OTHER GRAM NEGATIVE BACILLI

Future RMTs ☺

“Do your very BEST

while you have the time.”

“It is always better to suffer the

pain of DISCIPLINE today
than to suffer the pain of
REGRET tomorrow.”
Gram (-) organisms on Mac
Pseudomonas aeruginosa
 Obligate aerobe
 Motile (1 or 2 polar flagella) and rod-shaped
 Sweet / grape-like / corn-taco like odor
resulting from the production of 2-
aminoacetophenone
 Grows well at 37-42degC, its growth at
42degC will differentiate it from other
Pseudomonas spp.
 OXIDASE POSITIVE - dark purple
 Does not ferment carbohydrates(TSI : K/K
Gas (-), H2S (-) like Alcaligenes)
Pseudomonas aeruginosa
 Bailey and Scotts: Serrated and confluent
growth
 Produces blue-green, diffusible pigment:
 Pyocyanin - blue

 Pyoverdin – a.k.a. Fluorescein, green

 Produces infection of wound and burns,

giving rise to blue-green pus
 Meningitis, UTI, pneumonia, sepsis
Pseudomonas aeruginosa
 Mild otitis media/externa – Swimmer’s ears
 Skin lesion – Ecthyma Gangrenosum
 Hot tub or Whirlpool dermatitis in spa
 Keratitis associated with contaminated
contact lens care solution
 Respiratory infection in patients with Cystic
fibrosis
 Resistant to a number of disinfectants and
has been responsible for serious nosocomial
infections
 Nice to know!

 MILK BACTERIOLOGY
 Changes in milk color
◼ Pseudomonas aeruginosa: blue green milk
◼ Pseudomonas syncyanea: blue milk
◼ Flavobacterium synxanthum: yellow milk
◼ Serratia marcescens: red milk
 Other changes:
◼ Streptococcus lactis: causes souring of milk
◼ Bacillus subtilis: hay bacteria which causes proteolytic
action on coagulated milk
◼ Coliform bacteria: hydrolyzes casein (milk protein)
◼ Alcaligenes viscous: capsule-forming bacteria which
causes slimy or ropy milk
Burkholderia mallei
 Causes GLANDER’S DISEASE, a disease of
horses and similar animals transmissible to
humans
 Horses - pulmonary involvement
 Humans - fatal, begin as ulcer of skin and
mucous membrane followed by
lymphangitis and sepsis
Burkholderia pseudomallei

 Whitmore’s Bacillus
 Causes MELLIODOSIS (Vietnamese Time
Bomb Disease) , an endemic glander’s
like disease of animals and humans which
can be reactivated after many years
Vibrio
 Comma bacillus
 Gram (-) bacilli, comma shaped
 Facultative anaerobe, monotrichous
 OXIDASE POSITIVE to differentiate from
Enterobacteriaceae
 Halophilic except Vibrio cholerae and
Vibrio mimicus
Vibrio
Vibrio cholerae
 Choleragen toxin stimulates adenylate cyclase :
GI fluid loss, “rice-watery stool”
 Vibrio cholerae O1 and O139 - spread of disease
 Cultured on Alkaline Peptone Water and TCBS
 TCBS: Yellow colonies, Sucrose fermenter
 Associated with seafoods
 String Test
 Reagent: 0.5% Sodium desoxycholate

 (+) VIBRIO to differentiate from Aeromonas and

Plesiomonas shigelloides
Vibrio
Vibrio cholerae
Serogroups
 Inaba – Philippines

 Ogawa - India

 Hikojima - Japan
Vibrio Biotypes (Biovars)

CLASSICAL EL TOR
Red cell - +
hemolysis
VP - +
Polymyxin B Sensitive Resistant
Aggl’n with - +
chicken RBCs

Classical : Pandemics of the past

El Tor : Recent pandemics
Vibrio
Vibrio alginolyticus
 Clinical significance: wound and ear infections

associated with marine environment

 Halophilic

 Sucrose fermentation positive (Yellow colonies on

TCBS)
Vibrio parahemolyticus
 Clinical significance: gastroenteritis, usually

associated with contaminated seafood

 Halophilic, sucrose fermentation negative (TCBS:

green colonies)
Vibrio
Vibrio mimicus
 Clinical significance: gastroenteritis and ear

infections involving marine environment

 NONHALOPHILIC, sucrose fermentation

negative (TCBS: green colonies)

Vibrio vulnificus
 Clinical significance: septicemia and

wound infections involving marine

environment
 Halophilic; TCBS- green or yellow colonies
Haemophilus
 Nonmotile, non-sporeforming
 Facultative anaerobes
 Most spp. are oxidase and catalase positive
 Preferred incubation : 35-37 degC, 5-10% CO2
 Requirements:
 X factor (Hemin) - from the degradation of
Hgb; heat stable
 V factor (NAD/Coenzyme I) - produced by
some bacteria and yeast; can be obtained
from potato and yeast extract; heat-labile
 Haemophilus
SPECIES X FACTOR V FACTOR BETA D-ALA
REQUIREMENT REQUIREMENT HEMOLYSIS
H.influenzae √ √ - -
H.parainfluenzae × √ - +
H.hemolyticus √ √ + -
H.parahemolyticus × √ + +
H.aegypticus √ √ - -
H.aphrophilus × × - +
H.paraphrophilus × √ - +
H.ducreyi √ × - -
Haemophilus

