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1 MICRO 7 - Gram-Positive Bacilli - Dr. Sia-Cunco
1 MICRO 7 - Gram-Positive Bacilli - Dr. Sia-Cunco
Sia-Cunco
THE GRAM POSITIVE BACILLI JULY 8, 2013
OUTLINE
I. Gram Positive Spore Forming Bacilli Cultural characteristics
a. Bacillus o Inoculate on 5% Blood Agar Plate under aerobic conditions
i. B. anthracis o Produces large, raised, opaque, grayish white plumose colonies with
ii. B. cereus irregular fringe-like edge (Medusa head) with ground glass
iii. B. subtilis appearance.
iv. B. stearothermophilus o No hemolysis
b. Clostridium (anaerobic)
i. C. botulinum
ii. C. tetani
iii. C. perfringens
iv. C. difficile Medusa Head
II. Gram Positive Non-spore Forming Bacilli
a. Corynebacterium
i. C. diphtheriae
ii. C. pseudotuberculosis
iii. C. pseudodiphtheriticum and C. xerosis
b. Listeria monocytogenes
c. Erysipelothrix rhusiopathiae
d. Actinomycetes
May also be Acid Fast Figure 2 Medusa Head with ground glass appearance
e. Nocardia
Laboratory identification
GRAM (+) SPORE FORMING BACILLI
o Virulent strains produce ROUGH colonies when grown in the absence
BACILLUS
of CO2
Large ,aerobic, Gram (+) rods occurring in chains
o Virulent strains produce MUCOID colonies when grown under 5% CO2
Most are prevalent on soil, water, air and/or on vegetation
with NaHCO3
Typical organisms have square ends and are arranged in long chains with
o String of Pearls Test: B. anthracis produces colonies that resemble a
spores in the center of the bacilli
string of pearls when grown on a medium containing Penicillin (PCN).
Some members of the genus are the source of the antibiotics Polymixin B
B. cereus would be negative because it is not sensitive to PCN
and Bacitracin (Topical Antibiotics)
o Bacteriophage (a gamma phage) added to a diffusely inoculated plate is
expected to cause lysis only of B. anthracis. (No lysis of B.cereus)
Bacillus anthracis
o Inoculation of a mouse with organisms produces death in 2-5 days and
Causative agent of Anthrax, a disease of sheep and cattle and to a lesser organisms can be recovered in heart blood
extent horses, hogs and goats
Humans may acquire this in an agricultural setting or in an industrial
setting (processing of hides or animal hair with inhalation of spores).
Infection is usually initiated by the subcutaneous inoculation of spores
through incidental skin abrasions = Cutaneous Anthrax.
Also by inhalation of spore-laden dust (Pulmonary Anthrax)
B. anthracis spores may remain viable for many years in contaminated
pastures and in bones, wool, hair, hides, and other animal materials.
Potential use as a bioterrorism agent can be easily grown in large
quantities and the spores are resistant to destruction and can be
formulated into an aerosol for wide dissemination.
Virulence Factors
o Poly D-Glutamic Capsule (capsule usually interferes with phagocytosis)
o Exotoxin
Figure 1 Bacillus anthracis
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MICROBIOLOGY 1.6
Gradually accumulates in the infected animal with a maximum Nausea, vomiting, diarrhea and hematemesis and/or blood in
accumulation at the time of death the stool, eventual shock and death.
The toxin acts on the CNS producing respiratory failure and anoxia. o Anthrax infection provides permanent immunity.
o With 3 active proteins that act synergistically to produce the systemic
effects of anthrax. Individually, the three proteins have no known toxic
activity
Protective Antigen (PA)
Edema Factor (EF)
Lethal Factor (LF)
o The PA binds to cell receptors and is proteolytically activated
o PA mediates entry of EF and LF into the cell by endocytosis
o PA with EF forms the Edema Toxin which causes inhibition of
neutrophil function and elevation of intracellular cAMP, resulting in
impaired maintenance of water homeostasis and characteristic severe Figure 5 Black Eschar
edema usually seen in B. anthracis infections.
