BACILLUS

>aerobic
⮚ Sporeforming
⮚ > rod shaped organism
⮚ can be isolated from soil.
>Easily grown in BAP
>Motile
(peritrichous) except
B. anthracis
>Catalase
+
>Ferments glucose
>Starch Hydrolyzers
>Can survive as low as -5C and high as 75 C
>Survival in extreme temp. is due to
endospores
 
 1. Bacillus anthracis
>Anthrax bacillus
Gram (+) rods, chain-bamboo, square end
>non-motile, spore forming, zoonotic
Requires Thiamine (B1) for growth
The centerpiece for counter terrorism planning efforts
* Virulence factor:
Exotoxin (edema and lethal) and capsule-
D glutamate-bicarbonate medium
(enhance capsule formation)
 
Mc Fadyean’s reaction-
a capsular stain
Uses:
methylene blue
 Disease:
1. Malignant pustule-
black eschar
(cutaneous antrax)
CONTACTED
through animal contact and soil
2. Woolsorter’s –
respiratory infection
(rag pickers)
(pulmonary anthrax
3. gastroenteritis-
bloody diarrhea
(intestinal anthrax)
 Laboratory Diagnosis
 
1. Selective medium –
PLET
Polymixin Lysozyme EDTA Thallous acetate
2. Medusa head colony, inverted pine tree (gelatin medium)
3. Catalase
Positive
4. string of pearl test
USING: (0.5 units of penicillin) on BAP
5. Ascoli test- serologic examination
(pptn test)
Result (+)-
precipitin ring
6. PCR, fluorescence Ab test, ELISA
Best method of diagnosis
7. Penicillin susceptibility test (
+)
Uses:
10 unit PEN
 2. Bacillus cereus
>Fried rice bacillus
- spore rice grain
Best specimen for testing:
suspected food
* Virulence: Exotoxin (Cholera like Toxin)
Food poisoning
 2 forms
1. Diarrheal type
Ingestion of meat or poultry, and vegetables.
8-16 hours incubation period
Abdominal pain, watery diarrhea w/o fever
Production of heat labile enterotoxin
2. Emetic type
Ingestion of improperly stored rice/ reheated rice
9 hours is the average duration of illness
Abdominal cramps and profuse vomiting
Production of heat-stable enterotoxin
 B. anthracis B. cereus
Motility - +
Capsule + -
Hemolysis Gamma beta
Growth at 45C - +

Salicin - +
fermentation
Penicillin G S R
Gelatin HOH - +
and PEA
 3. Bacillus subtilis
>source of antibiotic such as Bacitracin
>opportunistic
>mistaken as Pseudomonas on culture media
To differentiate:
Pseudomonas- moist
B. subtilis- dry colony
>Gram (+) rod in chain; central spore
>common laboratory contaminant
>QC control in oven (hot air oven)
> causes eye infection among heroin addicts
BAP- large, flat, beta hemolytic, ground glass
 
Bacillus pumilus
 
On BAP- Large, moist, blister colony, may be B-
hemolytic
QC- radiation
 
 
Bacillus stearothermophilus- QC autoclave
> Flat sour spoilage; acid without gas
>thermostable
 
Anaerobic Gram positive Bacilli
Indicators of Anaerobic Bacteria
1. Foul odor
2. Presence of sulfur granule-
Actinomycetes spp. Propionobacterium
spp. Eubacterium nodalum
3. Brick red fluorescense- Prevotella or
Porphyromonas
4. Absence of superoxide dismutase (SOD)
 Clostridia
• Obligate anaerobic; sporfeforming g (+)
bacilli
• Motile w/ peritrichous flagella except C.
perfringens, C. ranosum and C. innocum
• Have swollen sporangia except C.
perfringens and C. bifermentans
• Non-encapsulated except C. perfringens
• Has a single hemolytic reaction except C.
perfringens- double hemolytic pattern
• CHO fermenters except C. tetani and C.
histolyticum
• Encountered in exogenous anaerobic
infections or intoxications
• Toxins usually gain access to the body
through ingestion or via open wounds.
• They produce collagenase (spreading
factor), hyaluronidase, lecithinase or
phospholipase (spreading-factor)
 3 types of Clostridium