D-ALA
 detects requirement for X factor
 Requires X factor: D-ALA (-)
 Do not require X factor: D-ALA (+);
enzymes that convert pophobilinogen and
porphyrin to hemin
Haemophilus
Haemophilus influenzae
 Formerly known as PFEIFFER’S BACILLUS

 Six serotypes (a, b, c, d, e and f); most

frequently encountered serotype in
infection: b
 Encapsulated strains are pathogenic; some
are non-encapsulated strains
 Main cause of meningitis in children <5years
old (1 month – 5y.o.)
 Associated with respiratory conditions

including epiglottitis
Haemophilus
Haemophilus aegypticus
 Formerly known as KOCH-WEEK’S BACILLUS
 Closely resembles Haemophilus influenzae biotype III
 Causes Pink-eye conjunctivitis
Haemophilus ducreyi
 Requires X factor only
 Smallest pathogenic bacteria
 Infective agent of CHANCROID or soft chancre,
venereal disease characterized by painful ulcers in
the genitalia
 Direct examination : Short bacilli in a school of fish
arrangement
Campylobacter
 S-shaped / Sea-gull’s wings
 Human pathogens: C.coli, C.jejuni (associated
with gastritis and diarrhea)
 Animal pathogens: C.fetus (abortions in animals;
No growth at 42 degC)
 Microaerophilic and capnophilic, motile, gram
negative rods that are associated with gastritis
and diarrhea
 Optimum temperature for growth: 42-43degC
 Motility : single polar flagellum, “DARTING”
 Culture medium : Campy’s BAP, Skirrow’s
medium
Helicobacter
 Associated with peptic ulcer
 The natural habitat is the human stomach,
where the organism is found in the mucus
secreting cells
 Current evidence may suggest a role
between H.pylori and peptic and
duodenal ulcers
 Histology staining and culture of biopsies
obtained from the stomach or duodenum
are recommended for the identification
Helicobacter
 Le(a-b+) : phenotype prone to
Helicobacter infection

 Helicobacter pylori - strong producer of

urease to withstand acidity of stomach
 Motile by 4 to 6 polar flagella
 Optimum temp : 35-37 degC
 Skirrow’s medium can also support its
growth
Bordetella
 Gram negative bacilli, aerobic, non-motile
except Bordetella bronchiseptica
 Bordetella pertussis - agent of WHOOPING
COUGH
 Three stages of pertussis (whooping cough)
 Catarrhal: general flu-like symptoms
 Paroxysmal: repetitive coughing episodes

 Convalescent: recovery phase

 Ideal specimen : Nasopharyngeal swab

Bordetella
 Culture medium
 Bordet-Gengou Medium : potato-glycerol
based medium enriched with 15-20%
defibrinated blood

 Regan-Lowe / Charcoal-Cephalexin
Medium : charcoal agar supplemented with
horse blood, cephalexin, and amphotericin B

 Colonies : Mercury droplets

Bordetella
 Bordetella parapertussis - pertussis-like
syndrome, mild form

 Bordetella bronchiseptica - inhabits

respiratory tract of canines (Kennel’s
cough); infrequently responsible for
chronic respiratory tract infections in
humans; MOTILE by peritrichous flagella
 Bordetella

UREASE MOTILITY NITRATE OXIDASE

B.pertussis
- - - +

B.parapertussis + - -
-
B.bronchiseptica +
+ + +

Source: Delost p.246

Brucella
 Associated with milk and dairy products
 Agents of brucellosis, undulant fever, Malta fever
 Small, nonmotile, aerobic, gram negative
coccobacilli or short rods
 Organisms are normal flora of the urinary tract and
GIT of sheep/goat (Brucella melitensis), cattles
(Brucella abortus), pigs (Brucella suis) and dogs
(Brucella canis)
 Humans acquire infections through the ingestion of
contaminated animal products, including meats
and milk; farmers can directly acquire infections
through direct animal contact
 Brucella
CO2 H 2S Growth in the presence of
THIONINE BASIC FUCHSIN

B.abortus + + Inhibited G
B.melitensis - - G G
B.suis - - G Inhibited
B.canis - - G Inhibited

Source: Delost p.247

Specimen : Blood, Bone marrow

Culture for Brucella : Castañeda bottles,
-Biphasic medium (agar & broth
Incubation : 3-4 weeks
 Legionella
 Gram negative bacilli
 Legionella pneumophila (Legionnaire’s disease, Pontiac
fever)
 Legionella micdadei (Pittsburgh pneumonia)
 Legionella bozemanni (Wiga agent of pneumonia)
 Naturally found in both natural and artificial water
sources; the bacteria have been found in ponds, creeks
and streams, wet soil, water-cooling towers for air-
conditioning and heating systems, shower heads, and
plumbing systems
 Specimen : Bronchial wash, expectorated sputum
 Stain : DIETERLE’s stain (brown-black)
 Culture medium : Buffered Charcoal Yeast Extract, Feeley-
Gorman
Pasteurella
 Pasteurella multocida -agent of pasteurellosis (“many
killings”)
 Carried in the oral cavity, respiratory and GIT of cats
and dogs
 Agent of shipping fever in cattles, a hemorrhagic
septicemia
 MOT to humans: contact with domestic animals that
harbor the bacterium; most frequent route of
infection is from bite or scratch of an infected animal,
usually a cat
 Wound infections are the most common type of
Pasteurella infection
 Will grow on ordinary media
Francisella
 Faintly staining, gram negative coccobacilli that
are non-motile and obligately aerobic
 Francisella tularensis - requires CYSTINE &
CYSTEINE
 Tularemia is a disease of the rodents, primarily the
rabbits
 Individuals who handle the infected animal,
acquire through direct contact with blood or
through animal bite or scratch
 Bacterium can also be inhaled or acquired
indirectly through insect vectors, primarily ticks
 Culture medium: Blood Glucose Cystine Medium
Gardnerella
 Gardnerella vaginalis - associated with
bacterial vaginosis