o LF with PA forms the Lethal Toxin which is a major virulence factor of Laboratory diagnosis of clinical manifestations
the organism and causes apoptosis and is responsible for tissue o Specimens for culture can be obtained from malignant pustule, sputum
necrosis. It causes death in infected animals and humans. or blood. Stained smears will show Gram positive blunt-ended bacilli
o LF also causes rapid release of inflammatory mediators that may that occur singly, in pairs, or frequently in long chains
contribute to the sudden death that occurs with anthrax. o Can be identified in dried smears by immunofluorescence techniques
o On Blood Agar Plate (BAP) – non hemolytic gray to white colonies with
Clinical infection a rough texture and ground glass appearance. The edge of the colonies
o Anthrax affects principally domestic herbivores (e.g. sheep, goats, and may have a fringed or Medusa Head appearance
horses) and is transmitted to humans by contact with infected animal o B. anthracis and B. cereus are similar in morphology. B. anthracis is non
products or contaminated dust motile, sensitive to penicillin and Gamma-phage positive.
o In the terminal stages of the disease in animals, bacilli are shed in large o Antibodies to the organism can be demonstrated by serology (ELISA)
numbers from all orifices which sporulate in the soil and remain a o Acute and Convalescent sera can be used to confirm infections.
source of infection
o B. anthracis spores may remain viable for many years in contaminated Treatment
pastures and in bones, wool, hair, hides, and other animal materials. o Penicillin not recommended because of inducible β-lactamase in B.
These spores are highly resistant to physical and chemical agents anthracis.
(recommendation is to autoclave it). o Cutaneous anthrax responds to Ciprofloxacin
o Human infection can occur by: o Multidrug therapy: Ciprofloxacin plus Rifampicin plus Vamcomycin is
Cutaneous Route – organisms gain access through wounds and recommended for pulmonary anthrax
multiply locally producing a dramatic inflammatory response. o Prophylaxis for potential exposure: Ciprofloxacin or Doxycycline for 4
Inhalation/Pulmonary – organisms are inhaled, multiply in the weeks together with 3 doses of vaccine. Give prophylaxis for 8 weeks if
lungs, spread to the draining hilar lymph nodes where no vaccine is given.
hemorrhagic necrosis may occur and goes into the blood stream. o Erythromycin is an alternative (but may cause GI irritation)
Ingestion – from infected meat producing gastric ulceration o A cell-free vaccine is available for workers in high-risk occupations
o From these invasion of the bloodstream follows producing profound
toxemia. Metastatic infections like meningitis may occur. Prevention
o The soil is contaminated by spores from the carcasses of dead animals.
Clinical Manifestations o Animals are infected by spores that enter injured mucous membranes
o Cutaneous Anthrax or injured skin. Humans by contact with animal carcasses.
Accounts for 95% of cases o Dispose of animal carcasses by burning; Autoclave animal products;
Begins 2-5 days after infection as a papule that develops within a Wear protective clothing when handling carcasses; Immunization of
few days to a vesicle filled with black fluid. Rupture leaves a black domestic animals with live attenuated vaccine.; Immunization of farm
eschar surrounded by an inflammatory ring reaction. This is personnel.
called a Malignant Pustule.
There is lymphadenopathy, fever & headache Bacillus cereus
The lesion is painless and is found in the hands, forearms or Cause of food-borne illness
head. Similar in morphology with B. anthracis
The organisms may invade regional lymph nodes and then the It is usually motile, not susceptible to PCN or the Gamma phage
general circulation, leading to fatal septicemia, meningitis and Types of diseases:
death. o Emetic Type of Disease
o Inhalational Anthrax or Wool-sorter’s Disease B. cereus is a soil organism that usually contaminates rice. When rice
Due to inhalation of spores by people who handle raw wool, is cooked and allowed to cool the spores germinates and starts
hides or horsehair producing the toxin.
Fever, malaise and cough which progresses to a severe infection Starts 1-5 hours after ingestion of reheated fried rice or pasta
with respiratory distress and cyanosis. Death occurs within 24 dishes.
hours. There is nausea, vomiting, abdominal pain and occasionally diarrhea.
o Gastrointestinal Anthrax Recovery occurs in 24 hours.
Rare form. B. cereus may be found in the normal stool so it is not diagnostic.
Due to ingestion of meat contaminated with spores Concentration of 100,000 bacteria per gram of food is diagnostic.
