Gram Positive Bacilli

Gram positive spore forming bacilli are the Bacillus and Clostridium species
These bacilli are ubiquitous and they form spores. They can survive in the environment
BACILLUSAEROBES
CLOSTRIDIAANAEROBES
BOTHmost do not cause disease and are not well characterized in medical microbiology

Bacillus anthracisAnthrax
---major agent of bioterrorism and biologic warfare
Bacillus cereusfood poisoning and occasionally eye or other localized infections
Clostridia---cause several important toxin-mediated disease
Clostridium tetanitetanus
Clostridium botulinumbotulism
Clostridium perfringensgas gangrene
Clostridium difficilepseudomembranous colitis
*other clostridia are also found in mixed anaerobic infections in humans

BACILLUS SPECIES
Includes aerobic, gram positive rods occurring in chains
Most members of this genus are Saprophytic organisms prevalent in SOIL, AIR and WATER, and Vegetation such as B. subtilis
and B. cereus
Some are insect pathogen
B. cereus can grow in foods and produce Enterotoxin or an Emetic toxin and cause food poisoning.
Some organisms may occasionally produce disease in immunocompromised humans (ex. Meningitis, endocarditis,
endopthalmitis, conjunctivitis, or acute gastroenteritis)
B anthracis causes anthrax, is the principal pathogen of the genus.

Morphology
Measure::1x3-4m,
Have square ends and arranged in long chains
Spores are located in the center of the nonmotile bacilli
Colonies of B anthracis are round and have a cut glass appearance in transmitted light
B. anthracisUncommon Hemolysis
Saprophytic BacilliCommon Hemolysis
*Gelatin is liquefied, an dgrowth in gelatin stabs resembles an inverted fir tree.

Growth Characteristics
Saprophytic bacilliutilize simple sources of Nitrogen and Carbon for energy and growth.
Spores are resistant to environmental changes, withstand heat and certain chemical disinfectants
for moderate periods, and persist for years in dry earth.
Animal products contaminated with antrax spores ex. Hides, bristles, hair, wool, bone.can be sterilized by autoclaving.

BACILLUS ANTHRACIS
Pathogenesis
ANTHRAX
Primarily a disease of herbivoresgoats, cattle, horses
Other animals like rats are relatively resistant to the infection
Humans become infected incidentally by contact with infected animals and their products.
In animals, the portal of entry is the MOUTH and the GI tract.
Spores from contaminated soil find easy access when ingested with spiny or irritating vegetation.
In humans, the infection is usually acquired by entry of the spores through::
injured skin (cutaneous anthrax)
mucous membranes (gastrointestinal anthrax)
inhalation of spores into the lungs (inhalation anthrax)
Spores germinate in the tissue at the site of entry, and growth of the vegetative organisms result in formation of a gelatinous
edema and congestion.
Bacilli spread via lymphatics to the bloodstream, and they multiply freely in the blood and tissues shortly before and after the
animals death.
B. anthracis that does not produce a capsule is not virulent and does not induce anthrax in test animals.
The poly-D-glutamic acid capsule is antiphagocytic. capsule gene is on a plasmid.


Anthrax toxin is made up of 3 proteins
1. Protective Antigen (PA)
2. Edema factor(EF)
3. Lethal Factor(LF)

Inhalation anthrax (woolsorter) disease
The spores form the dust of wool, hair, hides are inhaled. Phagocytosed in the lungs transported by the lymphatic drainage to
the mediastinal lymph nodes,
This Is followed by toxin production with the development of hemorrhagic, mediastinitis and sepsis, w/c are usually rapidly
fatal.
In anthrax sepsis, number of organism in the blod exceeds 10^7/mL prior to death



Pathology
In susceptible animals, the organisms proliferate at the site of entry.
The capsule remain intact and the organisms are surrounded by a large amount of proteinaceous fluid containing few
leukocytes from w/c they rapidly disseminate and reach the bloodstream
In resistant animals, the organism proliferate for a few hours by which time there is a massive accumulation of leukocytes.
The capsule gradually disintegrate and disappear. the organism remain localized.

Clinical findings
HUMANS95% of cases: cutaneous anthrax
--5% ARE INHALATION ANTHRAX
--gastrointestinal anthrax is very rare
Cutaneous anthrax generally occurs on exposed surfaces of the arms or hands, followed by face and neck
A pruritic papule develops 1-7 days after entry of the organisms or spores through a scratch
1
st
it resembles an insect bite
2
nd
papule rapidly changes into vesicle or small ring of vesicles that coalesce and a necrotic ulcer develops
Lesions are typically are 1-3 cm in dm. and have a central black eschar
Marked edema occurs
Lymphangitis and lymphadenopathy and systemic s/sx of fever malaise and headache may occur
After 7-10 days-eschar is fully developed.
Eventually it dies, loosens and separates
Healing is by granulation and leaves a scar
Takes weeks for the lesion to heal and edema to subside
Antibiotic therapy does not appear to change the natural progression of the disease
Cutaneous anthrax can lead to sepsisthe consequence of systemic infectionincluding meningitis and death.