 Gram-variable to gram negative bacillus,

previously known as Haemophilus vaginalis
and Corynebacterium vaginalis

 Bacterial vaginosis (BV) is characterized by

foul-smelling, grayish, vaginal discharge
Gardnerella
Gardnerella vaginalis
 Amsel and Nugent systems are used to

diagnosed BV
 Cytology / Pap’s : CLUE CELLS

 Whiff or Sniff test

 Vaginal discharge + 10% KOH

 (+) Fishy-amine like odor
 Organism can be isolated on: HBT
(Human Blood Bilayer Tween Medium)
Streptobacillus moniliformis
 Gram negative bacilli
 Normal flora of rodents (ex. RATS)

 Agent of:
 Rat-Bite Fever : from animal bite / scratch

(SM /SM - Spirillum minus)

 Haverhill
Fever : from ingestion of
contaminated milk
MISCELLANEOUS GRAM NEGATIVE
BACILLI
Capnocytophaga
 Capnophilic, fusiform, or filamentous
bacilli
 Characteristic motility: GLIDING
motility
Calymmatobacterium
granulomatis
 Agent of GRANULOMA INGUINALE or
DONOVANOSIS
 Encapsulated, pleomorphic gram negative
bacillus usually observed in vacuoles in large
mononuclear cells
 Groups of organisms are seen within
mononuclear cells; this pathognomonic entity is
known as Donovan body
 Closely related to Klebsiella
 Based on DNA-DNA Hybridization, it has been
recently proposed that the name of this organism
be changed to Klebsiella granulomatis
Eikenella corrodens
 Was first called Bacteroides corrodens
 Fastidious, capnophilic rod; part of the
gingival and bowel flora
 Corroding bacterium
 Characteristic in agar : Pits / corrodes
 Produces : bleach-like odor

 Associated with infections in insulin-

dependent diabetics and IV drug users
causing “needle-licker’s osteomyelitis”
Chryseobacterium
 Former Flavobacterium

 Yellow colonies on BAP

Chromobacterium violaceum
 Found in soil and water, has the ability to

produce violacein, a purple pigment

Cardiobacterium hominis
 Normally found in the upper RT and is

isolated as rare cause of endocarditis

GRAM POSITIVE BACILLI
Bacillus
 Gram positive bacilli that form spores
AEROBICALLY
 Catalase POSITIVE

Bacillus anthracis
 the most virulent; the largest pathogenic
bacilli, largest bacteria; agent of anthrax
 Cutaneous anthrax / Malignant pustule /
Black Eschar
 Woolsorter’s disease or pulmonary anthrax /
Inhalation anthrax
 Gastrointestinal anthrax
Bacillus
Bacillus anthracis - LAB
 Medusa head colonies on BAP

 From patient, encapsulated (polypeptide;

D-glutamic amino acid)

 From culture, bamboo pole arrangement
(large sporulated bacilli in a bamboo
fishing pole arrangement)
 On MHA with penicillin: string of pearls

 Colony lifted by a loop or needle : beaten-

egg consistency
Bacillus
Bacillus cereus
 Associated with food poisoning isolated

from several foods: rice, other cereals,

vegetables and milk
 Associated with vomiting and diarrhea

 Ideal specimen : FOOD

Bacillus

TESTS B.ANTHRACIS B.CEREUS

Catalase + +
Lecithinase + +
Motility (-) nonmotile +
Hemolysis Gamma Beta
Clostridium
 Gram positive bacili that form spores
ANAEROBICALLY
 Catalase NEGATIVE

Clostridium perfringens
 Formerly Clostridium welchii / Bacillus
aerogenes
 Cause of myonecrosis (gas gangrene)

and food poisoning - “pork poisoning” /

PIG BEL
 Associated with SULFHEMOGLOBINEMIA
Clostridium perfringens
 In solid media and thioglycollate broth, spores are
USUALLY ABSENT
 Double zone of hemolysis
 Inner: COMPLETE zone (Theta toxin)

 Outer: INCOMPLETE zone (Alpha toxin & Lecithinase)

 Lecithinase positive
 Reverse CAMP
 Streptococcus agalactiae on middle streak,
Clostridium perfringens on horizontal
 “BOW-TIE HEMOLYSIS”
 Nagler reaction
 (+) PPT’N around colonies on site without anti-toxin
and no ppt’n on side with anti-toxin
Clostridium
Clostridium botulinum
 Agent of food and wound botulism, as well

as infant botulism (“floppy-baby”)

 “CANNED-GOOD POISONING” - preformed

toxin
 Botulism results from the liberation of

botulism toxin, a powerful neurotoxin

associated with FLACCID PARALYSIS
 Spores : Oval and subterminal (oftentimes)
and center (sometimes)
 Lipase positive
Clostridium
Clostridium tetani
 Etiologic agent of tetanus, which results

from entry of the organism or spores into a

puncture wound
 Produces tetanospasmin, (neurotoxin)
associated with spastic contractions / lock
jaw / “Devil’s grin” or “Risus Sardonicus”
 Spores : Round and terminal (“tack-
head”, “drum-stick”, “lollipop”)
Clostridium
Clostridium difficile
 Normal flora of the GIT

