Gram Positive

Cocci
Jill J. Jaime
Catalase Test
Staphylococcus
aureus
• Catalase-positive spherical organisms, often
appear in grape-like clusters
• (+) Hemolysis
• Selective medium for the isolation of S. aureus
is one containing 7.5% to 10% sodium chloride
(NaCl) with mannitol
• Differentiated from other species of
staphylococci principally by its production of
coagulases, which are capable of clotting
plasma.
Staphylococcus aureus
• Part of normal flora - skin, eye, upper respiratory tract, gastrointestinal
tract, urethra, and, infrequently, vagina.
• Community-acquired infection with S. aureus that are oxacillin resistant
(CA-MRSA) have become more common.
• Virulence:
• Exotoxin TSST-1 - toxic shock syndrome
• Enterotoxins A to E – heat-stable toxins staphylococcal food poisoning
• Epidermolytic toxins A and B—cause skin erythema and separation, as seen in
scalded skin syndrome (Ritter Disease).
• Protein A – surface protein; high affinity for the Fc receptor on IgG molecules as well
as complement. This is a mechanism for S. aureus to directly bind immunoglobulins,
decreasing immune clearance
Staphylococcus aureus
• Virulence:
• Panton-Valentine leukocidin (PVL). – pore-forming and kills leukocytes, preventing
clearance; rare in hospital-acquired infection
• Polysaccharide capsule - inhibits phagocytosis; (slime layer or biofilm)
• Peptidoglycan - activates complement, IL-1, chemotactic to PMNs
• Teichoic acids - species specific, mediate binding to fibronectin
• Exotoxins: cytotoxins (alpha, beta, delta, and gamma)
• Protease, lipase, and hyaluronidase – destroy tissue function and aid in invasion
• Antibiotic Resistance
• mecA gene - responsible for production of a new penicillin-binding protein (PBP-2a
or PBP-2′); causes oxacillin resistance (CA-MRSA and HA-MRSA)
• mecC gene – mecA gene homologue
CoNS: Staphylococcus epidermidis
• Normal flora
• Nosocomial infections: bacteremia associated with indwelling
vascular catheters; endocarditis involving prosthetic cardiac valves
(rarely involves native valves); CSF shunts, prosthetic joints, vascular
grafts, postsurgical ocular infections, and bacteremia in neonates
under intensive care
• Virulence:
• Exopolysaccharide “slime” or biofilm;antiphagocytic
• Exotoxins: delta toxin
Which CoNS is associated with
honeymoon cystitis?
A. S. lugdunensis
B. S. saprophyticus
C. S. haemolyticus
D. Micrococcus
Other CoNS
• Staphylococcus haemolyticus and Staphylococcus lugdunensis -
involve implantation of medical devices
• Staphylococcus saprophyticus - community-acquired urinary tract
infections in young, sexually active females but is not commonly
associated with HAIs
• Staphylococcus schleiferi
• Micrococcus spp.,
• Kocuria spp. Common Contaminants

• Kytococcus spp.
Laboratory Diagnosis
• No special considerations for specimen collection and
processing
• Direct Microscopy - Staphylococci appear as gram positive
cocci, usually in clusters. Micrococci typically appear as
gram-positive cocci in tetrads.
• Media of choice:
• 5% sheep blood and chocolate agars. They also grow well in
broth-blood culture systems and common nutrient broths
• Selective: Mannitol Salt Agar, Phenylethyl alcohol (PEA) or
Columbia colistin-nalidixic acid (CNA) agars to eliminate G- (i.e.
fecal specimens)
• CHROMagar – contains Cefoxitin, selective and differential media
for the identification of MRSA.
Approach to
Identification
•Bound coagulase, or clumping factor, is
detected using a rapid slide test (i.e., the
slide coagulase test), in which a positive test
is indicated when the organisms agglutinate
when mixed with plasma
• S. lugdunensis and S. schleiferi may be (+) S may

•Extracellular (i.e., free) coagulase/ Tube

Coagulase Test - performed by inoculation of
a tube containing plasma and incubating at
35°C for 4 hrs. If no clot, extend to 24 hrs
Approach to
Identification
•Disk tests - used for the determination of
bacitracin (A) and furazolidone (FX)
resistance:
• A 0.04 U bacitracin-impregnated disk and a
100-mg furazolidoneimpregnated disk are
placed on the surface of a 5% sheep blood
agar streaked in three directions with a
cotton-tipped swab that has been dipped in a
bacterial suspension prepared to match the
turbidity of the 0.5 McFarland standard

•Microdase - for differentiating Micrococcus

spp. (+) from Staphylococcus spp (-)
Approach to
Identification
•Novobiocin - S. saprophyticus is resistant
•Latex Agglutination Test - detect clumping
factor and protein A
• Some also detect capsular polysaccharide,
which may improve the ability to detect
methicillin (oxacillin)-resistant S. aureus

•MALDI-TOF
•16S rRNA – Reference/research centers
 •Inducible resistance to Clindamycin –
facilitated by erm gene; can be evaluated by
Antimicrobial a modified Kirby-Bauer Test, “D zone test”

