MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS
GRAM-POSITIVE COCCI COAGULASE TEST
- Common isolates in the clinical microbiology
laboratory
Staphylococcus
- Spherical, non-motile grape like cluster
- Came from Greek term staphle meaning ‘bunch of
grapes’
- Non-encapsulated, non-spore-forming
- Aerobic or facultative anaerobe
- Catalase-positive, gram-positive
Catalase utilizes H2O2 to convert into H2O and O2
- Can ferment glucose except S. saprophyticus
- Colonies produced after 18-24 hrs of incubation are
medium sized (4-8 mm) and appear cream-colored,
white, or rarely light gold, and “butterfly-looking”
- Normal inhabitants of the skin and mucous
membranes of humans and other animals
3 CLINICALLY IMPORANT SPECIES
1. Staphylococcus aureus
2. Staphylococcus epidermidis
3. Staphylococcus saprophyticus
Staphylococcus aureus
- Mannitol Salt Agar or MSA:
Golden yellow pigment
- K Tellurite Medium:
Jet black colonies
- Blood Agar Plate or BAP:
Beta-hemolytic
Uses 10% sheep blood to exhibit hemolysis
- S. citreus – lemon yellow pigment
- S. albus – porcelain white pigment
Types of hemolysis:
 Alpha- partial hemolysis (green in color)
 Beta- complete hemolysis
 Gamma- non-hemolytic
BAGUISTAN, EVANKA MARIELLE D. | BSMLS3-1
Embag.
- Best single criterion for pathogenicity
- Positive result: coagulation of plasma
- Uses citrated plasma
The plasma came from the rabbit
- 2 methods:
Tube method: demonstrate free coagulase
Slide method: demonstrate bound coagulase
- Only S. aureus will be positive
- Reading within 4 hours prevent false-negative
results
- Reading within 20 prevent false-positive results
Reading should be done within 4-20 hours
CATALASE TEST
- Uses 3% Hydrogen peroxide (H2O2)
- Positive result: rapid effervescence of gas
- Staphylococcus species are catalase-positive
- Streptococcus species are catalase-negative
- The test is performed by emulsifying a colony of
staphylococcus in H2O2 and observing a bubbling
reaction as oxygen is liberated
Staphylococcal Virulence Factors
a. Protein A – limits host immune response
b. Ribitol
c. Coagulase – clotting; also catalyze fibrinogen to
fibrin
d. Catalase – convertion of H2O2 into oxygen and water
e. Beta-hemolysin – RBC destruction
f. Alpha, beta, delta toxins – cytotoxic; destroy cells
g. Staphylokinase – protease that can convert
plasminogen into plasmin
h. Lipase – metabolize lipids and fats
i. Gelatinase – hydrolysis of protein
j. Hyaluronidase – degradation of hyaluronic acid (for
DNA synthesis and spreading of bacteria)
k. Leucocidin – destroy WBC
l. Staphylococcal enterotoxins – common cause of
food poisoning
m. Nuclease – destroy nucleus of cells (host)
n. TSST-1, TSST-2 - Toxic Shock Syndrome Toxin;
responsible for systemic organ damage; induce
fever, vomiting, and toxic shock
MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS
MANNITOL SALT AGAR
- Ability of S. aureus to ferment mannitol and
tolerate 10% NaCl
- Positive result: golden yellow colonies
- pH indicator: phenol red
Diseases produced by S. aureus
- Pimples
- Carbuncles – skin disease; damages skin going to
hair follicle; boils
- Furuncles – more severe than carbuncles
- Folliculitis – infected hair follicle
- SSSS – Staphylococcal Scalded Skin Syndrome
o Peeling skin over large parts of the body
due to exfoliative toxin
- Staphylococcal food poisoning
o Enterotoxin
o Heat stable
o Symptoms: nausea, vomiting, and
abdominal cramps
o Caused by unhygienic preparation of foods
- Toxic shock syndrome – possible of not capable of
getting pregnant
- Impetigo
- Fasciitis
- Cellulitis
Staphylococcus epidermidis
- Resident flora of the skin
- Coagulase-negative; novobiocin sensitive
- Slime factor (form biofilm)
- Stitch abscess, mild UTI, endocarditis, bacteremia,
meningitis
Staphylococcus saprophyticus
- Common cause of UTI
Second most common cause (E. coli first)
- Coagulase-negative; novobiocin resistant
BAGUISTAN, EVANKA MARIELLE D. | BSMLS3-1
Embag.
 positive sa catalase test, staphylococci; kapag
negative, streptococci
 positive sa coagulase test, s. aureus; kapag
negative, s. epidermidis
 strep. mitis: catalase-negative; alpha-hemolytic
 strep. pyogenes: catalase-negative; beta-hemolytic
 strep. faecalis: catalase-negative; gamma-hemolytic
Staphylococcus lugdunensis
- Contain the gene mecA, which encodes oxacillin
resistance
- Important pathogen in infective endocarditis,
septicemia, meningitis, skin and soft tissue
infections, UTIs, and septic shock
- Endocarditis caused by S. lugdunensis is particularly
aggressive, frequently requiring valve replacement,
and infections
Other Coagulase-negative Staphylococci
- Species that are less commonly seen but
established as opportunistic pathogens:
o S. warneri, S. capitis, S. simulans, S.
hominis, and S. schleiferi
o Infections associated: endocarditis,
septicemia, and wound infections
- S. haemolyticus: commonly isolated CoNS
- S. pseudintermedius: common cause of pyoderma
in dogs and skin, ear, and postoperative infections
in dogs and cats, has been linked to human
infections
MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

