BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

Bacterial Cultures in Broth Media Antimicrobial Susceptibility Testing (AST)

a. Sterile (uninoculated broth) - note how clear Direct measures of antimicrobial activity are
the media is accomplished using:
b. Broth showing slight turbidity - some 1. Broth dilution
bacterial growth 2. Agar dilution
c. Broth showing significant turbidity - a lot 3. Disk diffusion
of bacteria growth for culture and sensitivity
d. Broth that hasn't been agitated (shaken) sensitivity: susceptibility to antibiotics

Blood agar - has intact RBC 1. TUBE OR MICROPLATE DILUTION (BROTH)

Three types of hemolysis: Serial dilution of antibiotic is prepared, to each
a. Alpha - partial tube, a uniform amount of inoculum is added
hemolysis; - Minimum Inhibitory Concentration (MIC);
greenish the lowest concentration of antibiotic that
b. Beta - complete inhibits bacteria, growth stops
hemolysis - Minimum Bactericidal Concentration
c. Gamma - no (MBC); lowest concentration of antibiotic
hemolysis that kills bacteria
inoculum: bacteria colony; different
concentration of antibiotic
bacteriocidal - kill
bacteriostatic - stops the synthesis of
bacteria; immune system will eliminate

2. AGAR DILUTION
Varying concentration of antibiotic is incorporated
to appropriate plating media
• combined agar mixture and antibiotic
• agar mixture – solution
• antibiotic – will harden
Petri dish divided into 4 quadrants
Last quadrant will have small amount of colony; can 3. DISK DIFFUSION (KIRBY BAUER)
get exact amount of colony • (MHA) MUELLER-HINTON AGAR: depth
Divided - semi-quantitative 4mm (3-5mm) pH 7.2-7.4
• STEPS (7)
Slant agar
• for better pigmentation Sources of Error
• for longer storage of bacteria 1. Used of mixed culture – will result to false
resistance of bacteria to antibiotic
2. Inoculum too light – not exact amount of
inoculum
3. Too heavy – more bacteria; overcome the
antibiotic = resist
4. Too much moisture on agar – overcome
resistance to antibiotic = falsely resistant
5. Very dry agar - falsely sensitive
6. Improper storage of disk
7. Reading and clerical error
• measure the zone of inhibition
• place tablet
• to know what medication to give
• THE BIGGER THE ZI = SENSITIVE
• THE SMALLER THE ZI = RESISTANT
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

• Increased concentration = increased LABORATORY

resistance 1. Gram Stain
• Basophilic: body temperature lover Staphylococcus – gram (+) COCCI in CLUSTERS
8. Deterioration of turbidity (grape-like COCCI)
Micrococcus – gram (+) COCCI in TETRADS
(packets of 4/8/16)

2. Growth on Blood Agar Plate

Staphylococcus - creamy white, pinhead colonies
with hemolytic pattern

Alpha prime hemolytic pattern - observed if petri

dish is from the fridge
Small zone of alpha pattern (inner) - green inside
surrounded by clearance
Large zone of beta pattern (outer) - clear color

3. Growth on Loeffler’s Serum Slant

S. aureus - golden yellow
S. citreus - lemon yellow
S. albus (epidermidis) - porcelain white

4. Growth on Mannitol Salt Agar

Inhibitor: 7.5%-10% NaCl
CHO: Mannitol
pH indicator: phenol red

S. aureus - utilize carbohydrate; yellow

S. epidermidis - non-mannitol fermenter; pink
1. E-test - antibiotic present on strips S. saprophyticus - variable; fermenter/non

5. Catalase test
NOTE: don't use colony/inoculum from BAP due
to pseudoperoxidase activity of hemoglobin

differentiate Staphylococcus from Streptococcus

(+) Staphylococcus, (-) Streptococcus

reagent: 3% Hydrogen peroxide (H2O2)

(+) result: can observe effervescence (bubbles)

6. Test to Differentiate Staphylococcus from

Micrococcus

Family Micrococcaceae
Genera
Clinically Relevant
1. Staphylococcus – pinhead
2. Micrococcus
Animals
1. Planococcus
2. Stomatococcus
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

