MICRO: GRAM (+) RODS

Dr. Fe Cabugao

GRAM (+) BACILLI

 Bacillus
 Clostridium
 Mycobacterium
 Corynebacterium
 Listeria

AEROBIC SPOREFORMING BACILLI: GENUS Bacillus

Bacillus anthracis
Morphology  Large, gram (+) rods with square or concave ends  corset-shaped
 Arranged in long chains like jointed bamboo or fishing rod appearance
 Sporeformer  oval, centrally-located, not swollen
 Capsulated  polypeptide D-glutamic acid
 Nonmotile

Cultural Characteristics  Obligate aerobe

 Non-fastidious  can grow on ordinary laboratory media
 Blood agar – colonies appear large, raised, opaque, with irregular finger-like edges
appearing like medusa heads, nonhemolytic
 “String of pearls” appearance – on solid medium containing 0.05 to 0.5 units of
Penicillin/mL
After 3-6 hours incubation, cells become large spherical and occur in chains on the
agar surface

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 Biochemical Characteristics  Ferments a variety of carbohydrates
 Gelatin is liquefied  inverted pine tree growth  (+) motility, only seen in some
strains
 Hydrolyze starch
 Vogues-Proskauer test (+)

Antigenic Structure Two major antigens identified:

 Capsular antigen – polypeptide
 Somatic antigen – polysaccharide antigen cell wall
Consist of D-galactose and N-acetyl glucosamine

Determinants of Pathogenicity  Polypeptide capsule (D-glutamic acid) – immunogenic and antiphagocytic

 Anthrax toxin – complex protein toxin produced in vivo responsible for signs and
symtpoms of disease, composed of:
o Protective antigen – binds to the cell, mode of action is unknown
o Lethal factor – kills cells by an unknown mode of action
o Edema factor – an exotoxin (adenylate cyclaase) responsible for binding and entry
of organism into the cell

Clinical Infections  Anthrax – primarily a disease in herbivorous animals

Human infection is rare
Generally an occupational disease, seen among industrial or agricultural workers
through contact with infected animals or animal products

 Pathogenesis:
o Spores in the environment  may be traumatically implanted, inhaled, or ingested
 germinate  produce anthrax toxin

 Clinical forms of infection in human

o Cutaneous Anthrax (skin) – malignant pustule
Painless ulcer with black neurotic lesion accompanied with localized edema
Most common  accounts for 95% of human infection
Acquired through direct contact with infected animals or animal products
Seen among people who handle horse hair, hide, animal skin
o Respiratory Anthrax (lungs) – Woolsorter’s Disease/Anthrax Pneumonia
Transmitted by inhalation of spores  lungs
Results in life-threatening pneumonia if not treated early
o Gastrointestinal Anthrax (GIT) – Milzbrand/Violent Enteritis
Acquired through ingestion of infected inadequately cooked meat or drinking
unpasteurized milk
Characterized by fever, abdominal pain, vomiting, and diarrhea

Laboratory Diagnosis  Bacteriological test – presumptive; smears from exudates, pus, sputum, or blood
 Culture
 Serologic
o ELISA
o Microhemagglutination
Treatment  Penicillin (drug of choice)
Prevention  Vaccination
o Animals  living spore vaccine
o Humans  protective antigen vaccine (high risk)

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 Bacillus subtilis Bacillus cereus
General  Hay bacillus  Same morphology as B. anthracis, except motile
 Ubiquitous organism (air, dust, brackish water)  Found naturally in rice and vegetables
 Common laboratory contaminant  Spores not killed by boiling
 Medical importance  antibiotic producer
Clinical Infections  Bacteremia  fatal  Foodborne gastroenteritis/Food poisoning –
 Infection among ICP resulting from ingestion of enterotoxin secreted by
 Eye infection the organism
Common source  fried rice
Frequently mistaken for Staphylococcal food
poisoning
Two forms:
 Emetic form – food poisoning with short
incubation period 4 hours after ingestion
 severe nausea and vomiting, lasts 8-10
hours
 Diarrheal form – food poisoning with long
incubation period 17 hours after ingestion
 abdominal cramps and diarrhea, lasts
20-36 hours
Treatment  B-lactam antibiotics  Aminoglycosides (Clindamycin/Vancomycin)

