Gram Postitve Coccus (GPC)

16th Shrawan 2081

 Bacterial Shapes
• Coccus (Plural - Cocci)
– Greek – kokkos – berry

• Bacillus (Plural – Bacilli)

– Rod shaped

• Spirillum
– Spiral shaped
 Coccus
• Each single coccus bacterium is an entire living
organism.
• Some species exist in groups of cells.
• The patterns they arrange themselves in are
given certain names based on the shape.
– Diplococci arranged in two-cell pairs
– Streptococcus arranged in chains
– Staphylococcus arranged in tetrad/clusters
• Which of the following is supposed to be
arranged in 8 or octet?
– COCCUS
– TETRAD
– DIPLOCOCCI
– SARCINA
 Gram Stain
• Simple staining technique
• Differential stain
– Differentiates between positive and negative
– Positive and negative based on cell wall
composition
 Gram Positive
• Peptidoglycan
– Glycan chains of GlcNAc and MurNAc cross linked
by peptide bridge.
• Teichoic Acid
– Polyribitol phosphate or glycerol phosphate cross
linked to peptidoglycan.
• Lipoteichoic Acid
– Lipid linked teichoic acid.
 MCQs
• Which of the following bacteria is not stained
by gram’s stain?
– Staphylococcus
– Streptococcus
– Pseudomonas
– Treponema
• Gram positive bacteria contain all of the
following except..
– Peptidoglycan
– Teichoic acid
– Lipoteichoic acid
– Periplasmic space
• Which of the following is important step in
gram’s stain?
– Crystal violet
– Gram’s iodine
– Decolorisation
– Safranine
 Coccus
• Gram Negative
– Anaerobic (Veillonella), Aerobic (Neisseria)
• Gram Positive
– Aerobic
– Facultative anaerobes
– Anaerobic
• Which of the following is gram negative
anaerobic bacteria?
– Veilonella
– Neisseria
– Staph
– strep
• Which of the following cocci is aerobic in
nature?
– Staphylococcus
– Streptococcus
– Veillonella
– Neisseria
FAMILY : MICROCOCCACEAE
GENUS: STAPHYLOCOCCUS, MICROCOCCUS,
STOMATOCOCCUS, PLANOCOCCUS

CATALASE POSITIVE

FAMILY: STREPTOCOCCACEAE
GENUS: STREPTOCOCCUS, ENTEROCOCCUS

CATALASE NEGATIVE
• All of the followings are catalase positive
except-
– Micrococcus
– Stomatococcus
– Planococcus
– Enterococcus
Staphylococcus
Gram positive cocci in cluster
Uniformly stained
Approximately 1micron size

Micrococci
Arranged in tetrads.
Non uniform staining
Modified oxidase positive
Normal flora
• Oxidase reagent-1% tetramethyl
paraphenylene diamine dihydrochloride
• Kovac’s reagent- para dimethyl amino
benzaldehyde
 STAPHYLOCOCCUS
• 33 species- 17 human specimen
• Coagulase positive
• Staphylococcus aureus

Slide coagulase
(Bound coagulase/ clumping
• Coagulase negative factor)
• Staphylococcus epidermidis
• Staphylococcus saphrophyticus

Tube coagulase
(free coagulase)
 General characteristics
• Species of the genus Staphylococcus divide in
random planes and tend to form irregular
clusters (Staph=grape-like clusters,
coccus=round)
• Micrococcus species first divide in parallel
planes and then perpendicular to that
resulting in predominance of tetrads
• Rothia species form short chains and small
clusters.
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• Which of the following is coagulase positive -
– Staph. epidermidis
– Staph. saprophyticus
– Staph. Citrus
– Staph. aureus
Staph clusters

Micrococcus tetrads

Rothia

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 General characteristics
• Staphylococci have a thick multilayered peptidoglycan
as the major component of their cell walls, the same
as all Gram-positive bacteria

• Staphylococcus species contain teichoic

acids in their cell walls

• Cell walls of Micrococcus & Rothia species do not

contain teichoic acids

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 General characteristics
• Members of the staph group are mostly non-
encapsulated and all clinically relevant ones are
nonmotile
• All Staph group organisms that grow in air are catalase
positive

• Rothia is catalase negative (or weakly positive).

