GRAM-POSITIVE BACILLI B.

AFB staining (sample from nasopharynx)

 bacilli appear as parallel bundles or pockets of
MYCOBACTERIA GROUP
cigar
 gram-positive
 acid-fast bacilli
CULTIVATION
 aerobic, non-motile, non-spore former
 non-culturable in artificial culture medium
o non-capsulated
 grow in the foot pods of mice and armadillo
 produce “much-granules”
Control: pre-immunization with BCG Isolation and
treatment of cases
Mycobacterium leprae
Treatment: MDT for 6 months
 also known as Hansen’s bacillus
 causative agent of Leprosy or Hansen’s disease
 incubation period is 2 to 4 years or up to 40 years Mycobacterium tuberculosis
 mode of transmission is Inhalation  Koch’s bacillus, Human tubercle bacillus, slender
beaded rods
Leprosy  could be stained with fluoro-chromatic dye, carbolfuchsin
 do not produce toxin but protected by intracellular
1. Lepromatous or Nodular type environment making them resistant to heat, drying and
 progressive and malignant chemical agents
 with numerous organisms in the lesions Ziehl-Neelsen staining
 Lepromen test (-)
2. Tuberculoid or Anesthetic type Lipase
 most important constituent of M.
 non-progressive/ Benign
tuberculosis
 few organisms in skin lession
 Lepromen test (+)  responsible for hydrophobic nature
 formation of cord factor (virulence factor)
 Mycolic acid (acidfastness)

Cultural Characteristics
 requires increase CO2 (8-12%)
 requires complex media
 Agar Base Media (Middle Brooke 7H-10)
 Egg Base Medium (Petragnani, LJM)
 growth is relatively slow (3-4 weeks
incubation)
 additives are used to suppress the growth
of other microorganisms
 colonies are dry, rough, granular
(Cauliflower like)

Colonies of M. tuberculosis

Lo¨wenstein-Jensen Medium
DIAGNOSIS: Epidemiology
A. Lepromen test
 a skin test using sterile extract from Lepromatous  Tuberculosis is endemic worldwide, less frequent in
nodule developed countries
 2 types: Early (Fernandez rection, 24-48 hours)  Seen from a worldwide perspective, tuberculosis is still a
Late (Mitsuda, 3-4 weeks) major medical problem.
 estimated that every year approximately 15 million persons b. Neutral red dye test
contract tuberculosis and that three million die of the 4. Animal inoculation test
disease. 5. Tuberculin test
 the main source of infection is the human carrier. a. OT (old original tuberculin)
 transmission of the disease is generally direct, in most cases b. PPD (Purified Protein Derivative)
by droplet infection.  positive test: past infection, close association
 Indirect transmission via dust or milk (udder tuberculosis in with infected patient
cattle) is the exception rather than the rule.
 incubation period is four to 12 weeks. Methods (tuberculin skin test)

Pathogenicity 1. Mantoux (injected intracutaneously)

 Initial droplet infection results in primary tuberculosis, 2. Von Riquet (scratching the skin)
localized mainly in the apices of the lungs. 3. Vollmer Patch Test (PPD soaked in a cloth then
 primary disease develops with the Ghon focus (Ghon’s placed over the skin)
complex), the hilar lymph nodes are involved as well. 4. Mono-percutaneous Test Tuberculin Time
 90% of primary infection foci remain clinically silent. (multiple puncture technique)
 10% with primary tuberculosis progresses to the
secondary stage (reactivation or organ Niacin Test – for slow grower
tuberculosis) after a few months or even years, (growth > 7 days)
characterized by extensive tissue necrosis
 specific immunity and allergy that develop in the course of
an infection reflect T lymphocyte functions
 allergy is measured in terms of the tuberculin reaction to
check for clinically in apparent infections with TB.

PREVENTION
 prophylactic BCG
 avoid contact with infected person
 Isolation and treatment of cases

Nontuberculous Mycobacteria (NTM)

 Mycobacteria that are neither tuberculosis nor
leprosy bacteria
 categorized as atypical mycobacteria (old
designation), nontuberculous mycobacteria (NTM) or
mycobacteria other than tubercle bacilli (MOTT)

LABORATORY DIAGNOSIS
Specimens: Sputum (early morning sputum) (inoculated in
Middle Brock 7H) biopsy material, pleural fluid, synovial fluid,
gastric content
1. Direct smear - AFB staining
2. Concentration technique (use of digesting
substance: 4% NaOH, 6% oxalic acid, 4% sulfuric
acid)
3. Virulence test
a. Serpentine cord formation
 CORYNEBACTERIA GROUP
 Gram-positive, aerobic, non-motile, non-sporeforming,
uncapsulated bacilli
 pleomorphic, club-shaped
 “Babes-Ernst” metachromatic granules (beaded
appearance)
 arrangement: X, V and L, palisade, Chinese letter or
character