Incubation period for inhalation anthrax6 weeks
Early manifestations
Marked hemorrhagic necrosis
Edema of the mediastinum
Substernal pain may be prominent
Pronounced mediastinal widening visible on x-ray chest films

Hemorrhagic pleural effusion follow involvement of the pleura
Cough is secondary to the effects on the trachea
Hematogenous spread to the known exposure
It is higher when the diagnosis is not initially suspected.

Animals acquire through ingestion of spores and spread of the organisms from the intestinal tract.
This is rare in humans
Abdominal pain, vomiting and diarrhea are clinical signs.

Diagnosis of Lab Test
Specimens::fluid or pus from a local lesion, blood sputum
Stained smears from the local lesion on the blood, from dead animals often show chains of long(+)
When grow on blood agar platesorganisms produce non-hemolytic
Gray to white colonies with a rough texture and around glass appearance
Comma shaped outgrowth (medusa head) may project from the colony.
Gram stain shows a large gram positive rods
CHO fermentation is not useful
In semisolid medium, anthrax bacilli are always nonmotile where as,
Related pathogenic organisms exhibit motility swarming
Virulent anthrax cultures kill mice or guinea pigs upon intraperitoneal injection.
Demo of capsule requires growth on bicarbonate containing medium in 5-7% carbon dioxide, lysis by a specific anthrax y-
bacteriophage may be helpful in identifying the organism
ELISAto measure antibodies against edema and lethal toxin but the test has not been extensively studied
Acute and convalescent sera obtained 4 weeks apart should be tested.
A (+) result is a few fold change or a single titer of greater than 1:32.

Resistance and Immunity
Based on the classic experiment of Louis Pasteur
In 1881, he proved that cultures grown in broth at 42-52 degrees Celsius for several months lost much of their virulence and
could be injected live into sheep and cattle w/o causing disease, subsequently such animals proved to be immune
Active immunity to anthrax can be induced in susceptible animals by vaccination with live attenuated bacilli, with spore
suspensions, or with protective antigens from culture filtrates.
The amount of protective antigen present per dose is unkown and all three toxins components (LF, EF, PA) are present and
absorbed to aluminum hydroxide.
The dose schedule is 0, 2 and 4 weeks then 6, 12, and 18 months.followed by annual boosters.

Treatment
Antibiotic are effective against anthrax in humans, but tx should start early
Ciprofloxacin is recommended for treatment; Penicillin G along with gentamycin or streptomycin
In the setting for potential exposure to B. anthracis as an agent of biologic warfare, prophylaxis with ciprofloxacin or
doxycycline should be continued for 4 weeks while three doses of vaccine are being given, or for 9 weeks if no vaccine is
administered.
Other Gram positive bacilli, such as B cereus are resistant to penicillin by virtue of B-lactamase production. Doxycycline,
erythromycin, or ciprofloxacin may be effective alternatives to penicillin.

Epidemiology, prevention, control
SOIL is contaminated with anthrax spores from the carcasses of dead animals.
Spores remain viable for decades.
Spores can germinate in soil at pH----6.5 at proper temp.
Infection through mucous membranes serve to perpetuate the chain of infection.
CONTROL MEASURES
1. Disposal of animal carcasses by burning or deep burial in lime pits,
2. Decontamination(usually by autoclaving )animal products
3. Protective clothing and gloves for handling potentially infected materials,
4. Active immunization of domestic animals with live attenuated vaccines.

BACILLUS CEREUS
2 distinct forms
1. Emetic typemanifested by Nausea, vomiting, abdominal cramps and occasionally diarrhea and is self-limiting
Fried rice
with recovery w/n 24 hours
it begin 1-5 hours after ingestion of rice and cereus spores germinate and the vegetative cells produce the
toxin during log phase growth and sporulation.
2. Diarrheal typeproduce toxins that cause disease that is more intoxication than food borne infection
Has an incubation period of 1-24 hours and is manifested by profused diarrhea with abdominal pain and
cramps, fever and vomiting are uncommon.
The enterotoxin may be preformed in the food or produced in the intestine.