- Post streptococcal infections: acute rheumatic

fever; acute glomerulonephritis
Streptococci - Scarlet fever, pyoderma, tonsilitis, endocarditis
- Gram-positive cocci in chain - Most common site of the disease is the pharynx
- Non-motile, non-spore former, non-encapsulated (strep throat)
- Facultative anaerobe - Streptococcus pyogenes
- Requires increase conc. of CO2 (capnophilic) - Throat swab to collect sample
- BAP: pinpoint hemolysis
- Has 29 recognized species
Infections caused by S. Pyogenes

- Tonsilitis
Classification of Streptococci - Erysipelas
a. Academic/Bergey’s - Scarlet fever
b. Smith and Brown o Strep. pyogenes (strep. scarlatinae)
c. Lancefield Classification o Pink tongue/strawberry tongue
o Dick’s test: test arm – toxin; control arm –
toxoid
ACADEMIC/BERGEY’S o Schultz-Charlton phenomenon –
determines whether the rashes is due to
TYPES 45°C 10°C EXAMPLES scarlet fever or not
Pyogenic No No Strep. pyogenes o Blanching phenomenon – injection of anti-
Yes No Strep. mutans erythrogenic toxin
Viridans
Strep. salivarius
Enterococcus Yes Yes Strep. faecalis
Lactic No Yes Strep. lacticola BACITRACIN TEST

- test to differentiate Group A beta hemolytic

SMITH AND BROWN CLASSIFICATION streptococci from non-Group A beta hemolytic
streptococci
- Based on hemolysis in BAP
- Uses Taxo A
o Alpha – Strep. pneumoniae
- Taxo A: 0.04 unit of bacitracin
o Beta – Strep. pyogenes
- Positive result: zone of inhibition
o Gamma – Strep. faecium

PYR TEST
REBECCA LANCEFIELD CLASSIFICATION - hydrolysis of L-pyrollidose naphthylamide by Group
- Based on the specific carbohydrate (CHO) antigen A beta hemolytic Streptococcus
known as polysaccharide C - Positive: cherry red color
- Viridans group has no classification since they
lacked M protein Group B Streptococci
o Group A
o Group B - Strep. agalactiae
o Group C - Normal flora of genital tract and GIT
o Group D - Neonatal sepsis, neonatal meningitis, post
puerperal fever
- Rapidly hydrolyze Na Hippurate
Group A Streptococci - Positive in CAMP test (Christie, Atskie, Munch,
Petersen): arrowhead hemolysis in BAP
- Pathogenic to man

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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