DIAGNOSIS:
1. Stained smear
a. (Grams, Methylene Blue)
2. Culture/Cultivation
 Loeffler medium
 BAP
Corynebacterium diptheriae  PAIS coagulated Egg medium
 Kleb’s Loeflers bacillus  Loefler’s serum slant (enhance
 causative agent of Deptheria pleomorphism)
 produces 3 varieties in Tellurite Agar  Tellurite Agar (best for colonial morphology)
1. C. deptheriae var. gravis a. Cystine Tellurite Blood Agar
2. C. deptheriae var. mitis b. McLeods Potassium Tellurite Agar
3. C. deptheriae var. intermedius
*mode of transmission Serological test/Toxigenicity
 droplet nuclei from infected patient a. Schick’s test (Susceptibility test)
 contact from coetaneous foci of infection  1 arm injected with 0.1 ml unheated toxin
 the other arm with heated toxoid
Pathogenesis  indicated by redness and swelling
 Deptheria is only caused by C. diptheriae infected Interpretations
with bacteriophage carrying toxigene (Lysogenic (+) no antibody, susceptible to infection
strains) (-) has antibody, protected from infection
 due to the powerful exotoxin (more poisonous than b. Elek test (neutralization/precipitation reaction)
that of cobra venum) c. Elek-Ouchterlony immunodiffusion test
 Extracellular toxin consists of two functionally distinct d. Animal inoculation
 fragments, A (toxic activity) irreversibly blocks
protein synthesis causing cell death and B (binding PREVENTION:
to receptors of target cells)  active and passive immunization
 supplemental antibiotics (eliminate the carrier state of
Pseudomembrane formation toxigenic strains during epidemics)
 due to production of serum exudate and cellular
infiltrate of mucous membrane in the pharynx Other species
(causes respiratory obstruction 1. Deptheroids
 Diptheria-like organisms
 non-pathogenic, found in the throat
 2. Corynebacterium minutissimum
 causative agent of superficial infection of the
auxiliary and pubic skin
 infected skin when exposed to UV produces
pink fluorescence
b. Pulmonary anthrax (5%)
 also known as “Wool’s sorters disease”
 caused by inhalation of spores
c. Intestinal anthrax (rare)
 obtained from ingestion of improperly
cooked infected meat (affect 6 hours after
ingestion)

DIAGNOSIS:
Specimens: blood, sputum, swab from pustules
1. Stained smear (Grams,
Immunofluorescence)
2. Culture (no special medium)
3. Animal inoculation
4. String of Pearl test
5. Ascoli test
 precipitation test
 ascoli is from the extract of infected
tissues

BACILLUS SPECIES PREVENTION:

 aerobic spore-forming bacilli  infected animals (cremation)
 prompt diagnosis
 isolation and therapy

Other species
1. Bacillus thuringensis
 used in biological control of mosquitoes
 source of Bt toxin for GMO’s
2. Bacillus cereus
 motile, non-capsulated, Beta-hemolytic
 can cause food poisoning (e.g. fried rice)
 two forms of infection
a) Emetic form
1. Bacillus anthracis o manifested by nausea, vomiting.
 important species, highly pathogenic Abdominal cramp (recovery: 24 hours,
 large, encapsulated, none-motile Gram (+) bacilli self- limiting)
 known as bamboo fishing rod appearance with o incubation period: 1 -5 hours
 centrally located spores o associated with fried rice
 disease of animals, humans are infected incidentally b) Diarrheal type
Pathogenicity o associated with meat dishes and
 causative agent of anthracis/anthrax sauces
 incubation period: 6 weeks o profuse diarrhoea with abdominal
 spread rapidly via lymphatics, then to blood cramps
 pathogenicity is due to presence of capsule and o incubation period: 1 – 24 hours
 toxin production o also associated with eye infection:
 three proteins of Anthrax toxin  Severe Keratitis
1. PA (protective antigen)]  Endophthalmitis
2. EF (Edema factor) 3. Bacillus steathermophilus
3. LF (lethal factor)  current name: Geobacillus steathermophilus
Clinical type of anthrax  used to determine the efficacy of autoclaving and
a. Cutaneous anthrax (95%) other sterilization procedures
 malignant pustule 4. Bacillus subtilis
 caused by skin contact with infected animals  also known as Hay bacillus, common
 may progress to systemic infection environmental contaminant
CLOSTRIDIA GROUP
 most species are motile with peritrichous flagella
 hemolytic and non-capsulated
 found in soil, feces of horses and other animals
Clostridium tetani
 with terminally located spore, motile
 drumstick or tennis rocket appearance
 Common source of infection: dirty pointed objects,
injured area with spores deposited on it, then
absorbed by the blood stream