 Important cause of antibiotic-associated

pseudomembranous colitis & diarrhea

 CLINDAMYCIN - first antibiotic associated

 Spores : Oval and subterminal

 Cultured on CCFA

(Cycloserine-Cefoxitin Fructose Agar)

 Clostridium

MOTILITY LECITHINASE LIPASE LACTOSE GLUCOSE

C.perfringens (-) (+) - (+) +
C.botulinum + - (+) - +
C.tetani + - - - (-)
C.difficile + - - - +
Corynebacterium diphtheriae
 KLEBS-LOEFFLER BACILLUS; agent of diphtheria
 Produces a powerful exotoxin that first attacks the
mucous membranes of the respiratory tract
 Results from inflammation and pseudomembrane
formation of the oropharynx leading to respiratory
obstructions
 Non motile, Nitrate reduction positive, Urease
negative, catalase positive
 Glucose, maltose fermented; Sucrose not
fermented
 Not all strains are toxigenic
Corynebacterium diphtheriae
 On BAP: colonies with narrow zone of Beta
hemolysis
 Babes-Ernst Granules / Metachromatic
/ Volutin granules
 Club-shaped (Coryne=club) : granules conc.
on one side of the cell
 Barbed or Dumbbell (granules on both sides)

 Pallisade or picket-fence

 Snapping form or Chinese character/letters

STAINS: LAMB, Burke’s Mod. of Gram Stain

Corynebacterium diphtheriae
CULTURE
 Loeffler’s Serum Slant (LSS)

 Pai’s coagulated egg

 Modified Tinsdale’s agar

 Tellurite agar : Gray-black colonies /

black colonies
Corynebacterium diphtheriae
COLONIAL TYPES OF C.DIPHTHERIAE
 Gravis type: 1 to 2mm colonies on blood

agar; largest colonial type

 Mitis type: fried egg appearance on blood

agar (clear colonies with white centers);

bleachlike odor on Tellurite medium
 Intermedius type: small colonies (0.5mm) on

blood agar; black colonies with gray

borders on Tellurite medium
Corynebacterium diphtheriae
TOXIGENECITY TEST
 In vivo : Animal inoculation (guinea pig)

 In vitro : ELEK’s test

 (+) Precipitation lines at 45 degrees angle

SCHICK’S TEST
 Susceptibilty test

Arm + diphtheria toxin

 (+) result: redness / erythema
DIPTHEROIDS
Corynebacterium jeikeium (formerly group
JK)
 Resistant to a number of antibiotics

 Associated with endocarditis, pneumonia,

and peritonitis
Corynebacterium pseudodiphthericum -
normal, throat
 Corynebacterium xerosis - conjunctiva

 Corynebacterium acnes - skin

 Corynebacterium minitissimum - erythrasma

Listeria monocytogenes
 Gram positive bacilli, CAMP positive
 H2S negative, Motile
 Found in the environment in soil, water, sewage
and decaying vegetation and in feces of
humans, swine and poultry
 Major source of infection is contaminated food
(cabbage, fruit, dairy products)
 Meningitis, pneumonia, abortion, stillbirth,
endocarditis, conjunctivitis and urethritis
 Perinatal human listeriosis (granulomatosis
infanseptica)
Listeria monocytogenes
 MOTILITY
 Hanging drop : Tumbling

 Semisolid medium : Umbrella-like / Inverted

Christmas tree-like growth
 Cultured on MCBRIDE MEDIUM
 Virulence Test : Anton’s Test / Ocular Test for
Anton
 Organism is inoculated to the conjunctival
sac of the rabbit
 Positive result : Purulent conjunctivitis
 NOTES!

LISTERIA CORYNEBACTERIA
Motility
+ -
Salicin
fermentation
+ -
Erysipelothrix rhusiopathiae
 H2S Positive, Catalase negative, non-
motile
 Causative agent of ERYSIPELOID, a
cutaneous inflammation of hand or fingers
(seal finger or whale finger)
 Veterinary infection and occupational
hazard for those handling meat, poultry
and fish
 Gelatin stab: Test tube brush / Bottle brush
growth
Lactobacillus acidophilus
 Normal flora of the mouth, gastrointestinal
tract (“Boas-Oppler bacilli”) and vaginal
canal (“Doderlein bacilli”)
 Nonpathogenic and has little clinical
significance
 Cultured on Tomato Juice Agar
Kurthia bessonnii
 Found in soil, opportunistic pathogen

Rothia
 Normal flora of the human mouth; rare
cause of abscess and endocarditis
MYCOBACTERIUM
 Mycobacterium tuberculosis
 Microscopically, the human tubercle bacillus is
slender, slightly curved rod shaped microorganism
measuring 0.2 to 0.6 micron in diameter and 1-4
micra in length.
 RESISTANCE: Highly resistant to drying. When
protected from sunlight, they remain in putrifying
sputum for weeks and in dried sputum for 6-8
months. Droplets of dried sputum in the air may be
infectious for 8-10 days. Organisms from culture
are killed within 2 hours when exposed to direct
sunlight; but in sputum, requires 20-30 hours
exposure before they are killed.
 Mycobacterium tuberculosis

 They are generally more resistant to chemical

disinfection than other vegetative organisms;
specifically difficult to disinfect in sputum in
which requires 24 hours exposure in 5% phenol.
However, they are easily killed by moist heat,
boiling for 10 minutes, pasteurization or steam
under pressure (autoclave).