LABORATORY IDENTIFICATION
Characteristics Bacillus anthracis Bacillus cereus
Hemolysis on BA - +
Motility - +
String of pearls appearance + -
Growth on PEA - +
Gelatin Hydrolysis - +
Susceptibility to Penicillin (10 U/mL) Susceptible (S) Resistant (R)

OBLIGATE ANAEROBIC SPOREFORMER: GENUS Clostridium Species of Medical Importance

 Composed of a large group of spore-forming gram (+) bacilli  Histotoxic clostridia  gas gangrene (clostridial myonecrosis)
 Majority are obligate anaerobes, a few are aerotolerant food poisoning
o Aerotolerant – microbes which are exclusively anaerobic, but o Species include: Clostridium perfringens/Clostridium welchii
are insensitive to the presence of O2. They live by fermentation Clostridium novyi
alone, whether or not O2 is present in the environment Clostridium septicum
 Found abundantly in the soil, dust, water and common inhabitant of Clostridium histolyticum
the GIT in humans and animals Clostridium sordelli
 Pathogenic species which secrete soluble potent toxin responsible Clostridium fallax
for disease  Tetanus bacillus  tetanus or lockjaw
 Culture medium: Fluid thioglycollate o Clostridium tetani
 Fatal food poisoning or botulism
o Clostridium botulinum
 Antibiotic Associated Pseudomembranous Enterocolitis
o Clostridium difficile

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Clostridium perfringens (also, Clostridium welchii)
General  One of the most important species
 25 – 35% of healthy individuals harbor the organism in the colon
 Found abundantly in soil, water, dust
 Invasive infection
 Cause myonecrosis
 Gas gangrene
Morphology  Short plump Gram (+) rod with blunt/square ends
 Size: 2-4 um length, 1-1.5 um width
 Sporeformer → large, ovoid, centrally located not swollen
 Capsulated
 Nonmotile

Cultural and Biochemical Characteristics  Aerotolerant

 Glucose & Lactose Fermenter
 Culture media isolation
o Sheep blood agar  “ Target hemolysis ”
 double zone hemolysis due to alpha and theta toxin.
 Theta toxin: narrow zone with complete hemolysis
 Alpha toxin: wider zone with incomplete hemolysis
o Milk medium  stormy fermentation
 due to fermentation of lactose (milk)
Target hemolysis on sheep blood agar
→ produce large amount of acid → protein (casein) coagulation
o Nagler’s test – neutralization of alpha toxin lecithinase with a specific antitoxin
 Toxin produced are : alpha, beta, delta, theta, epsilon, iota and enterotoxin

 Stormy fermentation on milk medium

due to protein coagulation and large
amount of gas production
 Control (left) and Test (right) o Animal inoculation – oral administration culture into mice  (+) flaccid paralysis

Antigenic Structure  5 Serotypes: A, B, C, D, E

 Type A – most important
 Type A, C, D – causative agent for human infection
Responsible for food poisoning produced by serotype A & C

Clinical Manifestations  Gas gangrene (or, Clostridial Myonecrosis)

o Severe tissue infection secondary traumatic injury skin with compromised oxygen
supply
o Toxin produced causes pain and massive tissue destruction (due to alpha toxin) with
production of gas and shock
o Condition associated:
 War or Gunshot Wounds
 Automobile or Motorcycle Accidents
 Septic abortion
 Compound fractures
Clostridial Cellulitis
o Spore (Soil)  implanted traumatized tissue  germinate & produce wide variety of

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 toxin  attack healthy muscle & surrounding tissues
 Food poisoning
o Due to enterotoxin secreted by organism in GIT  germinate 8-24 hours after,
development of signs and symptoms
o Characterized by diarrhea, abdominal pain, vomiting
o Self-limiting and resolve 24 hours
 Used as microbial indicator for fecal contamination of water

Laboratory Diagnosis  Bacteriological test – smear from tissue or exudate

 Anaerobic Culture of organism
o Blood Agar  double zone of hemolysis or target hemolysis due to alpha and beta
hemolysis
 Carbohydrate Fermentation test – (+) lactose and glucose fermentation

Treatment  Penicillin (drug of choice)

 Wound debridement is important
Prevention  Clean & debride wounds
 Penicillin given for prophylaxis

Clostridium tetani (also, Tetanus bacillus)

Morphology  Slender straight gram (+) rod
 Motile  peritrichous flagella
 Sporeformer  circular, terminal and swollen “plectridium spore” with drumstick or tennis
racket appearance