Group C Streptococci - Lobar pneumonia (rusty colored sputum),

meningitis, septicemia, otitis media
- Most animal pathogens
- Facultative anaerobe
- In humans: S. equisimilis
- Capnophilic
- In horses: S. equi
- Alpha hemolytic, dome shape colonies in BAP
- Infects Upper respiratory tract
- Produces Specific Soluble Substances (SSS) and
- Beta-hemolytic except S. dysgalactiae
soluble antigen
- Francis Test: skin test
Group D Streptococci

ENTEROCOCCAL VIRULENCE FACTORS

- Grows at 6.5% NaCl inhibited but not killed by
penicillin
- UTI, cardiovascular infection, and meningitis
- E. faecalis, E, faecium, E. durans
NON-ENTEROCOCCAL
- Only those strains of Streptococcus pneumoniae
which contain capsule are virulent and are able to
cause disease
- As with other encapsulated organisms, this
structure is highly phagocytic

- Inhibited by 6.5% NaCl and killed by penicillin

- UTI, endocarditis DISEASES
- S. bovis, S. equinus
- Upper respiratory tract infections
- S. pneumoniae is one of the most common
Toxins and Enzymes of Streptococcus organisms which causes middle ear infections (otitis
media)
- Streptokinase – fibrinolysin; lyse blot clot;
- Lower respiratory tract infections: pneumonia
responsible for rapid spread of the organism
- Invasive infections: bacteremia and meningitis
- Streptodornase – decrease viscosity of DNA
- Erythrogenic Toxin – exotoxin responsible for
scarlet fever
Tests to differentiate pneumococci
- Hyaluronidase – spreading factor enzyme
- Hemolysin (Streptolysin) – lysis of RBC PNEUMOCOCCI OTHER
STREPTOCOCCUS
BILE SOLUBILITY Bile soluble Insoluble
VIRIDANS STREPTOCOCCI INULIN Fermenter Non fermenter
FERMENTATION
- S. mutans, S. sanguis (found in oral cavity) NEUFELD Capsular swelling No swelling
- Able to form biofilms on tooth surfaces (plaque) QUELLUNG
- Acid metabolites are then important in the QUINIDINE Susceptible Resistant
formation of dental cavities (tooth decay) OPTOCHIN Susceptible Resistant
- Disease MOUSE Mouse dies Mouse alive
o They are of low pathogenic potential VIRULENCE TEST within 16-48 hrs
o They can cause disease when they are able
to infect previously damaged heart valves OPTOCHIN TEST
and cause endocarditis
- Test to differentiate Strep. pneumoniae from other
streptococci
Streptococcus pneumoniae - Uses Taxo P: ethylene hydrocupreine
- Positive result: zone of inhibition
- Gram-positive diplococci, lancet shape,
encapsulated

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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

colonization or infection despite adequate humoral

antibody response at the site of entry

GRAM-NEGATIVE COCCI
Neisseria gonorrhoeae
Neisseria
- Humans are the only known host
TAXONOMY - Coffee bean shape, kidney shape in pairs or
diplococci
- Although there are many species of Neisseria - Found intracellularly in PMNs
resident on human mucus membranes, only 2 - Fastidious, requires CO2 (candle jar)
species of these encapsulated, gram-negative
diplococci are normally pathogenic for humans
o N. gonorrhoeae Gonorrhea
o N. meningitidis
 N. sicca - Acute pyogenic infection of columnar and
 N. lactamina transitional epithelium of:
 N. flavescens o Urethra
 N. mucosa o Endocervix
 N. subflava o Anal canal
o Pharynx
o Conjunctiva
ANTIGENIC STRUCTURE - Incubation period: 2-7 days
- Newborn: prone to ophthalmia neonatorum
- Neisseria gonorrhoeae: the surface has not yet (Crede’s prophylaxis)
proven useful to devise an effective vaccine
- Pilin proteins have been targeted for potential
vaccines
COMPLICATIONS
- Neisseria meningitidis: this organism produces a
capsule that is used to divide the species into a Male
number of serogroups
- Acute urethritis
- Serogroups common in human disease include A, B,
- Purulent discharge
C, X, Y, Z, and W135
- 95% asymptomatic
- A safe and relatively effective anti-capsular vaccine
- AHU strains (arginine, hypoxanthine, uracil)
is available for groups A, C, Y, and W135
- Prostatitis
- Epididymitis