Source: Training Manual on Direct Sputum Microscopy

National Tuberculosis Reference Laboratory, RITM, DOH
Mycobacterium tuberculosis

 TB usually begins in the middle or lower lung,

other areas for dissemination include the spine
(Pott’s disease), long bones, heart, meninges,
and genitourinary system.
 Macrophages phagocytize the bacteria and
form multi-nucleated cells, which are
eventually surrounded by fibroid cells. Together
the cells form granulomatous lesions called
tubercles, which can be seen on chest X-rays.
The lesions calcify, at which point they are
called “GHON COMPLEXES”.
Mycobacterium tuberculosis
Skin test for TB - PPD (Purified Protein
Derivative) - heat killed ammonium
sulfate - precipitated organism is
injected intradermally.
(+) redness after 48-72 hours
Notes: Mantoux - Intracutaneous
Von Pirquet - scratch
Volmer - patch
Mycobacterium tuberculosis

ANTITUBERCULOSIS AGENTS COMMONLY

TESTED AGAINST M.TUBERCULOSIS
Primary Drugs: Pyrazinamide, Isoniazid,
Ethambutol, Rifampicin, Streptomycin
(PIERS or RIPES)
Secondary Drugs: Ethionamide,
Capreomycin, Ciprofloxacin, Ofloxacin,
Kanamycin, Cycloserine, Rifabutin
Methods for Decontamination and
Digestion of Mycobacteria

1. N-acetyl-L-cysteine (NALC) and 2-4%

NaOH
 NALC: digesting / mucolytic agent
 NaOH : decontaminating agent

2. Trisodium phosphate and benzalkonium

chloride (Zephiran)
3. Dithiothreitol and NaOH
4. QUATS
 Inactivated by ORGANIC substances
Specimen
1. Sputum - gargle with water or saline
2. Secretion - obtained by bronchoscopy
3. Blood
4. Urine
5. CSF : (+) Pellicle formation/web-like clots
after refrigeration for 12-24 hours
6. 3Ps: Pleural / Peritoneal / Pericardial :
Increased ADA (Adenosine Deaminase)
Media
 Requires ↑ PROTEIN
 Malachite green - inhibit the growth of

contaminating agents
1. Lowenstein-Jensen : most common
2. Petragnani – for heavily contaminated sx
3. American Thoracic Society
4. Middlebrook 7H10 and 7H11c
Clear agar: optimum for exam of colonies
 Culture maintained for 8 weeks

 Colonies: Tan to buff, nonpigmented colonies,

rough, dry, warty-looking; granular res.

CAULIFLOWERS
M. tuberculosis on LJ medium
M. gordonae (“tap-water bacillus”)
Stains
1. Ziehl-Neelsen (HOT method) : most
commonly done in the lab
2. Kinyoun (COLD method) : for
demonstration of AFB in tissues
3. Fite-Faraco’s : Hematoxylin is used as
counterstain
4. Auramine-rhodamine stain (Truant’s)
(+) Yellow fluorescent organisms on
black background
Stains
5. Spengler’s : for color blind individuals
(+) BLACK
6. Pappenheim’s
MTB : red
M.smegmatis : blue
7. Baumgarten’s
MTB : blue
M.leprae : red
Methods of Reporting
CDC Method to Report AFB
Number of AFB seen Report
0 Negative
1-2/300 fields +/-, Repeated on
second slide
1-9/100 fields 1+
1-9/10 fields 2+
1-9/field 3+
>9/field 4+
Methods of Reporting
National Tuberculosis Association Method

Number of AFB seen Report

1 to 2 per slide Report number and
request another sx
3 to 9 per slide Rare (1+)
10 or more per slide Few (2+)
1 or more per OIF Numerous (3+)
Methods of Reporting
NATIONAL STANDARD REPORTING SCALE
0 No AFB seen in 300 fields
+n 1-9 AFB / 100 VF (write actual
number)
1+ 10-99 AFB / 100 VF
2+ 1-10 AFB / OIF in at least 50 VF
3+ More than 10 AFB / OIF in at least
20 VF
NOTES!

 The National Standard Reporting Scale is

the method used in the Direct Sputum
Smear Microscopy (DSSM) of the TB-DOTS
program (Tuberculosis-Directly Observed
Treatment Shortcourse) of the Department
of Health (DOH).
Mycobacterium leprae
 Hansen’s disease or leprosy (not highly
contagious)
 Acid fast rod in nasal mucosa of patients
with nodular variety of Hansen’s disease
 Transmission occurs person to person
through inhalation or contact with
infected skin
 Armadillo is susceptible to Hansen’s
disease and has been used experimentally
Mycobacterium leprae
 Smears of tissue juice, AF stain for lepra
cells
 On Microscopic exam : Parallel or palisade
resembling CIGARPACKETS
 Cultured on Foot Pads of Mice
 Treatment : Sulfone dapsone
 Skin Test : Lepromin Test
 Fernandez or Early reaction: (+) 24 – 48 hours
 Mitsuda or Late reaction: (+) 3-4 weeks
NOTES!
 Mycobacterium bovis : TB in animals and
cattles
 Attenuated form in BCG (Bacillus Calmette-
Guerin)
 Mycobacterium avium: TB in birds / chicken
 Mycobacterium avium-intracellulare complex :
BATTEY Bacillus, ↑ in AIDS px
 Mycobacterium kansasii : Yellow bacillus
 Mycobacterium gordonae : Tap water bacillus
 Mycobacterium marinum : Swimming pool
granuloma
NOTES!
 Mycobacterium terrae : raddish bacillus
 Mycobacterium ulcerans : INERT bacillus;
mostly (-) biochemical reactions
 Causes BURULI ULCER
 Mycobacterium gastri : J bacillus
 Mycobacterium fortuitum-chenolei : grows
on MacConkey without Crystal Violet
Runyon Classification of
MOTT (Source: Delost)
Group I : Photochromogens
Pigment production
Time required for growth: 3-60 days
 Mycobacterium kansasii