Cultural Characteristics  Fluid Thioglycollate – broth culture medium

organism grows in the bottom of tube where oxidation-reduction potential is low
 Blood agar  swarming growth
colonies appear translucent finely granular with beta hemolysis
 Cooked meat medium  no digestion of meat (+)
small growth for 24 hours
Disease  Tetanus (Lockjaw) – organism is a soil sporeformer
 results from traumatic implantation of spore into tissues with low oxygenation (such
as via puncture wound, burn wound, unsterile, surgical areas , umbilical stump of
neonate)  spore germinate  grow & multiply  toxin production
(tetanospasmin)  released  goes to the CNS (anterior horn cell)  ganglion
receptors in the spinal cord  blocks release inhibiting mediator at spinal synapse 
peripheral nerve  spasm occurs

Clinical Manifestations
 Convulsion – due to contraction of voluntary muscles when toxin the spreads
hematogenously, resulting in spasms
 Spastic paralysis – beginning in the jaw area (trismus/lockjaw, risus sardonicus/sardonic
smile)
If left untreated, descends downward, causing paralysis of large muscles &
opisthotonus (rigid back spasm)
May lead to death from paralysis of throat & respiratory muscles

Risus sardonicus Opisthotonus

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 Laboratory Diagnosis  No microbiological or serological diagnosis
 Organism is rarely isolated from the wound site. So diagnosis is based on:
o Clinical appearance of patient – signs and symptoms of disease
o Anaerobic culture of contaminated tissue from infected wounds
o Biochemical test – confirmatory

Treatment  Tetanus Immune globulin (TIG) – at the wound site

 Tetanus antitoxin – given immediately to neutralize the toxin
 Penicillin – given in large doses
 Muscle relaxant
 Respiratory support
 Clean the wounds with soap and water

Prevention  Tetanus Vaccine

o Active immunization – routine
 Tetanus toxoid or inactivated toxin
 DPT – given to infants at 2, 4, 6 and 18 months, with booster dose every 10 years
o Wound Prophylaxis – involves proper wound care
o Passive immunization – Tetanus Immune Globulin (TIG)

Clostridium botulinum
Morphology  Gram (+), large, straight or slightly curved rod with rounded ends
 Motile  peritrichous flagella
 Sporeformer  oval, subterminal, swollen, racket-shaped appearance

Cultural and Biochemical Characteristics  Strictly obligate anaerobe

 Easily cultured in ordinary media
 Blood agar – translucent, circular, swarming colonies with beta hemolysis
 Ferment glucose only (does not ferment lactose)

Disease
GOOD TO KNOW:
 Botox – Type A botulinum toxin
 Blocks the release of Acetylcholine in the NMJ,
which is for muscle contraction.
 No muscle contraction occurs  leading to Flaccid
paralysis  wrinkle-free skin
Clinical Manifestations
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 Botulism / Fatal Food poisoning – due to ingestion of food containing preformed
botulinum toxin (neurotoxin)
Absorbed in GIT mucosa  bloodstream → acts on myoneural junction → blocking
the release of acetylcholine
Incubation period of 12-36 hours
Spores abundantly found in soil  contaminate vegetables and meat
When canned or vacuum-packed without adequate sterilization, spores survive 
germinate  produce toxin
Types of botulism:
o Foodborne (Toxicosis) – most common & most severe
results from ingestion of contaminated food containing neurotoxin
0
inactivated by heating @ 60 C
o Infant botulism – seen infants resulting from ingestion of spores found in dust 
GIT  germinate  toxin production
o Wound botulism – spores from the environment  enters the tissue 
germinate  production of toxin absorbed
 Symmetrical descending weakness & paralysis
 Diplopia, dysphagia  pharyngeal paralysis  respiratory failure
 Organism produces the most potent toxin known on earth, botulinum toxin
8 serotypes of botulinum toxin: A, B, C, D, E, F, G, H
 A, B, E  responsible for human botulism
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 Laboratory Diagnosis  Serology – demonstration of toxin from food, serum or stool of infected individual
 Culture – food or stool
 Mouse Protection test – mice are injected or inoculated with food extract + antitoxin
animal dies unless protected by antitoxin
Treatment  Antibiotics are usually not administered, because usually there are no bacteria and only the
toxin is present.
 Hyperimmune Human Globulin
 Supportive treatment
 Support the airway: Intubation, then attach to a mechanical ventilator
Prevention  Proper cooking or sterilization of all canned and vacuum-packed food
0
 Food should be heated adequately at 80 C for 5 mins to inactivate toxin
 Swollen or bulging canned must be discarded
 High risk food includes:
o Homemade canned food
o Spoiled vegetable
o Smoke or salted fish/meat