VIRULENCE FACTORS Female

a. Fimbriae – allow organism to adhere to target - Mostly asymptomatic

epithelial cells and establish colonization - PID → ectopic pregnancy
b. LPS – release of lipopolysaccharide endotoxin - Sterility
induces inflammation - Perihepatitis (Fitz-Hugh-Curtis Syndrome)
c. Outer membrane proteins – various outer
membrane proteins act as adhesions
d. IgA protease – for both pathogenic Neisseria, the SPECIMEN COLLECTION
production of IgA protease results in destruction of
mucosal IgA and therefore, allow persistence of - Male: urethra; Female: endocervix
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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

- 2-3 cm deep, rotate the specimen: anal canal 4-5 - Chromosome mediated spectinomycin resistant
cm deep
Ceftrixone: drug of choice for resistant strains
- Calcium alginate in cotton swab inhibitory  Dacron
or rayon swab
- Transport medium: Amies medium with charcoal,
JEMBEC, Transgrow, Bio Bag, Gono-Pak TREATMENT

- Penicillin G intramuscularly
- Ampicillin by mouth with probenecid
- If allergic to penicillin, tetracycline by mouth;
LABORATORY DIAGNOSIS spectinomycin IM
- If resistant to penicillin and Cephalosporins:
1. Direct Examination
1. Ceftriaxone
2. Culture – Selective Medium
2. Cefotasime
a. New York City Medium
3. Cefoxitin
b. Thayer Medium with VCN
c. Modified TM with trimetophrim
d. Martin Lewis (+anisomycin against yeast)
Neisseria meningitidis
e. GC lect
3. Presumptive test - Oxidase test uses the reagent: - Epidemic meningococcal meningitis
tetramethyl-p-phenylene-diaminedihydrochloride - CSF fever
or p-aminodimethylaniline monohydrochloride - Spotted fever
4. Definitive test - Can be found in the oropharynx and nasopharynx of
 CHO utilization test (CTA agar) 3-30% of normal individual
glu, mal, lac, suc - LPS: main virulence factor which is endotoxin
 Immunologic method (Co-agglutination complex
test)
 Nucleic acid probe test
Epidemic meningitis
 PCR and DNA amplification and
hybridization technique - Respiratory droplets
- Meningococcemia or sepsis may go without
meningitis
CHO UTILIZATION TEST
- Waterhouse-Friedrichsen syndrome (hemorrhage in
(N. gonorrhea, N. meningitidis, N. mucosa, N. lactamica, N. the adrenal glands)
sicca, Branhamella) - DIC, septic shock and death
N. gon N. men N. lac N. sic N. muc branha
BAP - - + + + + DISEASE: N. meningitidis
TMA + + + - - -
Glu + + + + + - - Dissemination may result in infection in a variety of
Mal - + + + + - organs including lung, joints, eyes, skin, and
Lac - - + - - -
Suc - - - + + -
meninges
NO3 - - - - + -
red’n
LABORATORY DIAGNOSIS
Resistance of N. gonorrhoeae
- Specimen: CSF, nasopharyngeal swab, blood, and
- Plasmid mediated penicillin resistance (PPNG) aspirates
- Chromosome mediated resistance (CMRNG) - Direct Examination: g/s intra and extracellular
- Plasmid mediated high level tetracycline resistance diplococci with halo; cytocentrifugation is necessary
(TRNG) - Culture: same as N. gonorrhoeae (small, gray,
convex, colonies)
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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS
- N. lactamica may mimic (+) with ONPG
Moraxella
TAXONOMY AND HABITAT
- Moraxella catarrhalis has gone through a number of
name changes and claims to disease states
- The organism was first described as Neisseria
catarrhalis, then Branhamella catarrhalis after Dr.
Sarah Branham, and now, Moraxella catarrhalis
- These organisms are found as common commensals
of the human upper respiratory tract
GROWTH AND METABOLISM
- These small diplococci (M. catarrhalis) and rods (M.
lacunata) are oxidase-positive aerobic or facultative
anaerobic organisms
- They are susceptible to drying, cold, sunlight, and
pH changes
- Growth conditions include enriched media and an
increased CO2 content for maximum recovery of
organisms
VIRULENCE FACTORS
- The enzyme beta-lactamase that hydrolyzes
penicillin antibiotics is produced by >50% of the
organisms
- This probably allows the organisms to persist and
complete with flora in the respiratory tract despite
antimicrobial challenge
DISEASE
- Although regarded as a commensal for years, there
is increasing evidence to link Moxarella with:
o Exacerbation of chronic obstructive
pulmonary disease leading to pneumonia
o Pneumonia in children
o Otitis media in children
GRAM-POSITIVE BACILLI
- Bacillus
- Corynebacterium
BAGUISTAN, EVANKA MARIELLE D. | BSMLS3-1
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- Clostridium
- Nocardia
- Erysipelothrix
- Listeria
- Lactobacillus
- Rothia
- Kurthia
- Propionibacterium
Bacillus
- All are motile except B. anthracis
- Motility is due to peritrichous flagella
- Widespread in nature
- Most common organisms in blood in a
contaminated blood transfusion
- Cell wall: meso-diaminopimelic acid
CLASSIFICATION ACCORDING TO ENDOSPORE FORMATION
Group I – ellipsoidal spores no swelling
Subgroup 1 (large cell >0.9 um)
B. anthracis, B. cereus, B. megaterium
B. thuringiensis
Subgroup 2 (<0.9 um)
B. subtilis, B. coagulans, B. licheniformis
B. pumilus
simple nutrional requirements
Group II – oval shape swollen sporangia
B. stearothermophilus, B. polymyxa
B. circulans
complex nutritional requirements
Group III – swollen circular sporangia
B. pasteurii, B. sphaericus, B. panthpthenicus
Bacillus do not grow in Columbia, Colistin, Nalidixic Acid
(CNA) agar
Bacillus anthracis (Anthrax Bacillus)
- Large encapsulated, NM, disjointed bamboo
appearance
- Medusa head colonies (cut glass appearance)
- Liquid medium: inverted fir tree
- Anthrax, Black bair, Silberian fever
- Glutamic acid capsule
- Polysaccharide somatic antigen
MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