 Mycobacterium asiaticum

 Mycobacterium marinum

 Mycobacterium simiae
Runyon Classification of
MOTT (Source: Delost)
Group II : Scotochromogens
 Mycobacterium scrofulaceum

 Mycobacterium szulgai

 Mycobacterium xenopi

 Mycobacterium gordonae

 Mycobacterium flavescens

 Mycobacterium thermoresistible
Runyon Classification of
MOTT (Source: Delost)
Group III : Nonphotochromogens
 Mycobacterium avium-intracellulare

complex
 Mycobacterium malmoense

 Mycobacterium haemophilum

 Mycobacterium terrae-triviale
Runyon Classification of
MOTT (Source: Delost)
Group IV : Rapid Growers
 Mycobacterium fortuitum-chenolei

complex
 Mycobacterium phlei

 Mycobacterium smegmatis
OTHER TESTS
1. Niacin
 All Mycobacteria produces NIACIN. Most

with enzyme that would convert niacin to

niacin ribonucleotide. MTB lacks enzyme
(accum.of NIACIN)
 Reagent : CYANOGEN BROMIDE ☺

 Positive result : Yellow (+) MTB

2. Nitrate reduction
A. Broth (+) Red/Pink
B. Strip (+) Blue
OTHER TESTS
3. Heat stable catalase
 Reagent : 30% H2O2, heated at 60degC

for 20 minutes
 Negative reaction for all members of the
Mycobacterium tuberculosis complex
which includes: MTB, M.bovis,
M.africanum, M.ulcerans, M.leprae, and
M.microti
OTHER TESTS
4. Tween 80 Hydrolysis
 Tween 80 (polyoxyethylene sorbitan

mono-oleate) is converted to oleic acid

by Tween 80 lipase.
 Most useful in the identification of
Mycobacterium kansasii
(+) Pink (-) Amber ☺
5. Arylsulfatase
 Uses Tripotassium phenolphthalein

 Positive: PINK (+)M.fortuitum-chenolei

OTHER TESTS
6. Pyrazinamidase
 In this procedure, pyrazinamide is

converted to pyrazinoic acid

 Positive for MTB and M.marinum

 Negative for Mycobacterium bovis &

Mycobacterium kansasii
7. Iron uptake
 (+) result : growth in 20% Ferric chloride

 Results : (+) M.fortuitum

(-) M.chenolei
OTHER TESTS
8. Urease
 Positive result : dark pink

 Positive for M.bovis, M.scrofulaceum,

M.gastri

9. Growth in 5% NaCl
 Positive for M.triviale
 NOTES!

To differentiate MTB M.bovis

1. Niacin
+ -
2. Nitrate
Reduction + -
FUNGUS-LIKE BACTERIA
Actinomyces
 Gram positive but neither acid fast nor
stained with fungus stain, anaerobic,
catalase negative
 Causes Chronic suppurative
granulomatous disease
 Agent of LUMPY JAW
 Colonies : MOLAR TOOTH COLONIES
 Sulfur granules in tissues
Nocardia
 Gram positive, PARTIALLY ACID FAST,
aerobic, catalase POSITIVE
 Nocardia asteroides is the most clinically
relevant species; other species include
Nocardia brasiliensis and Nocardia
otitidiiscavarium
 Primary pulmonary infection resembling
tuberculosis
 (+) UREASE
Tropheryma whipplei
 Phylogenetic analysis shows that this organism is
a gram-positive actinomycete that is not
closely related to any other genus known to
cause infection
 WHIPPLE’S DISEASE, found primarily in middle
age men, is characterized by the presence of
PAS-staining macrophages (indicating
mucopolysaccharide or glycoprotein) in almost
every organ of the body
 Bacillus is seen in macrophages and affected
tissue but has never been cultured
Spirillum minus
 Gram negative, helical, strictly aerobic
organism
 Causes rat-bite fever in humans and is
referred to as SODOKU FEVER
 Clinical signs and symptoms are similar to
those caused by Streptobacillus moniliformis
except that arthritis is rarely seen in patients
with Sodoku and swollen lymph nodes are
prominent; febrile episodes are also more
predictable in Sodoku
SPIROCHETES
BORRELIA, TREPONEMA AND LEPTOSPIRA
LAB
STAINS
 Levaditi

 Warthin-Starry

 Fontana-Tribondeau

CULTURE
 Kelly’s medium

 BSK (Barbour-Stoenner-Kelly’s)

Examination of Blood film

Borrelia
 Flexibly twisted organism resembling
STRETCHED SPIRAL
 Helically coiled bacteria transmitted
through arthropod vectors including
lice and ticks
Borrelia
Borrelia recurrentis
 Agent of louse-borne relapsing fever