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MICROBIOLOGY 1.6
Bacillus subtilis
Present in air, dust, brackish water and vegetable matter
Common Lab contaminant but can produce infections in the
immunocompromised host
Seen in bacteremias and eye infections in heroin addicts
Treatment: β-lactam antibiotics Figure 8 Palisade or “Fence” formation
Morphology
o Non spore-forming Gram (+) bacilli that appear in palisade or as
individual cells lying at sharp angles to each other in V or L formations
(Chinese character-like formations)
o Club shaped swellings and beaded and barred forms are common.
Babes-Ernst granules are responsible for the beaded appearance.
o Non motile
Determinants of Pathogenecity
o Capable of colonizing mucous membranes
o K Antigen
Heat – labile protein located at the surface of the cell wall.
Antiphagocytic property of K antigen allows the organism to colonize
cells
o Cord Factor: produces disruption of the mitochondria.
o Neuraminidase & N-Acetylneuraminate lyase: degrades N-
Acetylneuraminic acid residues from mucinous environments,
providing bacteria a ready source of energy.
o Exotoxin
Accounts for all the pathologic systemic effects
Produced by organisms infected with a bacteriophage carrying the
gene for toxin production Figure 11 Enlargement of regional lymph nodes in
Low iron conditions induce toxin expression, whereas high iron the neck with marked edema (BULLNECK)
conditions repress toxin production.
o Extrarespiratory Disease
Diphtheria is caused by the local and systemic effects of a single
The most common is Cutaneous diphtheria, due to secondary
exotoxin
infection of septic skin lesions
The toxin molecules are composed of fragments, A and B.
Appears as a chronic, spreading, non healing ulcer covered with a
Fragment B binds to susceptible cell membranes and mediates the
grayish membrane
delivery of fragment A to its target.
Strep. pyogenes and Staph. aureus are usually also present in the
Inside the cell, fragment A separates from fragment B and catalyzes
lesion. Lesions fail to heal until appropriate therapy is given for
a reaction between nicotine adenine dinucleotide (NAD+) and the
these organisms.
eukaryotic polypeptide chain elongation factor.
The toxin causes fatty myocardial degeneration resulting in
Inhibits eukaryotic protein synthesis, produces DNA cleavage and
circulatory collapse and accounts for more than half of case
cell lysis.
fatalities.
Cranial nerve paralysis may lead to paralysis of the soft palate with
Clinical infection
resultant difficulty of swallowing and nasal regurgitation of fluids.
o The primary habitat is the respiratory tract, and is transmitted by
The most common manifestation of peripheral nerve involvement
symptom-free carriers, by persons in the incubation stage and by
is Polyneuritis of the lower extremities producing a mild
convalescents.
weakness to paralysis.
o Transmission is via droplet inhalation or from discharges in
cutaneous infections.
o Less frequently spread by direct contact with an infected individual
or a contaminated fomite.
o There is NO lifelong immunity to diphtheria
o Presence in the blood of antitoxin due to infection or immunization
with toxoid confers immunity. Determined by:
Schick Test:
Injection of the volar forearm skin with 0.1ml of highly purified
toxin. Toxin treated with heat is injected in the other arm as
control.
Absence of immunity to diphtheria is characterized by an
inflammatory reaction that becomes maximal in 4-7 days Figure 12 Cutaneous Diphtheria due to secondary infection of septic skin lesion
o Respiratory Disease Diagnosis
Initial lesion on the tonsils and oropharynx and may spread to o Specific treatment must be started if the clinical picture is suggestive
the nasopharynx, larynx and trachea. of diphtheria.
There is exotoxin production that is absorbed in the mucous o Dacron swabs collected from beneath the membrane is taken from
membranes and causes necrosis of cells in the area. suspected lesions BEFORE antibiotics are started. Place swabs in semi
When the necrotic epithelium is embedded in fibrin, RBC & solid transport media such as Amies.