Clostridium difficile
General  Obligate anaerobe
 Spore-former found in soil and human GIT
 Associated with antibiotic-induced pseudomembranous enterocolitis
 Saccharolytic & weakly proteolytic
o Saccharolytic – capable of hydrolyzing complex carbohydrates, with the production of
acid and gas
 Part of the normal flora of GIT in 3% of general population

Pathogenesis
Clinical Manifestations
 Antibiotics (Clindamycin, Minocin, Ampicillin) → suppress growth of GIT normal flora →
allowing growth of organism → multiply → produce & secrete toxins:
o Polypeptide A enterotoxin – potent toxin that damages the intestinal mucosa
o Polypeptide B cytotoxin – found in the feces, causing changes in tissue culture cell
 Mild diarrhea associated with yellow-white pseudo-membrane plaques
 Abdominal cramps
 Fever

Antibiotic-associated Colitis
Laboratory Diagnosis
Treatment
 In order to label a patient as having pseudomembranous colitis, you have to satisfy the
following criteria:
o Detection of toxin from a stool sample
 Cytotoxicity test – Hela cell  (+) cytotoxic effect on cultured cell
o Should have an endoscopic observation of the pseudomembranes/microabscesses
o Diarrhea – watery or bloody
o Have been given certain antibiotics (all antibiotics are capable of causing
pseudomembranous colitis)
 Biopsy or Sigmoidoscopy
 Symptomatic – withdrawal of antibiotics
 Fluid replacement
 Metronidazole

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NON-SPOREFORMING ACID FAST RODS GENUS Mycobacterium
 Most distinctive property is the characteristic staining  Straight or slightly curved rods
o Ziehl-Neelsen  Gram (+) acid fast organisms  Mycolic acid
o Cold Kinyoun  Nonmotile
 Difficult to stain  Obligate aerobes
 Once stained, resist decolorization with acid-alcohol  Fastidious
 Often referred to as “acid fast bacilli”  Species:
 Contains a wide range of species, which are either saprophytic (soil) o Mammalian tubercle bacilli
or parasitic (humans)  M. tuberculosis
 M. bovis
o Leprae bacillus – M. leprae
o Atypical mycobacterium – Runyon’s anonymous group

Mycobacterium tuberculosis
General  Koch’s bacillus (1882)
 Two varieties:
Differences Mycobacterium tuberculosis Mycobacterium tuberculosis
variant hominis (human) variant bovis (animals)
Animal Pathogenicity
Rabbit - +
Guinea Pig + +
Growth in Lowenstein-Jensen Eugonic (grows luxuriantly) Dysgonic (sparse growth)
(L-J) medium at 37 C
Niacin test + -

Morphology  Slender, straight or slightly curved Gram (+) rod with rounded ends
 No characteristics cell arrangement – singly, irregular clusters, branching, or with
serpentine arrangement
 Some are beaded or banded  polyphosphate
 Acid fast  due to mycolic acid
 Much granules (Note: not much like “a lot”, they’re literally called Much granules)
 Gram (+) spherical bodies found in the sputum of TB patients
 Non-sporeformer, nonmotile, noncapsulated

Cell Wall Structure:

Three-layered 60% lipid substances:
 Mycolic acid – long chain fatty acid; contributes to acid fastness of the organism
 Wax D – enhance immune response to antigen found in experimental animals
 Phosphatides – play a role in caseation necrosis
Cultural Characteristics  Strictly obligate aerobe
 Optimum temperature = 37 C
 pH 7.9
 Can grow in very simple synthetic medium
 Media used for primary isolation from clinical material  Lowenstein-Jensen /
Middlebrook
 Incubated with 5-10% CO2
 Very slow growing, general time of 16-24 hours
 (+) cultures grow in 4-6 weeks  small, dry, scaly, cauliflower-like colonies
 Culture must be held for 6-8 weeks before being declared NEGATIVE
 Liquid medium  pellicle growth on surface of the medium
 Produce niacin (other mycobacteria don’t)
Resistance  Relatively resistant to acid and alcohol, drying and chemical disinfectants
 Cultures maintained at 37 C remain viable and virulent after storage for 12 years
 Direct exposure to sunlight  killed in 2 hours
 Bacilli in sputum  require 20-30 hours of exposure
 Dried sputum protected from direct sunlight  organism remains viable for 6-8 months
 Sensitive to heat and radiation