- Anthrax toxin: protective antigen, lethal factor blue color, rough, dry colonies
edema factor
B. cereus Food Poisoning

Laboratory Identification of all EMETIC FORM DIARRHEAL FORM

Bacillus spp. - Rice
- <6 hrs incubation
- Catalase (+) - Vomiting, nausea,
- VP (+) abdominal cramps
- Growth in aerobic jar, 50 and 65’C and 7% NaCl - 8-10 hrs duration
- NO3 reduction, hydrolysis of starch, casein - Heat stable
decomposition enterotoxin
- Meat, begetables
- >6 hrs incubation
- Diarrhea, nausea,
abdominal cramps
- 20-36 hrs duration
- Heat labile
enterotoxin

3 CLINICAL TYPES OF ANTHRAX

B. anthracis
1. Cutaneous anthrax – eschar formation in the skin; - non motile
20% mortality rate - non hemolytic
2. Pulmonary anthrax – Woolsorter’s disease; 90% - encapsulated
mortality rate; MOT: inhalation; commonly used in - non salicin
bioterrorism fermenter
3. Intestinal anthrax – violent enteritis; MOT: - can’t grow at 45°C
ingestion; 90% mortality rate - penicillin sensitive
B. cereus
- Motile
- Beta-hemolytic
- NE
- Salicin fermenter
- Can grow at 45°C
- Penicillin resistant

Laboratory Diagnosis for B. anthracis Bacillus subtilis

- Stained smear: MacFadyeaus’s method - Hay bacillus

- Culture: PLET (polymyxin, lysozyme, diNa EDTA, - Most common laboratory contaminant
thallou acetate) medium - Found commonly in soil
- Animal Inoculation Method
- String of Pearl test
- Ascoli test – diagnostic precipitin test Corynebacterium diphtheriae
- Kleb’s Loeffler’s bacillus
SNAIL LECTIN - Fac. anaerobe, catalase (+), NM, club shape
SOYBEAN LECTIN
(HELIX POMATIA) - Babes-Ernst granules, pallisade, pleomorphic
B. anthracis + - - Chinese character arrangement
B. mycoides + +