 
1. Neurotoxic-
C. tetani and C. botulinum
2. Histotoxic-
C. perfringens, C. septicum
3. Enteric-
C. difficile
 Clostridium perfringens
• Gas gangrene bacillus
• Formerly known as C. welchii
• Spores are seldom seen, but it is oval,
central to subterminal.
• Boxcar-shaped appearance on gram stain
• Dome-shaped, gray to white colonies on
BAP
• Characterized by double zone of
hemolysis (alpha and beta zones)
• Has characteristic “stormy fermentation of
milk” (using lithmus milk medium)
• Lecithinase (+)- detected using egg yolk
agar
• Nagler (+)- lecithovitallin rxn. Lecithinase
C on EYA- formation of ppt. around
colonies w/o antitoxin and no ppt. on
the other side w/ anti-toxin
• Reverse CAMP test (+)- arrow head zone
of hemolysis.
• Virulence factor: a-toxin and enterotoxin
• They produces deoxyribonuclease-
 Laboratory Diagnosis of Clostridium
perfringens
1. Chopped meat agar
growth (+) gas
2. BAP
Target or double zone of hemolysis
3. Nagler test –
lecithinase test
> due to alpha toxin, Lecithinase C and phospholipase C
 
Egg yolk agar + Neomycin= Mc Clung or Neomycin Eggyolk
Selective
Result:
+ opalescence on agar w/o anti toxin
No opalescence on agar w/ anti toxin
4. Reverse CAMP test
(+)
 Direct Nagler Test
- Using EYA plate + C. perfringens type A antitoxin
- Result: (+) inhibition of lecithinase reaction
produced by C. perfringens

C. perfringens type A antitoxin is not specific for

C. perfringens. Nagler test/Lecithinase test can
also be positive for C. bifermentans, C. sordellii,
C. perfringens, and C. novyi type A
 Positive: C. perfringens,
C. bifermentans
C. sordelli
C. noyvii type A

Lecithinase positive - A zone of opacity in the antitoxin-free half

only but not on another half due to neutralization of the alpha-
toxin.

Lecithinase negative - No reaction on the agar.

 Clinical infections
1. Gas Gangrene/Myonecrosis (eating
sore)
- Is a life threatening destruction of muscle
and other tissues: necrotizing infection of
skeletal muscles.
- Organisms contaminate wounds either
thru trauma, frostbite or surgery
- Pain, swelling, serous discharge,
discoloration and tissue necrosis
Hyperbaric oxygen therapy- high
concentration of O2 at increased pressure
2. Food poisoning/ Enteritis (Pig-bel)
- Ingestion of enterotoxin in contaminated
food.
- Diarrhea and crampy abdominal feeling
 Clostridium tetani
• Tack Head bacillus
• Soil and environmental inhabitants
• Produce heavily swarming anaerobically:
sporeforming bacilli
• With terminal spore and swollen sporangia
“drumstick/lollipop/tennis racket”
appearance
• On BAP, the colonies are with matte
surface, narrow zone of B-hemolysis.
• The endospores of C. tetani are found in
hospital environments, in soil and dust, and in
the feces of many farm animals.
• Virulence factor: tetanospasmin (neurotoxin)
Tetanospasmin- is an endopeptidase that
selectively cleaves the synaptic vesicle
membrane protein synaptobrevin:it causes
tension and cramping and twisting in skeletal
muscle surrounding the wound and tightness of
the jaw muscles
 Clinical infection/Disease
Tetanus- “trimus”/lock jaw (risus sardonicus smile)
- The organism (spore) establishes a wound
infection and elaborates the potent toxin that
mediates generalized muscle spasms.
Tetanus neonatorum- contaminated instruments
used for newborns
Symptomatology- muscular rigidity (jaws, neck and
lumbar region). Difficulty in swallowing, rigidity of
the abdomen, chest, back and limbs.
 Clostridium botulinun
• Canned good bacillus
• - most severe cause of food poisoning
• It is found in soil and aquatic sediments
• it is characterized by the presence of
subterminal spores; B hemolytic on BAP
• It is a proteolyticbacteria and has the
ability to oxidized one amino acid with the
second amino cid as the electron acceptor
(stickland reaction)
Virulence factor: botulism toxin- neurotoxin-
most potent toxin known to man.
Botulism toxin- Block release of acetylcholine