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 General Growth Characteristics
• Staphylococcus species are generally
nonfastidious
• They grow well on media without blood or
other special supplements
• Micrococcus species and R. mucilaginosa are
mildly fastidious and grow more slowly than
Staphylococcus sp.

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• Fastidious group-
– HACEK
• Haemophillus
• Actinobacillus
• Cardiobacterium
• Ekinella
• Kingella
 General Growth Characteristics
• Staphylococcus and Micrococcus tolerate a
high salt concentrations; they grow on
media containing 5%-7.5% NaCl (e.g.
Mannitol Salt Agar)
• Staphylococcus species are facultatively
anaerobic, as is Rothia. Micrococcus is an
obligate aerobe.
• Staphylococcus species produce a variety of
hemolysins and other toxins

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• Which of the following agar is selective for
staph. aureus?
– MSA
– XLD
– EMB
– CLED
 General Cultural Characteristics
• Some Staphylococcus species, most notably S.
aureus, produce a hemolysin that completely
lyses red blood cells of humans and some other
mammals (sheep blood). This is referred to as
“beta” hemolysis.

• Morphology (color, shape, surface, etc) of staph

colonies may change with age

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 Biochemical characteristic

• Production of catalase is a defining

characteristic of the Staphylococcus and
Micrococcus species
• The catalase reaction of Rothia is weak or
delayed and many are frankly catalase
negative

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 Pin head sized, beta hemolytic, white, round,
smooth, moist colonies on 5%sheep blood agar