WBC, a grayish pseudomembrane is formed over the tonsils, o Inoculate specimens on a
pharynx or larynx. Blood Agar Plate to rule out hemolytic strep
Removing the pseudomembrane would tear capillaries and Loeffler’s Slant, in 12-18 hours organisms with diphtheria-like forms
result in bleeding. can be identified by Gm stain
Clinical Findings: Tellurite Plate and incubate, in 36-48 hours the colonies can be
Sorethroat and fever followed by prostration and dyspnea identified
because of obstruction caused by the membrane o Gram stain
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MICROBIOLOGY 1.6
Use specimens from growth on cultures NOT from clinical specimens o Can grow at Refrigerator temperature, low pH and high salt conditions,
obtained from patients (Chinese character-like formation) so, it can survive food preservation methods making it an important
o Presumptive diphtheria isolate should be subjected to testing for food borne pathogen.
toxigenicity o Grows well on Sheep blood agar & Tryptose Agar
Modified Elek Method: An antitoxin disc is placed on an agar plate Produces Beta hemolysis on Sheep blood agar
and the culture to be tested is inoculated 9mm away from the disc. On Tryptose Agar colonies are blue green in color
After 48 hrs the toxin will be precipitated by the antitoxin from the o Catalase (+), ferments Glucose with the formation of acid, does not
disc as a whitish material between the disc and the growth ferment Mannitol
Polymerase Chain Reaction detects toxin on patient specimens
ELISA can detect toxin in clinical specimens
Immunochromatographic strip assay can also detect toxin on clinical
specimens
Animal Inoculation Test:
a)Guinea pig or rabbit is injected intracutaneously with the
organism.
b) 5 hours later, Antitoxin is injected intraperitoneally.
c) 30 minutes later, the animal is injected with the organism in a
control area. After 48 hours, if a toxigenic strain is present, the initial
injection site will have a necrotic area.
Treatment
o Treatment of choice is antitoxin, which should be given immediately on Figure 13 Tryptose Agar
the basis of clinical impression.
o The function of antitoxin is to neutralize the unbound toxin in the blood Serologic classification based on the O (somatic) antigen and H (flagellar)
o The patient must be tested for hypersensitivity before starting antigen yielded several serotypes.
treatment. o 1/2a, 1/2b, 4b : 95% of human isolates
o Treatment with penicillin G or erythromycin is recommended but o 4b causes most of the food-borne outbreaks
neither is a substitute for antitoxin.
o Antibiotics slow the spread of infection and, by killing the organism, Determinants of Pathogenicity
prevent further toxin production. o Facultative intracellular organism that invades and grows in
o Supportive care directed especially at respiratory and cardiac mammalian cells including macrophages, epithelial cells and fibroblasts.
complications is an essential part of the management of patients with o The organism enters the cytoplasm, grows and spreads to other cells.
diphtheria. o Normally, the immune system eliminates the infection before it
spreads (Cytotoxic T cells). If the immune system is compromised,
Prevention systemic disease may develop.
o Vaccination with diphtheria toxoid is highly effective & it is first o The organism binds to the host cell facilitated by adhesin proteins (Ami,
administered early in infancy with (DPT) at 2,4,6 months and booster at Fbp A & Flagellin Pr-)
18 months of age. o Internalin A on the cell wall of the organism interacts with E-cadherin
on the epithelial cells promoting Phagocytosis.
Corynebacterium pseudotuberculosis o The organism produces Listeriolysin O which lyses the phagolysosome
Produces an exotoxin and causes ulcerative lymphangitis in warm membrane allowing the organism to escape into the cytoplasm and
blooded animals undergo rapid division.
Causes chronic lymphadenitis in humans due to contact with infected o The organism becomes encapsulated by actin filaments. The filaments
animals. reorganize into a long tail extending from one end of the organism due
to the action of Act A.
Corynebacterium pseudodiphtheriticum and Corynebacterium xerosis o The tail propels the organism to the surface of the host cell, protrusions
Diphtheriticum is normal in the nasopharynx. Xerosis in the skin, mucous (filopods) are formed that can penetrate adjacent cells allowing the
membranes and conjunctica. bacterium to enter.
Infections occur in immunocompromised patients and those with o Since the bacterium is intracellular, antibodies are ineffective.
mucocutaneous defects o Its presence intracellularly in phagocytic cells also permits access to
Causes prosthetic valve endocarditis the brain and probably transplacental migration to the fetus in
Listeria monocytogenes pregnant women.