(flaccid paralysis)

Function on acetylcholine
mediator for muscle contraction
 Disease
1. wound botulism- spore on wound
2. infant botulism- grown in gut and honey bee
(floppy baby syndrome)
 
3. Clostridium tetani
> TERMINAL SPORE (tennis racket, drumstick)
- tack head bacillus
>assccharolytic
Virulence: Exotoxin (tetanolysin and tetanospasmin)
-binds to ganglioside receptors and inhibit neurons in CNS-
spastic paralysis
>lock jaw, Risus sardonicus, opisthotonus
 
Lab. Diagnosis
1. clinical findings- basis for Diagnosis
2. Terminal spore
3. Lipase test on EYA (+)
Lipase (+) organisms produce a colony that is covered with an
iridescent, multicolored sheen “appearance of gasoline on
water or mother-of-pearl”
(+)= C. botulinum, C. noyvii type A, C. sporogenes

 
Lipase (+) on EYA
 Clostridium difficile
• Causative agent of pseudomembranous colitis
• On g/s, it may produce chains up to 6 cells
aligned end to end
• Endospores may be oval and subterminal
• It has characteristics “horse stable” odor and
nonhemolytic- BAP
• Yellow, ground glass colonies- CCFA
• It is an infection control dilemma among
hospitalized patients.
 4. Clostridium difficile
• colon flora
• Can be activated by clindamycin
- Associated with
pseudomembranous colitis
Specimen: rectal swab/ stool
 
Laboratory Diagnosis
 
Direct detection of toxin (stool)
- EIA ; Cytotoxin assay
 
Culture: CCFA
Cycloserine cefoxitin fructose agar
Result:
Yellow horse manure odor
• It is acquired in the hospitals by individuals
receiving antibiotics.
• Virulence factors: toxin A (enterotoxin)
and toxin B (cytotoxin)
• Ferments fructose producing formic acid.
Specimen Collection and Transport
Specimens: Blood, CSF, abscess
- Material for anaerobic culture is best
obtained by tissue or by aspiration using a
needle and syringe.
- - food and fecal specimens suspected of
C. perfringens food poisoning should be
transported at 4 C
- Feces for C. difficile culture and toxin
assay should be liquid or unformed
- solid, formed or rectal swabs are adequate
to detect carriers but not to detect
enterocolitis.
- A crucial factor in the final success of
anaerobic cultures is the transport of the
specimens (lethal effect of oxygen)
- All specimen should be held at room
temperature. Refrigeration can oxygenate
the specimen.
1. Gram stain (+)
2. Culture-anaerobic blood agar,
thioglycollate, Egg yolk agar (EYA),
Cycloserine Cefoxitin Fructose Agar
(CCFA), peptone-yeast glucose broth
(PYG), Brucella/Blood Agar (BRU/BA),
PRAS
• Primary plates should be freshly prepared or
used w/in 2 weeks of preparation.
• Plates stored for longer periods accumulate
peroxides and become dehydrated
• Lipase (+) organisms produce a colony that is
covered with an iridescent, multicolored
sheen “appearance of gasoline on water or
mother-of-pearl”
• Swarming and double zone of hemolysis on
BRU/BRA is observable
• Reazurin or methylene blue- indicators for
anaerobic jars- colorless in absence of O2
 ANAEROBIC BACTERIOLOGY
Collection:
Needle aspiration
 
1. Reduced media- Anaerobic BAP
Schaedler Media, Bacteroides bile esculin
2. Laked kanaycin vancomycin BAP (LKVBA)
3. Anaerobic PEA, Egg yolk agar
4. Chopped meat, Peptone yeast extract glucose
5. Thioglycollate, Lombard Dowell agar
 Methods to promote anaerobiosis
>Gas pak jar or
Mcintosh Fildes jar
Brewer
Torbal
>Cooked meat medium/ chopped meat medium sealed with
petrolatum
>Anaerobic glove box and chamber
>PRAS pre-reduced anaerobically sterilized
(Roll tube of Hungate)
>Thioglycollate medium
Indicator:

resazurin red (boiling to remove O2)