Presence of B.cereus in a patients stool is not sufficient to make a diagnosis of B. cereus disease since the bacteria may be
present in normal stool specimen.
A concentration of 10^5 bacteria or more per gram of food is considered diagnostic
Important cause of eye infection, severe keratitis, endophthalmitis and panophthalmitis.
Associated with local and systemic infection including endocarditis, meningitis, osteomyelitis and pneumonia.
Presence of medical device or IV drug use predisposes to these infections

5 Bacillus Species
1. B. thuringiensis---coding for insecticidal compounds have been inserted into the genetic material of some commercial plants.
2. B. popilliae
3. B. sphaericus pathogen for insects
4. B. larvae
5. B. lentimorbus

Clostridium Species
Large anaerobic gram (+), motile rods
Decomposes proteins or form toxins and some do both
Natural habitat is SOIL or Intestinal tract of animals and humans, where they live as saprophytes
THEY CAUSE::
1. Botulism
2. Tetanus
3. Gas gangrene
4. Pseudomembranous colitis

Typical Organism
Spores are usually wider than dm. of the rods in which they are formed
Spore is placed centrally subterminally or terminally
They are motile and possess peritrichous flagella

Culture
Anaerobes and grows under anaerobic conditions
Few species are aerotolerate and will also grow in ambient air.
Clostridia grow well in the blood-enriched media used to grow anaerobes and on other media used to culture anaerobes

Colony Forms
Large colonyC. perfringens
Small colonyC. tetani
C. perfringens typically produces multiple zones of hemolysis around colonies.

Growth characteristics
1. Clostridia can ferment a variety of sugars, digest proteins
2. Milk is turned acid by some and digested by others and undergoes stormy fermentation

Antigenic characteristics
1. Clostridia share some antigens but also possess specific soluble antigens that permit grouping by precipitin test.

Clostridium Botulinum
It is found in SOIL and occasionally animal FECES
Types are distinguished by the antigenic type of toxin they produce
Spores are highly resistant to heat, withstanding 100 degrees C for several hours.
Heat resistance is diminished at acid pH or high salt concentration.

1. A, B, E (occasionally F)principal causes of human illness.
2. A, Bassoc. with a variety of food
3. Eassoc. predominantly with fish products
4. Cproduces limberneck in birds
5. Dcauses botulinum in mammals

Botulinum toxin is absorbed from the gut and binds to receptors of presynaptic membranes of motor neurons of the peripheral
nervous system and cranial nerves.
Proteolysisby light chain of botulinum toxin of the target SNARE proteins in the neurons, inhibit the release of acetylcholine at
the synapses resulting in lack of muscle contraction and paralysis.
SNARE proteins are synaptobrevin, SNAP 25 and syntaxin
Toxins of C. botulinum type A and E cleave 25,000-MW SNAP-25.
B toxin cleaves synaptobrevin
C. botulinum toxin are among the most toxic substance
Lethal dose for human is probably about 1-2 g
The toxins are destroyed by heating for 20 min. at 100 degrees C.

Pathogenesis
C.botulinum types A and B-cases of wound infection and botulism.
Most common offenders are spiced, smoked, vacuum packed, or canned alkaline foods that are eaten w/o cooking.
In foods, C. botulinum germinate under anaerobic conditions, vegetative forms grow and produce toxin.
Toxin act by blocking release of acetylcholine at synapses and neuromuscular junction, flaccid paralysis results.

Clinical Findings
Begin 18-24 hours after ingestion of toxic food with visual disturbances (in coordination of eye muscles, double vision)
Inability to swallow
Speech difficulty
Signs of bulbar paralysis are progressive
Death occurs for resp. paralysis or cardiac arrest.
GI symptoms are not regularly prominent.
NO FEVER
Infants
1
st
month develops poor feeding weakness and signs of paralysis.(floppy baby)
Infant botulinumone of the causes of sudden infent death syndrome
C. botulinum and toxin is found in feces but not in serum.

Diagnostic Lab. Test
Often demonstrated in seven
Toxin can be found in left overfood
Antigenic type is identified by neutralization with specific antitoxin in mice.
In infant Botulismtoxic can be demonstrated in bowel but not in serum
Toxin may be demonstrated by passive hemaglutination or radio immunoassay

Treatment
Trivalent (A, B, E) antitoxin must be proptly administered IV with precaution
Adequate ventilation must be maintained by mechanical .

Epidemiology, Prevention and Control
They are widely distributed in SOILoften contaminate vegetables, fruits
Canned foodsheat 20 min.before consumption
Toxoids are used for active immunization of cattle
Btoxin considered to be major agent, for bioterrorism and biologic walfare.