Modified oxidase test (Microdase test) Tube Voges-

Organism PYR
• differentiate Staphylococcus from Coagulase Proskauer
Micrococcus S. aureus
+ + -
• Negative - clear subsp. aureus
• Positive - blue S. intermedius variable - +
• Reagent: Tetramethyl-p-phenylenediamine S. hyicus variable - -
dihydrochloride with Dimethylsulfoxide S. scleiferi
subsp. + + +
CHO Oxidation Fermentation Test coagulans
• CHO: glucose
• pH indicator: Bromthymol blue
• if glucose is utilized = acidic (yellow) PYR TEST (L-Pyrrolidonyl-B-
• if not = alkaline (remain as green) naphthylamide test) is a
• medium: OF tube biochemical test used to detect the
ability of bacteria to produce
pyrrolidonyl aminopeptidase
enzyme.
Voges-Proskauer test is used to determine if an
organism produces acetylmethyl carbinol from
glucose fermentation.
Closed tube: sealed with VASPAR (vaseline and
paraffin) or Mineral oil
other pH indicator
• Andrade’s acid fuchsin - pale yellow to
pink
• Phenol red - red to yellow
• Bromthymol blue - green to yellow
• Bromcresol purple - purple to yellow

7. Coagulase test
most important pathogenic determinant of S.
aureus
a. slide method – detects cell bound coagulase
- clumping factor 8. DNAse test (DNA hydrolysis test)
- Reagent: Rabbit’s plasma
- Alternative rgt: human plasma • Medium: DNA medium with methyl green
- Rabbit’s plasma + inoculum = (+), clumping • If capable of DNA hydrolysis = clearance
b. tube method – detects free coagulants • (+) DNAse – S. aureus, Moraxella, Serratia
- reagent: 0.5 mL plasma of Rabbit’s
- rabbit + inoculum = (+), gel-like fibrin clot CoNS = NOVOBIOCIN SUSCEPTIBILTY TESTING
• Coagulase Negative Staphylococcus
Incubate 37 degree C for 4 hours and check every • Use: Novobiocin 5 mg
30 mins if there is a clot • S. epidermis – associated with bacterial
If no clump for 4 hours – add 20 hours for endocarditis following insertion of artificial heart
observation valves
- susceptible to novobiocin, 14 mm of zone of
S. aureus – the only coagulase (+) under inhibition
Staphylococcus genes
• S. saprophyticus – most common cause of
Coagulase – ability to convert fibrinogen to fibrin
UTI in young sexually active female
clot; bacteria will convert
- Resistant
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

PATHOGENIC DETERMINANTS OF S. AUREUS Lancefield

• protein A – to inhibit phagocytosis
Group A
• coagulase – fibrinogen→fibrin clot can coat
S. pyogenes
neutrophil to protect from phagocytosis
- can cause pharyngitis, tonsilitis, scarlet
• staphylokinase (fibrinolysin) – capable in
fever, impetigo, cellulitis, erysipelas,
producing and dissolving clot/dissolve fibrin clot
necrotizing fasciitis (flesh-eating bacteria)
= spread of infection
- associated with acute rheumatic fever – has
• lipase – hydrolysis of lipids, plasma = skin Aschoff Bodies (heart)
irritation; boils - acute glomerulonephritis
• hyalurodinase – capable of hydrolyzing
hyaluronic acid in tissue called as spreading Group B
factor AKA Duran Reynal Factor in Synovial S. agalactiae
fluid - associated with neonatal infection
• DNAse – degrades/hydrolyze DNA - habitat: vagina
• Exofoliatins – hydrolyzes tissue thru cleavage - neonatal: pneumonia, meningitis
of stratum granulosom (component of tissues); Group C
associated with Ritter Lyell Disease AKA • S. equisimilis
Staphylococcal Scalded Skin Syndrome • S. zooepidemicus
• LEUKOCIDINS – lyse neutrophil and • S. equi
macrophage • S. dysagalactiae
• hemolysin – capable of lysing RBC
• enterotoxins – - ONLY SXT SUSCEPTIBLE
- A and B = food poisoning - Trimethoprim/sulfamethoxazole
- F/toxic shock syndrome = desquamation of - Can cause severe pharyngitis followed by
palm and sole bacteremia
Group D
- Can cause UTI and good infection
FAMILY STREPTOCOCCACEAE
Enterococci – can grow in 6.5% NaCl (saline
• GRAM POSITIVE
solution)
• NONMOTILE • E. faecalis
• NON-SPORE FORMING • E. faecium
• FACULTATIVE ANAEROBES • E. durans
• CATALASE NEGATIVE • E. avium
Streptococcus – chains/in pairs, gram (-) cocci Non-enterococci – cannot grow in 6.5% NaCl
- Divided according to classification • S. bovis
• S. equinus