PRAS Medium
Anaerobic chamber
 Anaerobic chamber
Must contain:
1. Catalyst → Palladium pellets
2. Desiccant → Silica gel (turns blue to pink=FULL WITH
WATER)
3. Indicator → Methylene blue (blue to colorless=NO
OXYGEN) /Resazurin red (red to colorless=NO OXYGEN)
4. Gas
: 5% Hydrogen gas (to combine with Oxygen to form water)
: 10% CO2 (growth req. of anaerobes)
- a 10% increase in CO2 level lowers Oxygen levels by 18%
: 85-90% Nitrogen gas (Filler gas)
Gas Pak Jar
Gas Pak Jar
 Gas Pak Jar
Contains:
1. Gas-Pak envelope
Old: Add water to produce Hydrogen and CO2 - dangerous
if the Jar is opened near an open flame. (Presence of water
vapor)
New: Does not need water. Opened and put inside the jar
within 1 minute. Absorbs oxygen while releasing CO2. (No
water vapor present= easy viewing)
2. Catalyst → same with anaerobic chamber
Old: Catalyst working properly= water vapor will be present
3. Indicator → same with anaerobic chamber
Gas Pak Jar
Failure to produce anaerobiosis (removal of O2)
1. Poisoned catalyst= due to H2S gas produced
by anaerobes
Reusable catalyst=rejuvenated after every use
by heating in a 160° C oven for a minimum of 2
hours.
2. Crack in jar
3. Crack in lid
4. Broken “O”-ring
 ID-kanamycin Vancomycin Colistin
1. RRR- B. fragilis
2. SRS- Fusobacterium, B.ureolyticus, Veilonella
3. SSR- Clostridium, gram (+) cocci
4. RSR- Porphyromonas, P. anaerobius
5. RRS- Prevotella
 
CHARACTERISTIC ANAEROBE
Brick red fluorescence Prevotella, Porphyromona
Red fluorescence Veilonella
Pitting of agar B. ureolyticus
Double zone of hemolysis C. perfringens
Swarming C. tetani , C. septicum
Molar tooth colony, sulfur Actinomyces israelii
granules

Breadcrumb colony F. nucleatum

Horse odor (CCFA) C. difficile
 “Bamboo fishing rod”
• A. Bacillus anthracis
• B. Clostridium difficile
• C. Clostridium perfringens
• D. Listeria monocytogenes
Canned good bacillus
• A. Clostridium perfringens
• B. Clostridium botulinum
• C. Clostridium tetani
• D. Clostridium histolyticum
 Gram (+) anaerobic bacilli-
nonsporeforming
1. Actinomyces-
fungus like bacteria
Actinomyces bovis- lumpy jaw
Actinomyces Israeli- draining sinus tract with sulfur granules
(molar tooth colony)
2. Bifidobacterium dentium
3. Eubacterium lentum
4. Propionobacterium acne
5. Lactobacillus-
Tomato juice agar
6. Mobiluncus- vaginitis
(G. vaginalis) HBT agar
 Gram negative anaerobic bacilli
1. Bacteroides fragilis
needs 20% bile (black colony) beta lactamase
Normal flora of the GI
Colon bacillus
2. Porphyromonas asaccharolytica
black pigment, red fluorescence on UVL
3. Prevotella melaninogenica
black pigment, red fluorescence on UV
What is the difference ?
 
4. Fusobacterium nucleatum-
Breadcrumb colonies; fusiform rod
5. F. necrophorum-
Vincent’s angina
Aka. Gingivitis/gums disease/Lemierre’s disease
6.Bacteroides ureolyticus-
pitting of agar
 
 Gram positive anaerobic cocci
1.Peptostreptococcus anaerobius
• SPS sensitive
• Indole (-)
2. Peptostreptococcus asaccharolyticus
3. Peptococcus niger (staphylococcus like)
Catalase (+) black colony
 
 Gram negative anaerobic cocci
1. Veilonella parvula- oral flora
NO3 (+)
Jaw disease, Periodontitis
Fluorescence red UVL
2. Megasphera
3. Acidaaminococcus
 Sources:
Lecture notes of Dr. Emerson Bernardino
Mahon’s 5th Edition