Bacillus cereus Virulence Factor in Corynebacterium

species
- 2nd most virulent species of Bacillus
- Major virulence antigen VIRULENCE ACTIVITY ORGANISMS
o Heat labile enterotoxin FACTOR
Diptheria Exotoxin Blockage of C. diphtheria
o Heat stable enterotoxin
“tox” gene- CHON synthesis C. ulcerans
o Cerelysin
introducec to by inhibition of C. pseudoTB
o Phospholipase C lysogenic B- peptide
- gastroenteritis, psnophthalmitis, opportunistic corynephage translocation on
infection ribosomes
- Presumptive Dx: PEEMBA medium (polymyxin Dermonectrotic Sphingomyelinase C. ulcerans
pyruvate egg yolk mannitol bromthymol blue (+) toxin that increases C. pseudoTB
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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS

vascular  Pseudomembrane formation: composed of

permeability bacteria, lymphocytes, plasma cells, fibrin, dead
Hemolysin Undefined C. pyogenes cells, and covers the uvula, tonsils, and palate
activity hemolysis
of RBC
LABORATORY DIAGNOSIS

Cultivation of Corynebacterium  In vivo test: animal inoculation test

 Tinsdale: form brown halo which is caused by the Test animal: C. diphtheriae cell suspension
action of cysteine sulfhydrolase w/ the formation of Control animal: C. diphtheriae cell suspension +
FeSO2 in the medium diphtheria anti toxin
 Egg and Serum medium
Interpretation
o Pai’s coagulated egg
o Loeffler’s serum slant Test dead/Control alive = toxigenic diphtheria
 Cystine Tellurite Agar - BAP Test alive/Control alive = nontoxigenic organisms
Test dead/Control dead = toxicity not due to diphtheria

CYSTINE TELLURITE AGAR

 In vitro test: Elek test
- Black or gray color of colonies; differentiates o Gel diffusion test on Protease peptone agar
Corynebacterium into 3 major types
o Filter paper strip impregnated w/ antitoxin
HYDROLYSIS  Schick test: skin susceptibility test
TYPES COLONIES HEMOLYSIS
STARCH/
Test arm Control arm
GLYCOGEN
0.1 mL toxin 0.1 mL toxoid
Gravis Large, flat, Negative Positive
dark, gray, (+) redness/swelling (-) no reaction
irregular, Susceptible to diphtheria
striated, Daisy
head colonies Other species of Corynebacterium
Mitis Small, black, Positive Negative
glossy,  C. matruchotti: (Bacterionenia matruchotti),
convex, assume a “whip w/handle” shape w/ branching and
translucent, fragment filaments
fried egg  C. pseudotuberculosis: horse
appearance  C. ulcerans: respiratory pathogen in man and other
Intermidus Small, flat, dry Positive Negative higher animals
gray, or black
 C. minutissimum: erythrasma of skin and pubic,
on CTA;
cord red fluorescence
colorless on
BAP  C. J-K: adherence to handwashing, lipophilic
 Diptheroids: flora of throat, shorter w/ less
metachromatic granules
PATHOGENICITY

 Exhibits lysogenicity Differentiation between C. diphtheriae

 Nontoxigenic form (normal flora) and C. ulcerans
 Local infection
TESTS C. diphtheriae C. ulcerans
 Systemic infection
Urea hydrolysis Negative Positive
 Cutaneous: skin diptheria (urease)
 Endocarditis NO3 reduction Positive except Negative
for mitis var.