2 TYPES OF CLASSIFICATION Brown’s Classification

• LANCEFIELD CLASSIFICATION S. pneumoniae – causative agent of Lobar
- based on antigenic characteristic of a pneumonia
group-specific cell wall polysaccharide S. viridans – subacute bacterial endocarditis
- CHO, cell wall, antigenic determinants
- Single lance group = many ang under Viridans Strep
• BROWN'S CLASSIFICATION S. sanguis (sanguinis)
S. mutans
- according to the pattern of hemolysis S. uberis S. salivarius
- more than 20 groups classification S. constellatus
S. mitis
- first 4 (clinically significant) S. intermidius
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

ALPHA (bile esculin S.pneumonia, viridans, BACITRACIN AND SXT SUSCEPTIBILTY TESTS
hydrolysis +) some group D
BETA Group A, B, C Some Bacitracin (Taxo A)
group D S. pyogenes –
Some group D - sensitive, 10 mm zone of inhibition
GAMMA Most group D - can inhibit immune system to fight because
of M protein
LABORATORY TESTS - M protein binds to factor H needed in
complete pathway
1. GRAM STAIN
2. BAP Some group C exhibit sensitivity to taxo A
3. CATALASE
ORG BACITACIN
4. BILE ESCULIN HYDROLYSIS (TAXO A) SXT NOTES
5. PYR (Pyrrolidonyl Arylamidase) 0.04 ug
6. CAMP REACTION A PYR
sensitive resistant
7. Hippurate hydrolysis resistant
8. BACITRACIN AND SXT SUSCEPTIBILITY B Camp (+),
Resistant resistant
TESTS Hippurate (+)
C resistant resistant
Gram Stain D BEH (+),
S. pneumonia – lancet/bullet-shaped/flame- enterococci PYR (+),
resistant resistant
shaped diplococci growth in
6.5% NaCl
Blood Agar Plate D non PYR
white pinpoint colonies with hemolytic pattern enterococci resistant resistant (-), growth in
6.5% NaCl
Bile esculin hydrolysis
- determines if bacteria can grow in 40 % bile and
esculin hydrolysis Optochin (Taxo P)
- (+) reaction = brown black precipitate (Group D) - Has Ethylhydrocupreine HCl
- 10 mm zone of inhibition
PYR - S. pneumoniae
- Substrate: L-pyrrolidonyl arylamidase
S. pneumoniae Viridans
- Color developer –
Mouse virulence +, dead -, alive
P-dimethylaminocinnamaldehyde
Inulin fermentation + -
- (+) reaction = red color (Group D, A)
Bile solubility + -
Optochin (Taxo P)
Chemical name:
Camp Reaction sensitive resistant
Ethylhydrocupreine
- Christine-Atkins-Munch-Petersen HCl
- Differentiate group B from other beta hemolytic Neufeld Quellung + (capsular
-
group swelling)
- (+) reaction = enhanced hemolytic pattern in Strains of Strep produces C5a
agar
Group A hemolysis
- (-) – no enhancement of hemolysis
Streptolysin O Streptolysin S
Hippurate hydrolysis
Oxygen labile Oxygen stable
- Detects hydrolysis of sodium Hippurate to antigenic Non-antigenic
benzoic acid and glycine Responsible in Surface hemolysis
- (+) reaction – purple color subsurface hemolysis (agar)
- (+) – group B, Listeria monocytogenes,
Gardnerella vaginalis
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