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  Killed by 5% phenol in 24 hours
 Killed at pasteurization temperature
Pathogenicity  Tuberculosis (Koch’s disease) – chronic granulomatous disease involving the lungs, CNS,
kidneys, or bones
Mode of transmission:
o Inhalation – aerosols; most common
o GIT – ingestion of infected milk or milk products
o Disseminated from the CNS, kidneys, bones
Clinical findings: fever, fatigue, night sweats, weight loss, cough, hemoptysis
Determinants of Pathogenicity  Do not produce exotoxin and endotoxin
 Virulence of organism is attributed to:
o Trehalose dimycolate – cord factor
Responsible for serpentine growth or arrangement of organism
Can result to tissue damage due to local effect of organism and host defense
that may cause injury at site of infection
o Sulfatides – allows organism to survive intracellularly
Pathogenesis  Inhaled aerosols
Engulfed by alveolar macrophages
Bacilli
replicate
Macrophages die

 Infected macrophages migrate to local lymph nodes

 Develop Ghon’s focus – a primary lesion caused by MTB, usually subpleural, often in the
mid to lower zones
 Cell mediated immune response  stops cycle of destruction and spread
 Viable but non-replicating bacilli present in macrophages
Clinical Presentation

Primary Infection Type of TB

 When a host has first contact with the tubercle bacilli, the following features are usually
observed:
o An acute exudative lesion develops and rapidly spreads to the lymphatics and regional
lymph nodes. The exudative lesion in tissue often heals rapidly.
o The lymph node undergoes massive caseation, which usually calcifies.
o The tuberculin test result is positive (+).
 The primary infection type occurred in the past, usually in childhood, but now frequently
in adults who have remained free from infection and therefore, tuberculin negative in
early life.
 The involvement may be in any part of the lung, but is most often at the base.

Reactivation Type of TB
 The reactivation type is usually caused by tubercle bacilli that have survived from the
primary lesion.
 Reactivation tuberculosis is characterized by chronic tissue lesions, formation of tubercles,
caseation, and fibrosis.
 Regional lymph nodes are only slightly involved, and they do not caseate.
 The reactivation type almost always begins at the apex of the lung, where the oxygen

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 tension (PO2) is highest.
 These differences between primary infection and reinfection or reactivation are attributed
to: (1) resistance, and (2) hypersensitivity induced by the first infection.
Laboratory Diagnosis  After inoculation of specimen in the Mycobacterium Growth Indicator tube (MGIT)  kept
in BacTec for 42 days
o If any tube appears positive, it should be subcultured, acid fast stained, and treated as
a presumptive positive.
 Microbiological tests – demonstration of organism by acid fast staining
o Direct Smear Microscopy (DSM) – sputum specimen
Sensitivity: 40-70%
Specificity: 90%
 Ziehl-Neelsen staining – Hot or Cold (Kinyoun) method
 Fluorescence staining – Auramine-Rhodamine, Acridine Orange
 Culture on egg-based media – Lowenstein-Jensen medium  incubated with 5-10% CO2
Most commonly used media for primary isolation of M. tuberculosis
Before culture, specimen must undergo digestion and decontamination with N-acetyl-
L-cysteine NaOH
Colony characteristics: rough, tough, and buff-colored  it takes 4-6 weeks to get
visible colonies
 Biochemical testing
o Niacin test  (+) canary yellow
M. tuberculosis lacks the enzyme that converts niacin to niacin ribonucleotide
 Molecular techniques
o Direct detection of mycobacteria from specimens – Polymerase Chain Reaction (PCR),
Target amplification
o Identification of mycobacteria from culture – PCR-based sequencing, DNA and Gene
probes
o Advantages: Rapid procedure, 3-4 hours
High sensitivity, 1-10 bacilli/mL sputum
o Limitations: Very expensive
Cannot differentiate between living and dead bacilli
Require specialist training and equipments
 Tuberculin test – hypersensitivity test
Based on the fact that a person infected with TB develops hypersensitivity to the
protein of the organism
Place an important role in the control of TB
Purified Protein Derivative (PPD): Mantoux method, intradermal infection on the
volar surface of the forearm
Procedure: The test is done by putting a small amount of TB protein under the top
layer of the skin on the inner forearm. If the patient has ever been exposed to TB, the
skin will react to the antigens by developing a firm red bump at the site within 2 days
(48-72 hours).
(+) test  presence of 10mm induration
Determines hypersensitivity state of individual to pulmonary TB
Determines whether a person has ever had tuberculosis
Disadvantage: A tuberculin skin test cannot tell how long a person has been infected.
It also cannot tell if the infection is latent (inactive) or active and can be passed to
others.
 Direct detection by Nucleic Acid Amplification (NAA) – this test can reliably detect MTB in
specimens within hours, as compared to weeks for culture
Treatment  Multiple drug therapy – two or more drugs given together to prevent emergence of
resistant, mutated strains
 First line  Isoniazid (INH) – Rifampicin – Streptomycin – Ethambutol
Prevention  Isoniazid (INH) prophylaxis – for: asymptomatic patients, children exposed to
asymptomatic patient, children with (+) PPD skin test
 Bacillus Calmette Guerin (BCG) Vaccine – live attenuated strain of M. bovis with low
degree of virulence
 Pasteurization of milk