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Trehalose Negative Positive - In severe myonecrosis, toxins diffuse into adjacent

Fermentation areas and necrosis spreads throughout the area
- Infection requires damaged or dead tissue that
supplies the necessary anaerobic environment for
Clostridia spore germination and subsequent bacterial growth
 All Clostridia are motile except C. perfringens, C. - Tissue necrosis is a result of the various toxins
ramosum, C. inocuum produced by the organisms
 All Clostridia have swollen sporangia except C.
perfringens, C. C. bifermentans
 All Clostridia are lactose negative except: C. Clostridium tetani
perfringens and C. septicum  Spore is located terminally, and sporangia is swollen
 All Clostridia are non-encapsulated except for C. giving rise to drumstick or lollipop
perfringens  Toxins: tetanolysin; tetanospasmin
 All Clostridia are single hemolytic and sucrose  Clinical syndromes: trismus (lockjaw), risus
negative except for C. perfringens sardonicus, opisthotonus
 All Clostridia are dextrose positive except C. tetani
and C. histolyticum, the former does not ferment
any sugar
CLINICAL MANIFESTATIONS OF TETANUS
Classification of Clostridium
1. Generalized
1. Gas Gangrene or Histotoxic  bulbar; paraspinal muscles
o C. perfringens  ANS (fluctuation in BP, hyperthermia,
2. Toxigenic Group arrhytmias)
o C. botulinum  Tetanus neonatorum
o C. tetani  Prognosis: age, immune status, site of
C. difficile infection
3. Others 2. Cephalic
o C. novyi, C. septicum, C. bifermentans  Primary infection in head, particularly ears
 Isolated or combined cranial nerves (7th)
 Very poor prognosis
Clostridium perfringens
3. Localized
 AKA C. welchii, Bacillus aerogenes or gas gangrene  Involves muscles in area of primary injury
bacillus  May proceed to generalized disease
 Contamination of an open woumd by Clostridial  Favorable prognosis
endospores
 Necrotic enteritis, food poisoning, gas gangrene,
MECHANISM OF TETANOSPASMIN ACTIVITY
cellulitis, fasciitis
 Four major toxins: alpha, beta, epsilon and iota  Neurotransmission is controlled by the balance
toxins between excitatory and inhibitory
 Produces Nagler reaction: egg yolk agar neurotransmitters
 Double zone of hemolysis: BAP  The inhibitory neurotransmitter (GABA, glycine)
 Stormy fermentation of milk prevent depolarization of the post synaptic
membrane and conduction of the electrical signal
 Tetanospasmin does not interfere w/ the
Spore-Forming Anaerobes
production or storage of GABA or glycine, but rather
Diseases: Gas Gangrene (Myonecrosis) their release (presynaptic activity)

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MLS303 CLINICAL BACTERIOLOGY LECTURE | MIDTERMS
 In the absence of inhibitory neurotransmitters,
excitation of the neuroaxon is unrestrained
Virulence Factors – Clostridium tetani
- The tetanus toxin (tetanospasmin) is a neurotoxin
that binds to receptors on nerve endings and
prevents the release of neuroinhibitors responsible
for regulating muscle contractions. Muscles
contract uncontrollably and a rigid paralysis result
TREATMENT, PREVENTION, AND CONTROL
 Debridement of the primary wound
 Administration of penicillin
 Passive immunization w/ human tetanus Ig
 Immunization with tetanus toxoid
 DPT booster every 10 years with the tetanus toxoid
Diseases: Food Borne Disease
(C. perfringens)
- Clostridium perfringens (type A) is a common cause
of food borne illness in 3rd world countries
- The usual food source is meat contaminated with
Clostridial spores
- Through improper cooking or storage, the spores
germinate and grossly contaminate the food source
- Once ingested, the large number of organisms now
present in the gastrointestinal tract secrete an
enterotoxin which cause short lived abdominal pain
and diarrhea
(C. botulinum)
- Botulinum toxin is the most potent enterotoxin
known
- Botulism is an intoxication illness associated with
improperly preserved foods
- Although exceedingly rare, the usual source is
canned foods (an anaerobic environment) which
have been contaminated with the spores
FORMS OF BOTULISM
BAGUISTAN, EVANKA MARIELLE D. | BSMLS3-1
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 BOTULINUM TOXIN: a potent neurotoxin which is
the most potent toxin known to man
 PROTOPLASMIC PROTEIN: potent neurotoxin w/c
are synthesized during the growth of organisms &
released during lysis
o Classical Foodborne: intoxication caused by
preformed botulinal toxin in contaminated
food
o Wound botulism: rarest form, result from
the elaboration of botulinal toxin in vivo
after growth of C. botulinum in an infected
wound
o Infant botulism: in vivo production in
colonized infant
o Undetermined Classification
ACUTE FLACCID PARALYSIS
 Clinical hallmark of botulism
 Begins w/ bilateral cranial nerve impairment
involving muscles of the face, head, and pharynx
and then descends symmetrically to involve muscles
of the thorax and extremities
Virulence Factors – Clostridium botulinum
- The botulinum toxin (Botulism) is an extremely
potent neurotoxin that prevents acetylcholine
release from nerve endings resulting in flaccid
paralysis
Clostridium difficile
 Present normally in the colon of some individuals
 Transient or permanent fecal flora of many healthy
infants
 Antibiotic associated diarrhea
 Produces 2 toxins:
Toxin A = Enterotoxin
Toxin B = Cytotoxin
 Cytotoxicity assay: the single most effective test for
C. difficile
Diseases: pseudomembranous colitis (C. difficile)
- The disease is characterized by bloody diarrhea
which often progresses to severe inflammation of
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the mucosa and the elaboration of  Gram (+) bacilli