Erysipeloid GRAM NEGATIVE COCCI

- From raw wish
- Dermatitis of the hands only Neisseria
Same biochemical test
Moraxella catarrhalis
- Causative: Erysipelothrix rhusiopathiae
Scarlet fever test Gram negative characteristics
1. Dick’s test – screening -
Gram (-) diplococci
- Arm + antitoxin = (+) = redness/erythema -
Resembles coffee bean or kidney-shaped
2. Schultz-Charlton – diagnostic except for N. elongata
- Arm + antitoxin = (+) = neutralization/no Neisseria spp.
rash, blanching phenomenon - Obligate aerobes
- Capnophilic (attracted to 5-10 % CO2)
Rheumatic fever - Catalase (+)
- develop after developing group A - Oxidase (-)
- inflammatory disease that can affect brain, - Cause infection thru mucous membrane
skin, heart, joints - Pilli is present
Francis test – detects pneumococcal flesh-eating - Found in the normal flora except N.
bacteria (Necrotizing Fasciitis) meningitidis and N. gonorrhoeae

Procedure for CAMP test Neisseria spp.

Standard Method
• Firstly, make a single straight line streak of beta-
hemolysin producing Staphylococcus aureus
down the center of a blood agar plate,
• Then, inoculate a streak of the test organism
(beta-hemolytic streptococci to be identified)
perpendicular to the staphylococcal streak. Take
care not to intersect the staphylococcal streak.
Other method: disk method, spot rapid method
N. gonorrhoeae
Reverse CAMP test - Conjunctivitis of the newborn (Ophthalmia
Neonatorum of newborn)
• This test is used for the differentiation of - Disseminated – focal infection, cause cervix
Clostridium perfringens from other Clostridium disease
species. - Always pathogenic
• In this method, the CAMP factor produced by S.
agalactiae is used to detect the Clostridium N. meningitidis
perfringens from other Clostridium species, - Transmitted through respiratory droplets
that's why this method is termed as a reverse - Acute Purulent Meningitidis – life-threatening
CAMP test. o Accompanied by petechiae
o Associated with
Meningococcal/Meningococcemia
- Can cause hemorrhage in Adrenal gland
(Waterhouse-Friderichsen syndrome)
Meningococcemia
• Infection causes disseminated intravascular
coagulation (DIC) → ischemic & necrotic
lesions caused by blood clots +
BACTERIOLOGY | Midterm Staphylococcus, Streptococcus, Neisseria spp.

Hemorrhages
• by clotting disorders → subcutaneous
bleeding ("meningococcal rash")

Other Neisseria
endocarditis, meningitidis, bacteremia
Moraxella (Branhamella) catarrhalis
- commensal or pathogenic
- formerly called Neisseria catarrhalis or
Micrococcus catarrhalis
Utilization of sugar
Ophthalmia Neonatorum – antibiotic:
erythromycin/silver nitrate
Dacon swab/Rayon-tipped – preferrable swab if
Neisseria is suspected
Commercial media for N. species
- JEMBEC
- Bio-bag
- Gono-Pak
- Transglow
If blood specimen for cultivation of N. spp. Neisseria meningitidis: Gram-stained smear
– 0.025% concentration of SPS • Smears should be examined for at least 10
If SPS is not controlled – plate for 1 hour minutes
Direct gram-staining • Large no of PMNs - usually indicative of good
prognosis
Symptomatic
Symptomatic Males
Females Colonial appearance
neutrophil is present
presence of diplococci
with diplococci
DIAGNOSTIC PRESUMPTIVE

• Fastidious bacteria – needs special nutrients

(OA)
• Optimal growth temperature of Neisseria
- 35-37 °C
- Placed in candle jar environment because
carbophilic for 24-48 hours
• N. meningitidis/Moraxella catarrhalis – grow
swell on 5% BAP and Chocolate agar
• N. gonorrhoea – enriched chocolate agar