Pathogenicity
 produces toxins
a. Tetanus lysin (Hemolysin)
o responsible for the Beta-hemolytic
activity

Clostridium perfringens Open lower-leg fracture following Fully manifest case of

a traffic accident; the portal of tetanus in a patient
A. Gas gangrene group/Histotoxic clostridia
entry of C. tetani.
1. C. perfringens/ C. welchii
2. C. histolyticum
3. C. novyi b. Tetanospasmin (Neurotoxin)
4. C. septicum  responsible for all the symptoms of
5. C. sporogenes tetanus
B. Toxigenic group  action is inhibition of GABA causing
1. Clostridium tetani spasm of masseter muscles resulting
2. Clostridium botulinum to “locked jaw” or Tresmus
 also causes Tetanus neonaturum
Clostridium perfringens  common among newborn due to
 gangrene bacillus cutting of umbilical cord with
 centrally located spore unsterilized material
 produce five types of toxins  incubation period: 4 – 10 days
a) type A- infect humans
b) type B to E – domestic animals DIAGNOSIS
Specimen: swab from wound infection
Pathogenicity 1. Gram-staining
1. causes anaerobic cellulitis 2. Anaerobic culture
 fermentation of muscle sugar causing gas 3. Clinical picture and history of injury of
formation the patient
 (causing crepitus) in the tissues which PREVENTION
causes necrosis  vaccine (toxoids)
 common among soldier’s would infection  prophylaxis (use of anti-toxins
2. Gangrenous foot infection (Myonecrosis)
 common among diabetic patient Clostridium botulinum
3. Causative agent of food poisoning in dried food  canned goods bacillus
 due to exotoxin  common also in smoked food, left over foods and other
4. Necrotic enteritis processed food, contaminated vegetable/fruits
 incubation period: 18 – 96 hours
DIAGNOSIS:  widely distributed in soil
 Gram staining
 BAP - double zone of hemolysis Pathogenicity
 Naglers reaction  causative agent of botulism (fatal infection)
 determines the type of toxin produce  due to the botulinum toxin (a neurotoxin)
 a very potent toxin (100x than cobra venom)
  post-partum bacteremia, vaginal abscess, endometriosis
* effect of toxin: flaccid paralysis  can be detected in vaginal discharge by means of
 blocked the excitatory neurotransmitter, microscopy and culturing.
acetylcholine synapsis and neuromuscular junction  the microscopic analysis, so-called “clue cells” (vaginal
Symptoms: double vission, speech difficulty, inability to epithelia densely covered with Gram-labile rods) could
swallow and constipation be observed
Cause of death: Cardiac arrest  this bacterium can be cultured on blood-enriched agar
Respiratory paralysis incubated in an atmosphere containing 5% CO2.
 drug of choice is metronidazole.
Types of Botulism
1. Botulinal food poisoning
 ingestion of food containing pre-
formed toxins
2. Wound botulism
3. Infant botulism
 ingestion of spores
 causes SIDS (Sudden Infant
Death Syndrome)

DIAGNOSIS:
1. Stained smear LACTOBACILLUS SPECIES
2. Culture of contaminated food  giant rod-shape bacillus (Doderlein’s bacillus)
3. Serological test  normal flora of the vagina (L. acidophilus)
 also inhabits the mouth and GIT (colon)
PREVENTION:  produces large amount of lactic acid from CHO
 boil food for 15 minutes/ heat at least 80 fermentation when immune system is suppressed,
C causes meningitis and endocarditis

Clostridium difficile Lactobacillus

 commonly found in colon of some individuals
 causes Pseudomembranous colitis
 type of toxin produced
a. Toxin A
o enterotoxin, causes increase secretions of
electrolytes and fluids
b. Toxin B
o cytotoxin, causes damage to mucosa of
colon

Clinical course : fever, diarrhea, and spasmodic

abdominal pains

DIAGNOSIS:
 involves culturing the pathogen from patient stool -
detection of the cytotoxin in bacteria-free stool
filtrates on the basis of a cytopathic effect (CPE)
observed in cell cultures

Gardnerella vaginalis
 a Gram-variable, nonmotile, nonencapsulated rod
bacterium.
 natural habitat: vagina of sexually mature
women
 pathology: vulvovaginitis (vaginosis), found in over
90% of women showing the symptoms of this
infection