GPC in cluster Catalase positive

Coagulase test
Skin infections Furuncle, folliculitis, abscess, wound infection

Lipase, hyaluronidase,cytolytic toxins

Staphylococcal scalded skin syndrome

Exfoliatin / epidermolytic toxins

 Diseases Virulence factors

• Toxic shock syndrome TSST-1

• Food poisoning Enterotoxins
• Musculoskeletal (osteomyelitis,arthritis)
• CNS (meningitis,abscess)
• Respiratory (upper& lower)
• Endovascular (bacteremia, endocarditis)
• Urogenital tract infections
 Staph saprophyticus
• Infection only in female
• Honeymoon cystitis
• Novobiocin test
– Resistant- S. saprophyticus
– Sensitive-S. epidermidis, S. aureus
• Which species is frequently associated with
nosocomial infections transmitted via medical
devices inserted into the body?
– Staphylococcus epidermidis
– Streptococcus pyogenes
– Proproniobacterium acnes
– Bacillus anthracis
 s. aureus
• UTI
– Descending
– Lymphatics
– Never ascending
• A 2-day-old premature baby boy born to a 22-year-
old woman developed pneumonia. The mother
developed the symptoms of urinary tract infection
after the delivery. A blood sample was collected from
the newborn for laboratory testing, and within 24
hours, culture plates showed the growth of beta-
hemolytic colonies.
• Which of the following group B streptococci could be
the possible pathogen that can be easily spread from
the mother to the baby and cause pneumonia?
– a) Streptococcus pyogenes
– b) Streptococcus agalactiae
– c) Enterococcus faecalis
– d) Streptococcus bovis
• Which of the following bacteria is commonly
isolated pathogen from infection pharyngitis?
– a) Streptococcus pneumoniae
– b) Staphylococcus aureus
– c) Streptococcus pyogenes
– d) Mycoplasma pneumoniae
• All of the following are the characteristics
of Streptococcus pneumoniae, Except?
a) The colonies are diplococcus and
encapsulated
b) Alpha hemolytic (greenish zone) colonies
appear on blood agar
c) Found as normal flora in healthy individuals
d) It grows well in a high concentration of
glucose media
• Which of the following bacteria is the
common pathogen in otitis media?
a) Streptococcus pneumoniae
b) Streptococcus agalactiae
c) Streptococcus pyogenes
d) Streptococcus intermedius
• Which of the following cocci is a part of the
normal flora of the mouth?
• a) Streptococcus agalactiae
• b) Staphylococcus saprophyticus
• c) Staphylococcus epidermidis
• d) Streptococcus mutans
• What are the different infections caused by
Group A Streptococcus spp?
• All are correct except:
a) Pharyngitis
b) Impetigo
c) Scarlet fever
d) Meningitis
• Which of the following bacteria are examples
of Group D Streptococci?
a) Streptococcus mutans
b) Streptococcus bovis and Enterococcus
faecalis
c) Streptococcus pyogenes and Enterococcus
gallinarum
d) Streptococcus pneumoniae
• Each of the following statements is correct
about Enterococcus spp, Except?
a) They can grow in both aerobic and
anaerobic conditions
b) Resistant to high salt concentrations
c) The morphological characteristics resemble
that of S. pyogenes
d) The common cause of hospital-acquired
infections
• Name the cocci that is a part of the normal
flora of human intestines?
• Select the correct answer:
a) Enterococcus faecalis
b) Micrococcus luteus
c) Staphylococcus epidermidis
d) Streptococcus mutans
• Which of the following is NOT the virulent
factor, an important antigenic property of S.
pyogenes?
a) M protein
b) Streptolysin
c) Streptokinase
d) Endotoxins
• Which of the following antibiotics have been
found to be most effective for the treatment
of infections caused by Enterococcus faecalis?
a) Cephalosporin
b) Macrolide
c) Aminoglycoside with penicillin
d) Oral penicillin V with ampicillin
• Which of the following bacteria have been
found to be most resistant to the drug
vancomycin?
a) Enterococcus faecium
b) Streptococcus pyogenes
c) Staphylococcus aureus
d) Lactobacillus acidophilus
• A 30-year-old individual experiencing symptoms suggestive
of a urinary tract infection provided a urine sample to a
local healthcare center. She reported discomfort, frequent
urination, and observed the presence of blood in her urine.
• Which of the following pathogen is less frequently
isolated from the UTIs sample?
• a. Streptococcus mutans
• b. E.coli
• c. Staphylococcus aureus
• d. Streptococcus agalactiae
 Streptococcus

Gram positive cocci in pairs and chains

Alpha hemolytic Beta hemolytic Gamma hemolytic

(Viridans group) 20 Lancefield grps Enterococcus group
• How many cells should be present in a
bacteria to call as “chain”?
–2
–4
–5
–6
• Which of the following is referred as viridans
group?
– Alpha-hemolytic
– Beta-hemolytic
– Gamma-hemolytic
– Delta-hemolysis
• On which basis, Lancefield has classified
Streptococci?
– Alpha-hemolysis
– Beta-hemolysis
– Gamma-hemolysis
– Delta-hemolysis
• Enterococcus fall under-
– Alpha-hemolysis
– Beta-hemolysis
– Gamma-hemolysis
– Delta-hemolysis
 Viridans group
viridis : Green
• Streptococcus mitis, Streptococcus mutans,
Streptococcus salivarius, Strep sanguis
• Ordinarily nonpathogenic
• IE
• Dental caries
• S. sanguis- infective endocarditis
• S. mutans- dental carries
• Which of the following bacteria is involved in
infective endocarditis?
– Stap. aureus
– Streptococcus pyogenes
– Streptococcus sanguis
– Streptococcus pneumoniae
• All of the following bacteria are associated
with dental caries except-
– Strep mitis
– Strep salivarius
– Strep mutans
– Strep pyogenes
 Beta hemolytic Streptococci
• 20 Lancefield Group (based on specific C
carbohydrate antigen)
• A – V except I and J
• Important groups: A, B, C, D, F, G
• Group A Streptococcus pyogenes
• Group B Streptococcus agalactiae
• Group C Streptococcus equisimilis
• Group D Streptococcus bovis
 Cultural Characteristics
Streptococcus species tend to be more fastidious than
staphylococci and Enterococcus
SBA is ideal for growing Streptococcus as it contains various amino
acids, vitamins & minerals
Sheep’s blood has 3 advantages over other animal blood:
1. Haemophilus species will not grow on SBA due to the presence
of NADase which neutralizes extracellular NAD (“V” factor).