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Mycobacterium leprae (also, Hansen’s bacillus - 1878)
Morphology
Cultural Characteristics
Pathogenicity
Clinical Manifestations
 Straight or slightly curved acid fast rod
 Size: 1-8 um length, 0.3-0.5 um width
 Arranged singly, in large masses (globus) or packed inside cells  packet of cigarettes
arrangement
 Nonmotile, non-sporeformer, noncapsulated
 Cell wall structure resembles M. tuberculosis
 Efforts to culture the organism in-vitro were unsuccessful
 Can be grown experimentally in animals only (mice/armadillo footpads)  24-30 days
 Optimum temperature for growth is 26-30 C (psychrophile)
 Leprosy (Hansen’s Disease) – chronic communicable disease
Involves the cooler portion of the body: skin, eyes, nose, pharynx, larynx, nerves
Grows preferentially on the skin and superficial nodes
Man is the only natural host for infection
Obligate intracellular parasite that multiplies very slowly within mononuclear cells,
histiocytes of the skin, and Schwann cells in nerves
Incubation period varies from 3-4 years, to as long as 40 years
 Mode of transmission: prolonged contact with infected individuals, respiratory droplets
 Site of entry: skin and peripheral nerves, URT and nasal mucosa
Has two forms.
Tuberculoid Lepromatous
Scattered, raised skin lesions with palpable More widespread skin lesion (macular, popular,
thickening of peripheral nerves nodular) accompanied by thickening of earlobes,
forehead and nose  Leonine facies
Associated focal area of anesthesia Absent
Few acid fast bacilli seen in lesion, but difficult to Large numbers of acid fast bacilli present in
demonstrate involved skin
Disease is benign, with better prognosis More severe disease, with poor prognosis
Lepromin test (+) Lepromin test (-)

 Lepromin test – skin test used to determine the immunologic spectrum of patient
Employs the use of heat-killed suspension of M. leprae prepared from lepromatous
nodule and injected intracutaneously
Two types of reactions observed:
 Fernandez reaction (early) – resembles tuberculin reaction, appears in 24-48
hours
 Mitsuda reaction (late) – presence of indurated nodule which develops after 3-4
weeks
Determinant of Pathogenicity  Reaction of the body to the organism
Laboratory Diagnosis  No specific immunologic/serologic test for leprosy diagnosis
 Evaluate patient especially hypoesthetic skin lesions
o Microscopic demonstration of acid fast bacilli – from skin scrapings, nasal mucosa, or
biopsy materials
o Culture – footpads of mice
o Phenolase test – demonstrate phenolase from lepromatous nodule produced by M.
leprae
o Polymerase Chain Reaction (PCR)
 Lepromin Skin test – to classify the stage of leprosy based on the lepromin reaction
Procedure: A sample of inactivated leprosy-causing bacteria is injected just under the
skin, usually on the forearm. The injection site is labelled and examined after 3 days,
and again after 28 days, to see if there is a reaction.
Treatment  Multiple drug therapy (MDT)  combination of 2 or more drugs given together to prevent
emergence of drug-resistant, mutated strains
 Dapsone, Rifampicin, Clobazamine
Prevention  Early detection and isolation of acute cases
 Prophylactic chemotherapy – for individuals in close contact with patients
 Active immunization with BCG