Clostridium Tetani
Causes tetani
Distribution in the soil and feces or horse
Several can be distinguished by specific flagellar antigens.
Neurotoxin, tetanospasmin

TOXIN
Toxin produce tetanospasmin (MW 150,000) that is cleaved by bacterial protease into 2 peptides (MW 50, 000) and 100, 000
linked by disulfide bond.
Initially, toxin binds to receptors on the presynaptic membrane of motor neurons.
Migrates by the retrograde axonal transport system to the cell bodies of these neurons to the spinal cord and brainstem.
Toxin diffuses to terminals of inhibitory cells, including both glycinergic interneurons and aminobutyric acid secreting neurons
from the brainstem.
Toxin degrades synaptobrevina protein required for docking of neurotransmitter vesicles on the presynaptic membrane
Inhibitory glycine and y-aminobutyric acid is blocked
Motor neuronsnot inhibited
Hyperreflexia, muscle spasm and spastic paralysis result

Pathogenesis
C. tetani is not an invasive organism
Infection remains strictly localized in the area of wound, burn, injury, umbilical stump, surgical suture.
Disease is almost entirely a toxemia,

Aided by::
1. Necrotic tissue
2. Calcium salts
3. Assoc.pyogenic infection
Toxin released from vegetative cells reaches CNS cells and rapidly becomes fixed in the spinal cord and brainstem.

Clinical Findings
1. Incubation period 4-5 days to weeks
2. Characterized by tonic contraction of voluntary muscles
3. Muscular spasm often involve first the area of injury and infection and then muscles of the jaw.( trismus, lockjaw)
4. Other voluntary muscles become involved resulting in tonic spasm
5. Patient is fully conscious and pain maybe intense.

Diagnosis
1. History of injury
2. Only 50% of patient with tetanus have an injury for which they seek medical attention
3. Differential diagnosis is strychnine poisoning
4. Anaerobic culture may yield C. tetani

Prevention and treatment
Prevention is all important
Depends upon:
1. Active immunization w/ toxoids
2. Proper care of wounds contaminated with soil
3. Prophylactic use of antitoxin
4. Administration of penicillin
IM administration of 250-500 units of human antitoxin (tetanus immune globulin) gives adequate systemic protection (0.01 unit
or more per mL of serum) for 2-4 weeks.
Active immunization with tetanus toxoid should accompany antitoxin prophylaxis.
Pt. who develop symptoms of tetanus should receive muscle relaxants, sedation and assisted ventilation-3000-10,000 units.
The efficacy of antitoxin for treatment is doubtful except in neonatal tetanus.
Surgical debridement is vitally important because it removes the necrotic tissue that is essential for proliferation of the
organisms
Hyperbaric oxygen has no effect
Penicillin strongly inhibits the growth of C. tetani and stops further toxin production.
Antibiotics may also control assoc.pyogenic infection.
For previously immunized individual sustains a potentially dangerous wound, additional dose of toxoid should be injected to
restimulate antitoxin production.

Control
Tetanus is totally a preventable disease.
Universal active immunization with tetanus toxoid should be mandatory
Tetanus toxoid is produced by detoxifying the toxin with formalin and then concentrating it. Aluminum salt adsorbed toxoids
are employed.
3 injections comprise the initial course of immunization, followed by another dose about 1 year later.\
Initial immunization should be carried out in all children during the 1
st
year of life.
A booster injection of toxoid is given upon entry to school.
Boosters can be placed 10 years apart to maintain serum levels of more than 0.01 unit antitoxin per mL.
In young children TT is often combined with diphtheria toxoid and pertussis vaccine.

Clostridia that produce invasive Infections
Clostridium perfringens and related clostridia can produce invasive infections including myonecrosis and gas gangrene if
introduced into damaged tissue.
An enterotoxin of C. perfringens is common cause of Food POISONING.

Toxins
Invasive clostridia produce variety of toxins and enzymes that result in s spreading infection. Many of these toxins have lethat,
necrotizing and hemolytic properties.
Alpha toxin of C. perfringens type A is a lecithinaselethal action is proportionate to the rate at which it splits lecithinan
impotant constituent of cell membranes) to phosphorylcholine and diglyceride.
The theta toxin has similar hemolytic and necrotizing effects but is not lecithinase.
DNase and hyaluronidase, collagenase that digests collagen of subcutaneous tissue and muscle are also produced.
When more than 10^8 vegetative cells are ingested and sporulate in the gut, enterotoxin is formed.
Enterotoxin is a protein (MW 35, 000) that may be a nonessential component of the spore coat.
It induces intense diarrhea in 6-18 hours.
The enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of fluids and electrolyts in diarrhea.
Much less frequent symptoms include Nausea, Vomiting and Fever.
This illness is similar to B. cereus and tends to be self-limited.