2. Sheep’s blood gives a distinctly clear beta hemolysis that is

obviously different from alpha hemolysis
3. Human blood can contain growth inhibiting antibodies, etc that
prevent recovery of organisms from samples

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• Which blood is best for preparation of blood
agar?
– Rabbit
– Horse
– Sheep
– Human
 Cultural Characteristics
• Streptococci are fermentors and therfore form small colonies
(some species form “pin-point” colonies)
• Most species form colonies that are translucent, convex, shiny
• Streptococcus agalactiae (group B streptococci or GBS) tend to
grow larger colonies (and therefore more opaque) than other
Streptococcus species
• Colonies of GBS range form 1mm-1.5mm in diameter in 18-24h

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 Clinical Significance
• S. pyogenes (GAS) causes several diseases.
– *Streptococcal pharyngitis (…+ otitis media & sinusitis)
– *Scarlet fever
– *Rheumatic fever
– *Necrotizing fasciitis
– Streptococcal toxic shock syndrome
– Meningitis
– Glomerulonephritis
– Impetigo
– Cellulitis
…and more

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 Streptococcus pyogenes
Pharyngitis

Lipoteichoic acid

Cellulitis, impetigo, erysipelas

Streptokinase (fibrinolysin)
Hyaluronidase

Necrotizing fascitis

Streptococcal pyrogenic exotoxin

 S. agalactiae (GBS)
– *Neonatal meningitis / septicemia
– Cystitis and pyelonephritis
– Postpartum endometritis
– Postpartum septicemia
– Suture site infections in women following
caesarian section

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Bacitracin sensitive
• What is the potency of bacitracin used for
identification of strep pyogenes?
– 0.04 unit
– 0.4 unit
– 0.1 unit
– 10 unit (acts as a growth factor of Haemophillus
spp.)
Group B Streptococci

CAMP Test positive

Diseases
Puerperal sepsis, neonatal infections
• Which of the following bacteria contains
CAMP factor?
– GAS
– GBS
– Stahylococcus
– Enterococcos
• Which of the following is CAMP test positive?
– GBS
– GAS
– Listeria monocytogenes
– Both a and c
 Enterococcus
Oval, in pairs or in chains
Present in intestine, genital tract
Grow in the presence of 40% bile,6.5%NaCl, pH 9.6, 45C

Diseases:
Urinary tract infections
Endocarditis
Biliary tract infections
Septicaemia

Resistant to penicillins and cephalosporins

 Streptococcus pneumoniae

Flame shaped or lanceolate appearance

Diplococci with apposing broad ends
Capsulated
Coloniies are alpha hemolytic
dome shaped –umbonate / draughtman / carrom coin appearance