 High fever, muscle and bone pain,

confusion
 Vector: Human louse (Pediculus

humanus)
Borrelia
Borrelia burgdorferi
 Agent of LYME DISEASE

 Transmitted by tick vectors (Ixodes or deer ticks)

 Three stages:

1. Stage one: appearance of lesion: Erythema

chronicum migrans (“Bull’s eye”)
2. Stage two: dissemination through blood;
affected areas may include the bones, CNS,
heart and liver
3. Stage three: neurological abnormalities,
arthritis and skin lesions
“Bull’s eye” rash in Lyme dse
Borrelia
LAB (Lyme Disease)
 Culture : Kelly’s medium

 Serological tests : ELISA, Western blot

Borrelia parkeri / Borrelia hermsii

 Tick-borne relapsing fever

 Vector : Ornithodords ticks

Treponema
 Tightly twisted organism resembling
CORKSCREW

Treponema pallidum subsp. pallidum

 Venereal syphilis / Great Pox / Evil Pox /

Italian / French / Spanish Disease

 Trasmitted by sexual contact, direct blood

transmission or transplacental route

 Dies on refrigerator after 3 days
NOTES!
Mother to fetus transmission (transplacental)
 Congenital syphilis

 HUTCHINSON’S TRIAD

 Notched teeth
 Keratitis

 Deafness
Treponema
STAGES
 Primary syphilis: hard chancre (painless

and firm) Secondary syphilis:

Condylomata lata -wart-like lesions in moist
areas of the body; highly infectious,
systemic dissemination
 Latent syphilis: absence of clinical

symptoms
 Tertiary syphilis: gummas / gummatas,

neurosyphilis
Treponema
LAB
 Primary syphilis: DF

 Secondary syphilis: highly contagious,

systemic dissemination
DF, Sero tests
 Latent syphilis: (+) Sero tests → ONLY

indication
 Tertiary syphilis: Sero tests
Treponema
Treponema pallidum subsp. pertenue
 Yaws (chronic nonvenereal disease of skin and
bones)
 Transmission: traumatized skin comes in contact with
an infected lesion
Treponema pallidum subsp. endemicum
 Bejel (lesions in oral cavity, oral mucosa, skin, bones
and nasopharynx)
 Transmission: Mouth to mouth by utensils
Treponema carateum
 Pinta (ulcerative skin disease)
 Transmission: traumatized skin comes in contact with
an infected lesion
NOTES!
Bejel / Nonvenereal endemic syphilis
 Primary lesion: oral cavity
 Secondary lesion: oral mucosa
 Tertiary lesion: skin, bones, nasopharynx
NOTES!
TREATMENT
 1st: Heavy metals (Arsenic)

 Arsphenamine, Salvarsan, 606

 DOC: Penicillin

JARISCH-HERXHEIMER REACTION
 Large quantities of toxins are released as

bacteria dies during treatment

NOTES!
 Other treponemes such as T.vincentii,
T.denticola, T.refringens, T.socranskii, and
T.pectinovorum are normal inhabitants of
the oral cavity or the human genital tract.

 Acute necrotizing ulcerative gingivitis, also

known as Vincent’s disease, is a
destructive lesion of the gums.
 Leptospira
 Tightly twisted organism with one or
both ends bent into a HOOK

Leptospira biflexa
 Nonpathogenic, found in water and soil
 Leptospira
Leptospira interrogans
 Cause of human and animal leptospirosis, a
zoonosis
 Parasitic on vertebrates other than humans,
including rodents, cattles, dogs, cats, raccoons,
and oats
 Shed in the urine of those animals and humans
acquire the infection through direct contact with
urine of animals who carry the organism
 Involves the kidney, liver and CNS
 Severe form of leptospirosis: WEIL’S DISEASE
PRINCIPAL LEPTOSPIRAL
DISEASES
Leptospira interrogans serovar.

 icterohemorrhagiae : WEIL’s disease

 canicola : Infectious Jaundice
 autumnalis : Fort Bragg fever / Pre-tibial
fever
 grippotyphosa : Marsch fever
 hebdomadis : 7-day fever
 mitis/pomona : Swineherd’s disease
Leptospira
Culture
 Blood is the most sensitive specimen during

early infection; urine should be cultured

after second week

 Media include Ellinghausen-McCullough-

Johnson-Harris and Fletcher’s, Stuart’s
 Maintain for 6-8 weeks
CHLAMYDIA, MYCOPLASMA and
RICKETTSIAE
Chlamydia

 Former BEDSONIA (large virus)

 Obligate intracellular

 Infectious particle: Elementary bodies

 Metabolically active: Reticulate bodies
Chlamydophila psittaci
 Agent of PSITTACOSIS / ORNITHOSIS, a
disease of birds, parrots, parakeets and
cockatoos

 Humans may acquire the infection by

inhalation of contaminated aerosols or
fomites or through person-to-person
transmission
Chlamydophila pneumoniae
 Associated with respiratory tract infections
 TWAR Strain (Taiwan Acute Respiratory Strain)
Chlamydia trachomatis
Subtypes Clinical Syndrome

A, B, Ba, C Endemic trachoma (multiple or persistent

infections that ultimately lead to blindness) /
TRIC-Trachoma Blindness Inclusion
Conjunctivitis
D-K Urethritis, cervicitis, pelvic inflammatory disease,
epididymitis, infant pneumonia, and
conjunctivitis
L1, L2, L3 Lymphogranuloma venereum
NOTES!
LAB
 Culture → McCoy’s cells