Pathogenesis
Spores reach tissue by contamination areas (soil, feces) or from the Intestinal tract.
The spore germinate at low oxidation-reduction potential, vegetative cells multiply, ferment carbohydrates present in tissue and
produce gas.
The distention of tissue and interference with blood supply, together with the secretion of necrotizing toxin, favor the spread of
infection.
Tissue necrosis extends providing opportunity for increased bacterial growth, hemolytic anemia and severe toxemia and death.
In gas gangrene-clostridial myonecrosisa mixed infection is the rule.
C. perfringens occurs in the genital tract of 5% of women.
C. sordellii has been reported to cause a toxic shock syndrome after medical abortion with mifepristone and intravaginal
misoprostol.
Endometrial infection with C. sordellii is implicated.
Clostridial bacteremia is a frequent occurrence in patients with neoplasms
C. perfringens type C produces a necrotizing enteritis (pigbel) that can be highly fatal in children.
Immunization with type C toxoid appears to have preventive value.

Clinical Findings
Contaminated woundthe infection spread in 1-3 days to produce crepitation in the subcutaneous tissue and muscle, foul-
smelling discharge, rapidly progressing necrosis, fever, hemolysis, toxemia, shock and death.
Treatment is with early surgery(amputation) and antibiotic administration.
Infection results only in anaerobic fasciitis or cellulities.
C. perfringens food poisoning usually follows the ingestion of large numbers of clostridia that have grown in warmed meat
dishes.
The toxin forms when the organism sporulate in the gut, with the onset of diarrhea-usually w/o vomiting or fever-in 6-18 hours.
The illness last only 1-2 days.

Diagnostic Laboratory Test
The prsesence of large gram-positive rods in Gram-stained smears suggest gas gangrene clostridia, spores are not regularly
present.
Material is inoculated into chopped meat-glucose medium and thioglycolate medium and onto blood agar incubated
anaerobically.
A clot torn by gas in 24 hours is suggestive of C. perfringens.
Lecithinase activity is evaluated by the precipitate formed around colonies on egg yolk media.
Final identification rest on toxin production and neutralization by specific antitoxin.
C. perfringens rarely produces spores when cultures on agar .

Treatment
Prompt and extensive surgical debridement of the involved area and exision of all devitalized tissue, in which the organisms are
prone to grow.
Administration of antimicrobial drugs, PENICILLIN
Hyperbaric Oxygen may be helpful in clostridial tissue infections-it detoxify patients rapidly.
Antitoxins are available against C. perfringens, noyvi, histolyticum, and septicum usually in the form of immune globulins.
Polyvalent toxin(containing antibodies ) can be used.
Food poisoning00symptomatic care

Prevention and Control
Early and adequate cleansing of contaminating wounds and surgical debridement, together with administration of antimicrobial
drugs directed against clostridia, e.q penicillin.

Clostridium Defficile and diarrheal disease.
Pseudomembranous colitis
Diagnosed by detection of one or both C difficile toxin in stool and by endoscopic observation of pseudomembranes or
microabscesses in patients who have diarrhea and have been given antibiotics
The diarrhea may be watery or bloody and the patient frequently has associated abdominal cramps, leukocytosis and fever.
Most common are ampicillin and clindamycin.
The disease is treated by discontinuing administration of the offending antibiotic and orally giving either metronidazole or
vancomycin.
Antibiotics result in proliferation of drug-resistant C difficile that produces two toxins.
Toxin Apotent enterotoxin that also has some cytotoxic activity, binds with the brush border membranes of the gut at
receceptor sites.
Toxin Bis apotent cytotoxin.
Both toxin are found in the stools of patients with pseudomembranous colitis.
Not all strains of C difficile produce toxin
Tox genes apparently are not carried on plasmid or phage.

Antiibotics-associated diarrhea
Administration of antibiotics frequently leads to a mild to moderate form of diarrhea, termed antibiotic-associated diarrhea. The
disease is generally less severe than the classic form of pseudomembranous colitis
25% of cases of Antibiotic0associated diarrhea may be associated with C. difficile.

Clinically important Gram positive bacilli
Spore forming
1. Bacillus- (O2)
2. Clostridium -no (O2)

Non spore forming
1.Listeria
2. Erysipelothrix
3.Corynobacterium
4. Propionibacterium

Acid-fast bacilli Non-acid-fast branching filamenous bacilli
1.Mycobacterium 1.Actinomyces
2.Nocardia