Diseases
Pneumonia, meningitis, otitis media, sinusitis
• Ethyl hydrocuprein hydrochloride- 5ug
• Lab diagnosis
– Optochin sensitivity test
– Bile solubility test
• Which of the following correctly describes the
microbiology of streptococcus pneumoniae?
• A. Gram positive, catalase positive, alpha haemolytic
• B. Gram positive, catalase negative, alpha
haemolytic
• C. Gram positive, catalase negative, beta haemolytic
• D. Gram negative, catalase negative, alpha
haemolytic
• HCAP stands for ?
• A. Hospital-community acquired pneumonia B.
Hospital-community associated pneumonia C.
Health care - associated pneumonia
D. Health care - acquired pneumonia
HAP-hos acq pneu
VAP-ven ass pneu
• Which of the following is the most common
route for bacterial pneumonia ?
A. Aerosolization
B. Contiguous extension
C.Microaspiration of oropharyngeal secretions
D. Hematogenous spread
• Release of which of the following results in
fever ?
• A.Interleukin (IL) 1
• B. Interleukin (IL) 8
• C. Granulocyte colony-stimulating factor
• D. All of the above
• To be adequate for culture, sputum sample should
have ?
• A. >25 PMN & <10 squamous cells per low power
field
• B. >25 PMN and <20 squamous cells per low power
field
• C. >50 PMN and <10 squamous cells per low power
field
• D. >50 PMN and <20 squamous cells per low power
field
• Methicillin resistance in S. aureus is
determined by ?
• A. ermB gene
• B. mecA gene
• C. gyrA gene
• D. parC gene
CA-MRSA has which type of SCCmec element ?
• A. Type I
• B. Type II
• C. Type III
• D. Type IV
HA-MRSA has all type of SCCmec element,
except?
• A. Type I
• B. Type II
• C. Type III
• D. Type IV
• Which of the following bacteria appears in
pair and short chain?
– Strep pneumoniae
– Strep pyogenes
– Staph aureus
– Enterococcus
• In gram stain, which of the following bacteria
appears a lanceolate or flame shaped?
– Strep pneumoniae
– Strep pyogenes
– Staph aureus
– Enterococcus
• In culture, which of the following bacteria
shows draughtsman/carom coin colonies?
– Strep pneumoniae
– Strep pyogenes
– Staph aureus
– Enterococcus
• Which of the following bacteria is Not is a part
of the normal human microbiota?
• a. Streptococcus agalactiae
• b. Staphylococcus aureus
• c. Streptococcus pyogens (A)
• d. Enterococcus faecalis
• e. Staphylococcus saprophyticus
• Majority of strains of Staphylococci are highly
resistant to which of the following antibiotics?
• a) Cephalosporins
• b) Clotrimazole
• c) Amphotericin
• d) Penicillin
• Name the constituent found in the cell wall of
gram-positive cocci?
• a) Lipopolysaccharide
• b) Teichoic acid
• c) Mycolic acid
• d) Endotoxins
• Which of the following statements is Not true
about the hemolysins produced by
Streptococci?
• a) Produce streptolysin O and S
• b)An important virulence factor
• c)Commonly produced by S. agalactiae
• d) Observed around the colonies on blood
agar
• Which of the following is the common method
for identification of Streptococcus
pneumoniae?
• a. Coagulase test
• b. Optochin sensitivity test
• c. Urease test
• d. Indole test
Quellung reaction :capsular swelling

Optochin sensitive

Inulin fermenting
Bile soluble
• Which of the following bacteria shows
quellung reaction?
– Strep pneumoniae
– Strep pyogenes
– Staph aureus
– Enterococcus
• Which of the following bacteria is optochin
sensitive?
– Strep pneumoniae
– Strep pyogenes
– Staph aureus
– Enterococcus
• PYR test (Pyrrolidonyl Aminopeptidase/arylamidase)
– Positive test is shown by
• S. pyogenes
• Enterococcus spp-Group D beta hemolytic
• CoNS- Staph hemolyticus
• Some enterobacteriaceae- E. coli
• Pyrrolidonyl arylamidase enzyme hydrolyzes the L-
pyrrolidonyl- β-naphthylamide substrate to produce a
β-naphthylamine, which can be detected in the
presence of N,N-dimethylaminocinnamaldehyde
reagent by the production of a bright red precipitate.
 ASO test (Antistreptolysin O)
• Diagnosis of acute rheumatic fever, higher
levels are usually found.
• Titres are often low in glomerulonephritis
• Titres higher than 200 are indicative of
streptococcal infection
 Anti-DNAase test
• Retrospective diagnosis of streptococcal
pyoderma
• Titres higher than 300 are usually significant.
VRE (vancomycin resistant enterococci)
• In Penicillin resistant strain, synergism of
aminoglycosides does not occur.
• Vancomycin is used in such case.
• VRE- genes responsible are Van A, Van B, Van
C, Van D, van E
– Mechanism responsible is alteration in D-alanyl-D-
alanine chain in the cell wall.
• Streptococcus sanguis- most often responsible
for endocarditis
• Dental caries- most by S. mutans
• Dick test-strep. pyogenes
• Shick test-c. diphth