 Cytology

 Immunologic tests

 FREI TEST - delayed hypersensitivity

skin test for LGV
Mycoplasma
 Gram negative, pleomorphic (vary in
shape)
 Smallest free-living organisms, found in
several animals and plants

 Originally known as pleuropneumonia-

like organisms (PPLOs), since they were
first discovered causing pleuropneumonia
in cattles
Mycoplasma pneumoniae
 EATON’S AGENT, frequent cause of
community-acquired pneumonia and
tracheobronchitis in children and young
adults

 Primary atypical pneumonia / Walking

pneumonia
Genital Mycoplasmas: Mycoplasma
hominis and Ureaplasma urealyticum

 Can colonize adults asymptomatically

and are also cause of nongonococcal
urethritis in males

 Mycoplasma hominis has also been

identified as an agent of salpingitis and
postpartal fever in females
NOTES!
MYCOPLASMA Culture
 Shepard’s / SP4 / A7B / E-Agar
 “Fried-egg” colonies

Serological tests
 Cold Agglutinins: Anti-I
 ↑ Ab titer of Streptococcus MG
Rickettsia
 Include the genera Rickettsia, Ehrlichia,
Coxiella and Rochalimea
 Gram negative, obligately intracellular
bacteria
 Infections are spread through insect
vectors such as lice, fleas and ticks
 All Rickettsia, except Coxiella, cannot
survive outside the animal host or insect
vector
Rickettsia
 Signs of infection include fever, headache,
characteristic rash that first appears on the
wrists and ankles
 Other manifestation of infection include
conjunctivitis, pharyngitis, and mild
respiratory distress
 All require tissue culture medium EXCEPT
Rochalimea quintana
 Rickettsia
GROUP SPECIES INFECTION TRANSMISSION
Spotted R.rickettsii Rocky Mountain Ticks
Fever Spotted Fever
R.akari Rickettsial pox Mites
R.australis Australian/ Ticks
Queensland tick
typhus
R.conorii Boutonneuse fever, Ticks
Mediterranean and
Israeli spotted
fevers; Indian tick
typhus, Kenya tick
typhus
 Rickettsia

GROUP SPECIES INFECTION TRANSMISSION

Typhus R.prowazekii Epidemic typhus Lice
Sporadic typhus Flying squirrels
Brill-Zinsser dse Reactivation
of latent
infection
R.typhi Murine typhus/ Fleas
Endemic typhus
Scrub typhus R.tsutsugamushi Scrub typhus Mites, chiggers
O.Tsutsugamushi

Q fever Coxiella burnetti Q fever Ticks, aerosol

 Rickettsia
GROUP SPECIES INFECTION TRANSMISSION
Ehrlichiosis E.chaffeensis Human Ticks
monocyte
ehrlichiosis
E.phagocytophila Human Ticks
E.ewingii granulocyte
ehrlichiosis
Neorickettsia Sennetsu fever Ticks
sennetsu (former
E.sennetsu)

Rochalimeae Rochalimea Trench fever Lice

quintana
NOTES!
 WEIL-FELIX TEST: used in the diagnosis of
Rickettsial infections
 Sourceof antigen:
Proteus vulgaris: OX-2 & OX-19
Proteus mirabilis: OX-K
Results:
OX-2(+), OX-19 (+), OX-K (-): RMSF, epidemic,
endemic typhus
OX-2(-), OX-19 (-), OX-K (+): Scrub typhus
OX-2(-), OX-19 (-), OX-K (-): Rickettsial pox, Q fever,
Trench fever
Bartonella
 Bartonella spp. originally were grouped
with the members of the family
Rickettsiae

 Short, gram negative, rod-shaped,

fastidious organisms that are oxidase-
negative and grow best on blood-
enriched media or cell co-culture
systems
 Bartonella

Organism Habitat MOT Clinical

(reservoir) Manifestations
B.quintana Uncertain; Human Trench fever
possibly small body Chronic
rodents, louse bacteremia,
humans endocarditis,
bacillary
angiomatosis,
chronic
lymphadenopath
y, pericarditis
B.bacilliformis Uncertain, Sand flies Carrion’s disease /
humans Oroya/Veruga
Peruana
Carrion’s dse
 Bartonella
Organism Habitat MOT Clinical
(reservoir) Manifestations
B.henselae Domestic Domestic Cat-scratch
(primary CA of cats cat by bites disease
CSD) and / or Bacteremia,
scratches, endocarditis,
cat fleas bacillary
angiomatosis,
peliosis hepatitis,
neuroretinitis
B.clarridgeiae Domestic Domestic Bacteremia, cat-
cats cat by bites scratch disease
and/or
scratches
B.elizabethae Rats Fleas Endocarditis
Afipia felis
 Associated with cat-scratch disease
(CSD), rare role in this disease

 Despite its rare isolation, indirect evidence

suggests that the organism may be more
commonly linked to CSD than is currently
appreciated due to lack of appropriate
laboratory methods for detection
 “A DREAM IS A PICTURE OF A
POSSIBILITY, AND WE SHOULD HAVE
A STRONG HEART AND A PREPARED
MIND TO TURN THAT INTO A
REALITY.”

~PIA ALONZO WURTZBACH

Miss Universe 2015
#RMT2019