GRAM POSITIVE BACILLI  Fever

 Pseudomembrane formation in
- Non-spore forming
the throat
- Spore forming
o Fibrin network – embeds the
NON-SPORE FORMING bacteria
o Contains necrotic host cells
- Genus Corynebacterium o Can extend to the throat
- Genus Listeria downwards (larynx or
SPORE FORMING trachea) - obstruction of the
air passage – suffocation
- Genus Bacillus  “bull-neck” – swelling of the neck
- Genus Clostridium  Mortality rate: up to 30% -- from
NON-SPORE FORMING: systemic complications

CORYNEBACTERIUM DIPHTHERIAE SYSTEMIC COMPLICATIONS

- Klebs-Loeffler’s bacillus  Polyneuritis

- General Characteristics: o nervous tissues (PNS)
o Gram (+) bacillus o paralysis
o Non-motile  Myocarditis
o Non-spore forming o Inflammation – myocardium
o Non-encapsulated of the heart
o Pleomorphic – show variation in shape: o Heart failure
 Coccoid/long bacilli  Cutaneous Diptheria
 Club shaped – “kory” –irregular  Lower mortality rate
swelling on one end  Can be a source of pharyngeal
o show metachromatic granules/ volutin diphtheria
granules: - Pathogenesis:
 phosphates o Diphtheria Toxin
 take up a color – red-purple w/  Lysogenized strains
methylene blue  Infected by a phage (ß-phage)
 Babes-Ernst granules  ß-phage- DNA  “tox gene”
o Chinese letter arrangement/ cuneiform  inhibits protein synthesis of host cells
o Facultative anaerobe  cause of systemic complications
o Catalase (+)  toxin is absorbed in the blood
o Oxidase (+)  vaccination
- Habitat & Transmission  trivalent form – DTP (Diphtheria,
o Habitat: Tetanus, Pertussis)
 Man only  diphtheria toxoid
 Nasopharynx  given: 2nd, 4th and 6th month of
 Immunized individuals – carrier state age
 Human skin  booster dose: 2 years old
o Transmission: - Schick’s Test
 Droplet o 2 purposes:
 Direct contact  Determine susceptibility
- Disease:  Susceptible: absence of antitoxin
o Diphtheria  Immune: circulating antitoxin
 Pharyngeal Diphtheria / Respiratory  Determine hypersensitivity
Diphtheria o Intradermal test:
 2 arms of the patient:
  1: test arm – inoculate small  Radial striations
amount of diphtheria toxin (can  “Daisy-head”
be inactivated by heating – 70°C)  Non-hemolytic
 2: control arm – inoculate  Can hydrolyze starch &
diphtheria toxoid (intradermal glycogen
injection) o C. diphtheriae var mitis
o Positive result: reddening & swelling  Medium(1-2mm)
o Negative result: no reddening & swelling  Convex
o Pseudo reaction: reddening & swelling,  Black
subsides in 4-5 days  Regular edges
 “coulee-hat”
Toxin Toxoid Interpretation
 Weakly ß-hemolytic
 Can’t hydrolyze starch
Skin 36 h 120 h 36 h 120 h susce hyper
response ptible sensit & glycogen
ive o C. diphtheriae var
intermidius
Positive - + - -
 Pinpoint(0.5mm)
 Flat
Negative - - - -
 Gray
Pseudo-  “frog-eggs”
+ - + -
rxn  Non-hemolytic
Combine  Can’t hydrolyze starch
+ + + -
d and glycogen
 Highly pleomorphic
 Tinsdale Agar
- Laboratory Diagnosis  Beef/bovine serum
o Microscopic Examination  Cystine-sodium thiosulfate-
 G + bacilli tellurite
 Pleomorphic  Selective: tellurite – inhibit G-
 Presence of metachromatic granules bacteria
 Chinese letter  Differential: tellurite reduction
o Cultural Method  Cystinase activity on cystine 
 BAM brown halo
 Colonies:  Loeffler’s serum medium
o White  Beef/bovine serum
o Entire  Coagulated eggs to solidfy the
o Convex medium
o Variable hemolysis  “poached eggs” colonies
 Cystine tellurite blood agar (CTBA)  Enhances 2 morphologic
 Selective & differential: cystine & characteristics:
tellurite o Metachromatic granule
 5% rabbit blood o Pleomorphic
 Colonies: black- tellurite  Pai medium
reduction tellurium  Coagulated eggs w/ H2O +
 3 biotypes: glycine
o C. diphtheriae var gravis  Metachromatic granule formation
 Large(2-4mm) and pleomorphisin
 Flat - Identification
 Dark gray o Toxigenicity test – ability to produce toxin
  Elek’s Test- in vitro – C. diphtheriae - C. xerosis – nasopharynx, skin, conjunctiva -
 Elek’s medium – rabbit serum normal flora
agar o Opportunistic
 Filter paper strip w/ diphtheria o Disease:
antitoxin  Respiratory tract infection
 (+) – v shaped white precipitin  Endocarditis
lines at 45°angle. – C. diphtheriae - C. minutissimum
– toxin producing o Erythrasma – superficial skin infection
 In vivo toxigenicity test o Red rashes
 Utilize a lab animal  Axillary skin
 Shave hair  Inguinal skin
 Put 2cm square marks on the o Wood’s lamp: brick red fluorescence/
back coral red
 Inoculate  Long UV radiation
 (+) – necrosis at the site of  Black light
injection – toxigenic