Facultative intracellular Gram (+) coccobacilli that occur in short chains of
3-5 organisms. Causes Listeriosis Clinical Infection
o Found in plant-soil environment and animal feces
Can assume a palisade formation in stained preparations.
o Food is the most common vehicle for transmission
Actively motile. “Tumbling-end-over-end motility” which distinguishes it
Raw vegetables, dairy products, raw hotdogs, undercooked chicken
from the non motile C. diphtheriae
o Human to human transmission
The disease affects primarily pregnant women, newborns, and adults with
Transplacentally through the umbilical vein
weakened immune systems.
During delivery through contact with infective secretions
Cultural Characteristics
o Aerobic to microaerophilic (can survive with little oxygen)
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MICROBIOLOGY 1.6
Pathogenesis Prevention
o The major portal of entry is the GIT. The disease may not occur in o Should center on the elimination of animal reservoirs (sheep, cattle,
immunocompetent individuals after ingestion of contaminated food or goats and pigs)
may develop gastroenteritis with fever and diarrhea. o Listeriosis can be prevented in infants by prompt recognition and
o Intraluminal bacteria gain access to the circulation when the host treatment of the mother.
defense is altered, followed by seeding of various sites.
Erysipelothrix rhusiopathiae
Clinical Manifestations Non motile, nonencapsulated, Gram(+) bacilli
o More than half of patients have underlying disorders like malignancy,
alcoholism, diabetes, etc. Cultural Characteristics
o In the healthy adult, infections are usually asymptomatic, or at most o Microaerophilic
produce a mild influenza-like disease. o Heart Infusion Agar with rabbit’s blood is suitable for isolation
o Adult Infection o On BAP, colonies are small, round, grayish white in color and shows
Meningitis is the most common form of listeriosis in the adult alpha hemolysis
especially in cancer patients and renal transplant recipients o Catalase(-), oxidase (-), indole (-)
Primary Listeria bacteremia usually affects patients under 50 o On TSI agar, hydrogen Sulfide is produced turning the TSI butt black
years of age. May produce endocarditis and pneumonia.
Produces Hyaluronidase (Spreading Factor) and Neuraminidase (increases
In pregnant women, even though the most usual symptom is a
ability of the organism to attach to host receptors)
mild influenza-like illness without meningitis, infection of the fetus
is common and can lead to abortion, stillbirth, or delivery of an Clinical Infection
acutely ill infant. Widely distributed in land and sea animals worldwide including birds.
o Neonatal Infections It occurs on the surface slime of fresh and seawater fishes, in the sewage
Genital tract infections in the pregnant with infection of the of abattoirs, and in the feces of infected animals
offspring. History of mild influenza-like illness. The disease is prevalent in abattoir employees, butchers, fish handlers and
those who have contact with animal products
Granulomatosis infantiseptica Infection is via direct inoculation of a wound with animal products
Most common E. rhysiopathiae infection in humans is Erysipeloid.
Early form
o Occurs on the fingers by direct inoculation on the site of a cut (“seal
Results from infection in utero and appears within 2 days of finger” or “whale finger”)
birth o Severe pain and swelling occur. The lesion is raised and violaceous in
May cause abortion, premature birth, stillbirth or death within a color. Pus is usually not present as differentiated from Staph infection.
short period after birth. Resolves in 3-4 weeks
o May also cause a diffuse cutaneous disease and bacteremia with
If the infant is born alive, septicemia develop within a few hours
endocarditis.
followed by fetal distress, pneumonia, diarrhea, seizures, and
o Dx: Culture of aspirated material from border of lesion.
maculopapular lesions on the legs and trunk o DOC is PCN. Erythromycin is an alternative
Late Form of Neonatal Listeriosis.
th
Occurs on the 5 day of life
Infection is acquired during or after birth rather than in utero
Meningitis is the usual presentation.
Immunity
o No second clinical infection with Listeria has been observed in patients
cured of Listeriosis
Laboratory Diagnosis
o Gram Stain of infected material and look for typical pleomorphic Gm (+)
bacilli
Blood, CSF, Amniotic fluid and Genital tract secretions
o Tryptose Agar medium is excellent for cultivation and propagation
o Accurate identification is based on combination of Beta hemolysis,
catalase production, and tumbling motility.
o Serology is unreliable because the organism cross-reacts with other
Gm(+) organisms
Treatment
o Penicillin G or Ampicillin
o Trimethoprim-Sulfamethoxazole or Erythromycin for patients allergic to
PCN.
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