaline (pH >4.6) foods (fruits, sausage, fish products)
“bulging” cans
 Wound botulism (3%)
 is caused by a nerve toxin produced from an infected wound

 CLINICAL FINDINGS
- symptoms begin 18-24 hours after ingestion of the toxic food
- visual disturbances (incoordination of eye muscles, double vision)
- inability to swallow & speech difficulty
- respiratory paralysis & cardiac arrest
- GI symptoms not regularly prominent
- (-) fever
 - conscious until shortly before death
- high mortality
- Patients who recover do not develop antitoxin in the blood
- Infant botulism: poor feeding, weakness, signs of paralysis (floppy baby), may be a cause of sudden infant death
syndrome

Contamination of traumatized areas (soil, feces) or from the

intestinal tract

Spores germinate at low oxidation-reduction potential

Spread of infection (distention of tissue, interference with

blood supply, necrotizing toxin, hyaluronidase)

Increased bacterial growth, hemolytic anemia, severe

toxemia, DEATH

 DIAGNOSTIC LABORATORY TESTS

- toxin can be demonstrated in serum from the patient and may be found in leftover food
- mice injected intraperitoneally dies rapidly
- type of toxin is identified by neutralization of specific antitoxin in mice
- organism may be grown from food remains & tested for toxin production
- infant botulism: organism & toxin are found in feces but not in serum
- Toxin maybe demonstrated by passive hemagglutination or radioimmunoassay
 TREATMENT
- potent antitoxins have been prepared in horses
- trivalent antitoxin (A,B,E) is promptly administered intravenously (to neutralize toxin not yet internalized in
neurons)
- adequate ventilation must be maintained by mechanical respirator
 EPIDEMIOLOGY, PREVENTION & CONTROL
- Spores are widely distributed in soil
- Often contaminate vegetables, fruits & other materials
- home-canned foods are of highest risk for contamination string beans, corn, peppers, olives, peas
- smoked fish, vacuum-packed fresh fish
- tomatoes or tomato sauce are sufficiently acidic to prevent growth
- Canned or preserved foods must be sufficiently heated to ensure destruction of spores or must be boiled for 20
minutes before consumption
- Toxoids for active immunization of cattle

Clostridium tetani (Tack-head bacillus)

 Spore located terminally and sporadium is swollen thus giving the characteristic features: drumstick, tennis racket/
lollipop appearance

 Causative agent for tetanus (Wasserman Takaki Phenomenon)

 Toxins produced:
o Tetanolysis responsible for beta hemolytic in BAP
o Tetanospasmin neurotoxin, responsible for tetanus
 Binds to the ganglioside of the brain
  Clinical Manifestation
o Trismus/ Lock Jaw: spasm of muscle with Risus sardonicus/sardonic grin spasm of fascial muscle

o Opisthotonus: arching of the back

Clostridium perfringens - GAS GANGRENE/ MYONECROSIS

 most common among the 30 species that produces invasive infection when introduced in damaged tissues (90%)
 may also produce enterotoxin that causes food poisoning
 can be found as a normal component of:
 decaying vegetation
 marine sediment
  the intestinal tract of humans and other vertebrates, insects
 Soil
 Capsulated
 Nonmotile
 Exhibit double zone of hemolysis
 Does not usually form spores when grown in laboratory
 Also called Clostridium welchii, Bacillus aerogenes capsulatus
 TOXINS: Produce large variety of toxins which have lethal, necrotizing and hemolytic properties
- Alpha toxin of type A Clostridium perfringens (Lecithinase)
Splits lecithin (an impt component of cell membranes) to phosphorylcholine and diglyceride
- Theta toxin - Has hemolytic and necrotizing effect
- Dnase
- Hyaluronidase - collagenase that digest collagen of subcutaneous tissue and muscle
- Enterotoxin - meat dishes
- >108 vegetative cell are ingested
- a protein (MW 35,000) that may be a non-essential component of the spore coat.
- Induces intense diarrhea in 6-18 hours
- Action involves marked hypersecretion in the jejunum and ileum, with loss of fluids &
electrolytes in diarrhea
- less frequent symptoms: nausea, vomiting, and fever
- tends to be self-limited
- Opalescence (due to alpha toxin) on serum egg yolk agar (Naglers Reaction/Lecithinase test)

-
Clinical Findings:
 Contaminated wound:
 crepitation in the subcutaneous tissue & muscle
 foul-smelling discharge
 rapidly progressing necrosis
 Fever
 Hemolysis
 Toxemia
 Shock
 Death
 Food poisoning:
 Onset of diarrhea within 6-18 hours after ingestion of large inocula frown in warmed meat dishes
 No vomiting & fever
 Last only 1-2 days
Diagnostic Laboratory Tests:
 Specimens:
 material from wounds, pus, & tissue
 Gram-stained smears:
 presence of large gram-positive rods
 spores not regularly present
 Culture:
 inoculated into chopped meat-glucose medium & thioglycolate medium & unto blood agar plates.
 incubated anaerobically
 Culture:
 a clot torn by gas in 24 hrs when the growth from one media is transferred into milk
 Identified by chemical reactions (various sugars in thioglycolate, action on milk), hemolysis, colony form
 lecithinase activity evaluated by the precipitate formed around colonies on egg yolk media
  Final identification rests on toxin production and neutralization by specific antitoxin
 Reverse camp test

Treatment
 prompt and extensive surgical debridement of the involved area, excision of all devitalized tissue
 administration of antimicrobial drugs, particularly penicillin
 hyperbaric oxygen
 Antitoxins
 food poisoning due to enterotoxin requires symptomatic care only
Prevention and Control
 early & adequate cleansing of contaminated wounds
 surgical debridement
 antimicrobial drugs
 antitoxins

Clostridium difficile – PSEUDOMEMBRANOUS COLITIS

 Carried asymptomatically as part of the large intestinal flora of 50% of all healthy neonates during the first year of life;
carriage rate decreases to <4% in adults
 Primary cases occur via endogenous mode in precolonized patients exposed to antibiotics
 Secondary cases occur via exogenous transmission of spores in the hospital environment and by the hands of health care
attendants
 C. difficile is ordinarily suppressed by the normal colonic flora, preventing overgrowth
 Broad-spectrum antibiotics suppress normal flora
 Cultured in anaerobic chamber on CCFA (Clycloserine Cefoxitin Fructose Agar), colonies have horse-manure odor
 Produces 2 toxins – toxin A and B
 Toxin A (potent enterotoxin)
 Toxin B (potent cytotoxin)
 Detection of one or both Clostridium difficile toxins in stool and by endoscopic observation of pseudomembranes or
microabscesses in patients who have diarrhea and have been given antibiotics
 Plaques and microabscesses may be localized to one area of the bowel
 Diarrhea may be watery or bloody
 Abdominal cramps, leukocytosis, fever
 Most commonly associated antibiotics:
 Ampicillin
 Clindamycin
 Treated by discontinuing administration of the offending antibiotic & orally giving metronidazole or vancomycin
 PREVENTION:
 Limit use of broad-spectrum antibiotics
 Handwashing
 Removing gloves before attending to another patient
 Enteric precautions