DIPHTHEROIDS
LISTERIA MONOCYTOGENES
- Other Corynebacteria
- Only pathogenic Listeria
- Mimic C. diphtheriae
- In animals: monocytosis = increase
- C. ulcerans
monocytes (30-35%)
- C. pseudotuberculosis
- General Characteristics:
- Habitat:
o G+ bacilli – coccobacilli- short bacilli
o Found in animals
o Non-spore forming
- Transmission:
o Non-encapsulated
o Human can acquire through contact w/
o Non-motile @ 35-37C
animals
o Motile @ room temp.: 22-28C
o Consumption of milk from infected
o Aerobic
animals
o Catalase (+)
- Disease:
o Oxidase (+)
o Mild pharyngitis
- Habitat:
o Diphtheria-like disease
o Primary: found in nature (soil, water)
 Some strains can produce
o Secondary: found in animals & plants
diphtheria-like toxin
- Transmission:
- Cultural Method
o Food-borne: consumption of infected
o BAM
animals
 Some strains are hemolytic (ß-
o Person-to-person: vertical transmission
hemolysis)
 Pre-natal, placenta
o Tinsdale agar
 During birth
 Brown halo
o Fecal
o Reverse CAMP test
- Can survive:
 Arrow head zone of no hemolysis
o Refrigeration temp
 Ability of the organisms to produce a
o High salt conncentrations
phospholipase enzyme (C/D) that
- Disease:
inhibits staphylococcal ß-hemolysin
o Listeriosis
- C. diphthericum
 Adults and children
o Nasopharynx – normal flora
 Immunocompromised
 Opportunistic
 Renal transplants
  Cancer px  McBride Agar – selective medium
 Elderly (partially)
o Meningitis and septicemia – common  High salt concentration – LiCl2
manifestation  Glycine
o Pregnant women – mild flu-like disease  Phenylethanol
o Prenatal Listeriosis  Blood agar medium
 Granulomatosis infantseptica o Other tests
 Early onset  Motility test
 Transplacental  Semisolid medium
 Granuloma formation  Umbrella zone of growth
o Abortion  Anton test
o Still birth  Pathogenicity test
 Meningitis and septicemia  Animal inoculation
 Late onset – 2-3 weeks  Instillation of a 24h broth culture
- Pathogenesis:  conjunctiva of the lab animal
o Internalin – A/B  (+) purulent conjunctivitis w/in
 Surface proteins 24-36h
 Binds w/ receptors on the epithelial
SPORE FORMING:
cells – E-cadherin
 Adhesion - G- bacilli
o Listeriolysin-O – destroys the phagosome - Genus: Bacillus, Clostridium
membrane  escape from the - spore, location of spore, characteristics fo
phagosome  multiply in the cytoplasm sporangium
o Phospholipase - oxygen requirement
o Act A – induces actin polymerization
 Actin filaments form on the surface Bacillus – sporangia – not swollen,
of the organisms  move to host aerobic/facultative anaerobic
cell membrane  cell-to-cell transfer Clostridium – swollen sporangium, anaerobic
 Intracellular existence
- Facultative intracellular BACILLUS:
o Phagocytes Bacillus anthracis
o Within non-phagocytic cells
- Lab Diagnosis - anthrax bacillus
o Microscopy - General Characteristics:
 G+ bacilli / coccobacilli o G+
 Wet mount: demonstrate motility o Large
 Tumbling motility o Square ends