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ATYPICAL MYCOBACTERIA (Runyon’s Anonymous Group)
 Strictly saprophytic Group II: Scotochromogens
 Can produce severe and fatal disease in humans  Produce pigments only when placed in a dark environment
 Classified into four groups according to:  Slow-grower, requires 7 or more days to produce visible colonies
o Rate of growth  Mycobacterium scrofulaceum
o Pigment production Scrofula  cervical lymphadenitis in children
 Laboratory Diagnosis
o Bacteriological – acid fast staining Group III: Non-Photochromogens
o Culture  Do not produce pigments under any circumstances
o Niacin test  (-) negative  Slow-grower, requires 7 or more days to produce visible colonies
 Mycobacterium avium-intracellulare
Group I: Photochromogens Opportunistic organism causing infection among
 Produce yellow-orange pigmented colonies only when exposed to immunocompromised hosts (cancer, AIDS, organ transplant
light patients)
 Slow-grower, requires 7 or more days to produce visible colonies
 Mycobacterium kansasii Group IV: Rapid-Growers
Disease produced resembles tuberculosis except symptoms are  Rapid grower, produce visible colonies in less than 7 days
mild  Rarely cause human infections
 Mycobacterium marinum  Saprophytic in soil and water
Produce granulomatous skin infection  swimming pool  Mycobacterium fortuitum
granuloma  Mycobacterium chelonae

NON-SPOREFORMING, NON-ACID FAST BACILLI

GENUS Corynebacterium
 Corynebacterium diphtheriae (Kleb-Loeffler’s bacillus)
 Corynebacterium pseudodiphthericum (Bacillus hoffmani)
 Corynebacterium xerosis
 Corynebacterium acne
 Corynebacterium minutissima

Corynebacterium dipththeriae
Morphology  Pleomorphic rod  straight or slightly curved, club-shaped
 Palisading, or Chinese letter arrangement
 Metachromatic (Babes Ernst) granules – responsible for beaded/banded appearance due
to polymerized metaphosphate; serve as a source of energy
Visualized using Loeffler’s methylene blue or Neisser stain
 Nonmotile

Cultural Characteristics  Facultative anaerobe, grow best aerobically

 Fastidious – in blood or serum
 Culture media:
o Loeffler’s Coagulated Serum – primary medium for isolation
Grayish white, glistening colonies  after 12-24 hours incubation at 37 C
o Cysteine Tellurite Blood Agar – selective, differential medium
Gray to black colonies
Can differentiate 3 colony types of C. diphtheriae:
 Gravis – large, flat, gray to black colonies with dull surface, non-hemolytic
 Mitis – small, black, glossy colonies, hemolytic
 Intermedius – flat, dry, convex colonies, non-hemolytic

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 Resistance  Resistant to light, dessiction, and freezing
 Dried pseudomembrane fragments can survive for 14 weeks
 Susceptible to disinfectants
 Readily killed after exposure to a temperature of 100 C for 1 minute or 58 C for 10 minutes
Disease  Diphtheria – human (natural and reservoir host) infection
An infection of local tissue of the URT with production of toxin which causes
systemic effects on the heart and peripheral tissues
Childhood disease  1 to 10 years old
Modes of transmission:
 Person to person – directly or indirectly
 Aerosol or droplet transmission – most common
Source of infection:
 Asymptomatic carrier
 Exposure to infected person during incubation period of the disease

Pathogenesis Inhalation  URT (Initial lesion in tonsil, oropharynx)

 
Multiply Nasopharynx
(non-invasive) 
 Pseudomembrane formation (dead cells, fibrin, toxins)
Secrete exotoxin
(Diphtheria toxin)

Bloodstream  disseminated to the kidneys, heart, CNS

 Incubation period of 1-7 days

 Signs and symptoms include: low-grade fever, chills, mild sore throat, cervical
lymphadenitis (bullneck appearance)

Local Manifestations Depends on the site of the lesion.