pseudomembrane and microabscesses  Isolated from fresh and spoiled meat, water, feces,
- The pathology of the disease is a result of both toxin milk, air from abattoir,
A (enterotoxin) and toxin B (cytotoxin)  Endocarditis

Kurthia zopfii
Listeria monocytogenes
 Small, gray, rhizoid colonies with a medussa head
 Increased in monocytes in the blood of an infected
appearance in agar (yeast extract NA) under LPO
animal
 Catalase (+), oxidase (-)
 Small gram (+) coccobacilli showing a typiacl
diphtheroid palisade  Gelatin slope: featherlike growth
 Motility at room temperature is called “tumbling Bacillus Kurthia
motility” but rarely at 35’C Endospore + -
 Umbrella shape motility in a butt medium CHO Ferm. + -
 Colonies are smooth, transluscent, slightly raised Gelatin liquefaction + -
with a distinct blue green color. (Columbia CNA)
 Anton’s test- 1-2 gtt of bacterial suspension into the
eyes of the Guinea pig
Rothia dentocarsiosa
 Normal flora of mouth and oropharynx
 Cat scratch disease
CLINICAL SYNDROMES OF LISTERIA  Non motile, w/ filamentous shape, catalase (+), NO3
MOT: contaminated foods and animal products reduction (+), and hydrolyzes esculin

 Neonatal diseases
o early onset disease
o late onset disease
o granulomatosis infantiseptica
Differentiation of Listeria,
 Meningitis in adults
Erysipelothrix, and Corynebacterium
 Primary bacteremia
 Endocarditis TESTS Listeria Erysipelothri Corynebact
x erium
Hemolysis beta alpha Variable
ERYSIPELOTHRIX RHUSIOPATHIAE BAP
Molitity + - -
 “rhusio” red; “pathiae” disease
 Erysipeloid; zoonotic infection Catalase + - +
 Pleomorphic, microaerophilic, NM H2S on TSI - + -
 BAP, CAP: alpha hemolytic, lampbrush or test tube
brush colonies
Lactobacillus spp.
 Colonies are barely visible for at least 48 hrs
 Tellurite medium: black colonies  Microaerophilic organisms Gram (+)
 TSI: H2S (+) but catalase negative  Organic factors are necessary
 Normal indigenous flora of vagina
 Acid pH is maintained by normal flora
Kurthia spp.
 Active fermenters
 K. zopfii and K. gibsonii (1986)  Homofermenters: 1 prod. of fermentation
 Aerobic, non-sporulating, motile 37’C, non-motile  Heterofermenters: more products other than lactic
20’C acid
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 Lactobacillus acidophilus (Doderlain bacillus)

 L. vulgaricus = yogurt production
 L. shirota strain: yakult milk

Actinobacillus
actinomycetemcomitans
 Gram (-), nonmotile, facultative anaerobe
 Normal oropharyngeal region
 Juvenile periodontitis and subacute endocarditis

BAGUISTAN, EVANKA MARIELLE D. | BSMLS3-1

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