 Head-over-heels motility o Long serpentine chain
 Peritrichous flagellation o “bamboo fishing rod”
o Cultural Method o Non-motile
 Cold enrichment technique o Spore: ovoid, sub terminal, not swollen
 Increase/ enhance isolation o Encapsulated: glutamic acid
 Ability to survive in cold o Aerobic
temperature - Habitat and Transmission
 BAM – 5% sheep blood o Found in nature – in soil as spores
 Small colonies o Pathogen of animals (herbivores)
 Blue-gray o Germination  toxemia  necrosis
 Narrow zone of inhibition o In humans: zoonotic
- Disease:
 o Human anthrax  BAM
 Cutaneous Anthrax  Large
 Spore contamination of wounds  Flat
 95% cases  Non-hemolytic
 Non-painful papule  Irregular edges
o Vesicle w/ edema fluid  “Medusa-head” colonies
 Black crater w/ edema  PLET Agar – Polymixin-Lysozyme-
o “black eschar” – lesion on EDTA-Thallous-Acetate
skin  Polymixin – inhibit G-
o Malignant papule  Lysozyme –
o Mortality is lower: 20%  EDTA – chelating agent
untreated individuals  Thallous acetate – inhibit G- & G+
o If untreated  toxemia  Blood Agar
 Pulmonary Anthrax  Selective
 Inhalation of spores  Mucoid – increase CO2 (5-20&) –
 Woolsorter’s disease incubation
 Germination  produce toxin  Bicarbonate agar
 Mortality rate: 100% in untreated  Defibrinated sheep/ horse blood
cases o Identification Tests
 Gastrointestinal Anthrax  Gelatin hydrolysis
 Ingestion of spores  Slow liquefaction
 Violent enteritis  “inverted fir-tree”
 <1% mortality  String-of-pearls test
- Pathogenesis  Ability of Bacillus anthracis to
o Capsule – important in early stages grow on penicillin –
o Anthrax toxin – late stage peptidoglycan
 Plasmid encoded  MHA – 10U penicillin
 Heat-labile o Place a coverslip on
 PA – protective Ag, binding protein, inoculation site
binds to receptor on host cell o Incubate: 3-6h
membrane  Bacilli  coccoid chains (string of
 LF – lethal factor, necrosis, hypoxia pearls arrangement)
 lysis of host cell  Ascoli Test
 EF – edema factor – fluid  Thermoprecipitin
accumulation  Heat-extracted Ag – capsular Ag
- Anthrax vaccine – subunit vaccine  Phage Typing
o PA – protective Ag  Susceptibility of B. anthracis to a
o Only given to high risk groups – lab specific phage (gamma-phage)
workers  Plaque – bacterial lysis
o Military personnel
Bacillus cereus
- Lab Diagnosis
o Microscopy - Causative agent of food poisoning
 G+ bacilli w/ square ends
 Spores (spore staining) EMETIC TYPE DIARRHEAL TYPE
 Capsular staining -Ingestion of -meat
 Glutamic acid – McFadyean heated/fried rice -vegetables
reaction – polychrome methylene -boiling – insufficient to -dairy products
blue (pink-purple) kill spores -ingestion of the spores
o Cultural Method -10-12h incubation
 -spores  C. baratii
germination toxin C. butyricum
-1-6h Difficile group C. difficile
Heat-stable enterotoxin Heat-labile enterotoxin (miscellaneous group)
Emetic syndrome: Abdominal pain
-nausea, vomiting diarrhea
Clostridium perfringens