 Unilateral or bilateral serosanguinous (blood and serous fluid) discharge from the nose
 Excoriation of the upper lip
 Toxemia is minimal
 Redness and swelling over the fauces
 Exudates on the tonsils coalesces to form grayish white pseudomembrane
Skin lesions  Regional lymph nodes are inflamed
 Sore throat and dysphagia

Determinants of Pathogenicity  Invasiveness

o Surface K antigen
o Cord factor – enhances the virulence
o Neuraminidase
o N-acetylneuraminate lyase
 Exotoxin (diphtheria toxin) – inhibits protein synthesis in the host cell
Responsible for signs and symptoms of the disease
Most important determinants of pathogenicity

Antigenic Structure  K Antigen – heat-labile, responsible for the type specificity of the organism
 O Antigen – heat-stable, responsible for cross-reactivity of the organism

 Schick test – susceptibility test to determine the presence of diphtheria toxin

Inject 0.1 mL of purified toxin intradermally on the volar aspect of the forearm  48-
72 hours after, (+) reaction  localized inflammatory reaction with induration at the
site of inoculation
Patient susceptible to diphtheria will have no circulating antibody against the disease

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 Laboratory Diagnosis
Treatment
Prevention
OTHER SPECIES OF Corynebacterium
C. pseudodipthericum
 Normally found in nasopharynx (diphtheroid)
 Non-toxigenic, non-pathogenic
 Usually implicated in opportunistic infection
 Can grow on ordinary medium
GENUS Listeria
Listeria monocytogenes
General
Morphology
Cultural Characteristics
Determinants of Pathogenicity
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 Bacteriological – swab from nasopharynx, nose and throat  Gram stain
Staining with Loeffler’s methylene blue  demonstration of metachromatic granules
 Culture
 Schick test
 Toxigenicity test – in vivo: Frasser and Weld, in vitro: Eleks
 Diphtheria antitoxin – to neutralize the toxin in the blood
20,000 to 50,000 units 1m/IV
 Penicillin G/Erythromycin – antibiotics
 Active Immunization with diphtheria toxoid (DPT) – given in 4 doses, during infancy at 6-8
weeks
C. xerosis C. minutissima
 Isolaated from human conjunctiva  Infection usually involves the pubic and
 Responsible for xerosis (conjunctivitis) axillary area
 Erythrasma – a superficial skin infection that
causes brown, scaly patches
 Widely distributed in nature, has some animal reservoirs
 Produce human infection with protean manifestations, such as:
o Meningitis
o Infection of the genital tract of pregnant women
o Neonatal infection either before or after delivery
 Facultative intracellular organism
 Can invade and grow in a variety of mammalian cells including macrophages, epithelial
cells, fibroblasts
 Ability to enter the cytoplasm of the cell  grow  spread
 Small gram (+) rods arranged in short chains, end over end, tumbling arrangement
 Size: 0.4-0.5 x 0.5-2.0 um
 Actively motile  peritrichous flagella
 Noncapsulated, nonsporeformer
 Aerobe or facultative anaerobe
 Catalase (+)
 Growth occurs over a wide temperature range, from as low as 2.5 C
 Specimen from mixed flora should be refrigerated first before inoculation
 Culture media:
o Tryptose Agar (MacBride medium) – blue-green translucent colonies on oblique light
o Sheep BA – small, glistening, with zone of beta hemolysis
 Endotoxin-like substance in the cell wall (lipopolysaccharide) – with anti-phagocytic
property
 Hemolysin-soluble substance secreted during growth – plays an important role in the
pathogenesis of the infection
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 Mode of Transmission  Direct contact – among high-risk individuals (veterinarians, agriculturist, industrial
workers)
 Oral – ingestion of contaminated food, raw vegetables, drinking unpasteurized milk
 During delivery – contact of infective secretion
Disease  Neonatal infection
o Granulomatous infantiseptica
o Meningitis – acquired during or after birth
 Adult infection
o Meningitis – most common
o Septicemia
Laboratory Diagnosis  Isolation of the organism from blood, CSF, amniotic fluid, genital tract secretions  Gram
staining
 Culture – Tryptose medium
 Animal Pathogenicity test – Anton/Ocular Rabbit test
Treatment  Penicillin G
Prevention  No vaccine available
 Control of infection includes:
o Elimination of animal reservoir
o Avoid contact with infected animals

GENUS Erysipelothrix
Erysipelothrix rhusiopathiae
Morphology  Slender, straight or slightly curved Gram (+) rod
 Nonmotile
 Nonsporeformer
 Microaerophilic
 Blood agar – alpha hemolytic

Disease  Erysipeloid – acquired by traumatic inoculation of the organism

Laboratory Diagnosis  Microscopy – Gram staining
 Culture

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