- Genneral Characterisrics:
- Lab Diagnosis:
o C. welchii – old name
o Microscopy
o Welch’s bacillus
 G- bacilli
o G+ bacilli
 Motile
o Non-motile
 Non-encapsulated
o Encapsulated
o Cultural Method:
o Spore: central/sub central, swollen
 BAM
sporangium
 Large colonies
 Eccentric
 ß-hemolytic
o Box car appearance
 (v) growth @ 45°C
o Anaerobic
 (+) growth in PEA (Phenyl Ethyl
- Habitat and Transmission
Alcohol)
o Soil – spores
o Tests
o GIT – man & animals
 Gelatin hydrolysis
o Man – through the skin / via ingestion
 (+) salicin fermenter
- Disease:
 Penicillin – Resistant
o Gas gangrene
 (-) phage lysis
 80% - caused by C. perfringens
Bacillus subtilis  Spores enter through traumatic
breaks on the skin
- Hay bacillus
 Sharp objects  impair blood flow
- G+ bacilli in chains
 hypoxia  anoxia  Myonecrosis
- “Laboratory contaminant”
 Edema
- Cultural Method:
 Discoloration of tissues
o BAM:
 Bubbles are formed – filled with air/
 Large colonies
gas
 Spreading
 Blood filled exolate
 Irregular margin
- Pathogenesis:
 Ground glass appearance
o α-toxin – lecithinase, phospholipase C
 ß-hemolytic
 disruption of the host cell membrane
CLOSTRIDIUM  Theta(Θ)-toxin – cytolysin, heat labile
 Responsible for vascular damage
- swollen sporangium  Collagenase, protease, lipase –
Gas Gangrene/ Histotoxic C. perfringens damage to tissues
group C. novyi  CHO fermentation – gas and acid
C. septicum prod’n
C. histolyticum  Swelling of the tissues
C. sordellii FAMILY ENTEROBACTERIACEAE
C. bifermentans
Tetani group C. tetani - Ubiquitous – found anywhere
Botulinum group C. boutinum - Entero – intestines (large intestines/ colon)
 - Enteric bacteria - Citrobacter
- Enterobacteria Opportunistic – other
- Oxidase (-) Serratia – late LF
- NO3 reduction (+) Hafnia
- Ferment glucose

EDWARDS-EWING CLASSIFICATION

TRIBES GENERA
Escherichia
I Escherichieae
Shigella
II Edwardsielleae Edwardsiella
III Salmonelleae Salmonella
IV Citrobatereae Citrobacter
Klebsiella
Enterobacter
V Klebsielleae Pantoea
Hafnia
Serratia
Proteus
VI Proteeae Morganella
Providencia
VII Yersinieae Yersinia
VII Erwinieaae Erwinia

Normal Flora Enteric Pathogens

Eschricia Salmonella
Klebsiella Shiegella
Enterobacter Yersinia enterocolitica
Hafnia
Serratia
Proteus
Providencia
Morganella
Citrobacter
Edwardsiella
Pantoea

COLIFORMS NONCOLIFORMS
LF- produce gas w/in NLF- cannot produce
48h gas w/in 48h

Normal enteric flora: Enteric pathogens:

- Escherichia - Salmonella
- Klebsiella - Shigella
- Enterobacter - Yersinia
- Pantoea enterocolitica