Susceptibility
•Inducible plasmid-encoded β-lactamase –
penicillin resistance; confirmed by Penicillin
Disk Diffusion Test or PCR for blaZ gene
•Resistance to the penicillinase-resistant
penicillins (methicillin, oxacillin, nafcillin):
mecA gene - encodes an altered
penicillin-binding protein, PBP2a.
•Vancomycin-intermediate susceptible S.
aureus (VISAs, MIC 4-8 mg/mL)
•Vancomycin-resistant S. aureus (VRSA)
are currently defined by the identification of
an MIC16 mg/mL. Tx: Linezolid and
Daptomycin
 Which is the only cephalosporin that
can treat MRSA isolates
A. Ceftazidime
B. Ceftriaxone
C. Ceftaroline
D. Cefoperazone
 Streptococci
Hemolysis Lancefield Species
Group
β A Streptococcus pyogenes
B Streptococcus agalactiae
C Streptococcus dysgalactiae
D Enterococcus spp.
α or γ D Enterococcus spp.
D Streptococcus bovis
complex (reclassified into many new
species, as described in text)
None Viridans group ∗
α None Streptococcus pneumoniae
Group A Streptococcus
• Streptococcus pyogenes – ß hemolytic
• Protein F mediates epithelial cell attachment
(fibronectin binding)
• Hyaluronic acid capsule inhibits phagocytosis
• Several enzymes and hemolysins that
contribute to tissue invasion and destruction,
including streptolysin O, streptolysin S,
streptokinase, DNase, and hyaluronidase.
• Streptococcal pyrogenic exotoxins (SPEs)
mediate production of rash (i.e., scarlet fever)
or multisystem effects that may result in death
• C5a peptidase-destroying complement
chemotactic factors
Group A Streptococcus
• Streptococcus pyogenes – ß hemolytic
• M protein is antiphagocytic (.100
serotypes)
• Rheumatic Fever - Cross-reactions of antibodies
produced against streptococcal antigens and
human heart tissue;
• APGN - Deposition of antibody-streptococcal
antigen complexes in kidney results in damage
to glomeruli
• Lipoteichoic acid - permits bacterial
adherence to the respiratory epithelium
• Streptococcal superantigens (Sags) -
thought to be responsible for the toxic
shock–like syndrome caused by strains of
S. pyogenes
Other ß-hemolytic Streptococcus
• Streptococcus agalactiae– ß hemolytic
• Neonatal sepsis, pneumonia, and meningitis.
• All pregnant women at 36 0/7 to 37 6/7 weeks of gestation should have vaginal/rectal
specimens collected and processed for detection of GBS (NAAT or Culture)
• Uncertain; capsular material interferes with phagocytic activity and complement cascade
activation
• Groups C, F, and G betahemolytic streptococci
• Similar types of acute infections in adults as described for S. pyogenes and S. agalactiae, but usually involve
immunocompromised patients
α and γ hemolytic Streptococcus
• Streptococcus pneumoniae - α hemolytic
• Infection: pneumonia, meningitis (especially in infants and the elderly), spontaneous bacteremia (in
persons who do not have a spleen), otitis, sinusitis, and spontaneous peritonitis
• Polysaccharide capsule that inhibits phagocytosis is primary virulence factor
• Pneumolysin has various effects on host cells, and several other factors likely are involved in eliciting a
strong cellular response by the host
• Secretory IgA protease
• Viridans streptococci - α or γ hemolytic
• Slowly evolving (subacute) endocarditis, particularly in patients with previously damaged heart valves;
bacteremia and infections of other sterile sites do occur in immunocompromised patients
• S. mutans – dental carries
• S. bovis - associated with malignancies of the gastrointestinal tract
• Enterococcus - urinary tract infection in hospitalized persons
• Other: Leuconostoc , Pediococcus , Rothia , Gemella , Aerococcus , Lactococcus , and other less
rarely isolated species.
• Aerococcus (A. urinae and A. sanguinicola ) - known urinary tract pathogens.
Laboratory Diagnosis
• No special considerations for specimen collection and
processing.
• Detection:
• Latex Agglutination or ELISA – S. pyogenes
• Two-plate culture method - to increase recovery for
diagnosing streptococcal pharyngitis: sheep blood
agar and SXT blood agar
• Antigen Detection on incubated LIM broth (contains
antimicrobials on vaginal N flora) – S. agalactiae
• Latex Agglutation Kits - detection of beta-hemolytic
streptococcus Lancefield groups A, B, C, F, and G
from primary culture plates
• PCR – for detection of
Christie-Atkins-MunchPetersen (CAMP) factor (cfb
gene) and C5a peptidase gene (scpB) to detect
group B streptococci.
Question: why do they form chains while Staphylococcus
form clusters?
Cellular division occurs along a single axis; thus they grow in chains or pairs. . In contrast, Staphylococcus divide along
multiple axes, which results in a cluster of cells.
Laboratory Diagnosis
• Media of Choice
• Will grow on standard laboratory media such as 5% sheep blood and
chocolate agars, and nutrient broths
• Columbia agar with colistin and nalidixic acid (CNA) and phenylethyl
alcohol agar (PEA) – inhibits G(-); selective
• Abiotrophia and Granulicatella will not grow on blood or chocolate agars
unless pyridoxal (vitamin B6) is supplied
• CHROMagar Strep B – for S. agalactiae; can be a subculture from LIM
brith
• Bile Esculin Agar – ability to hydrolyze esculin in the presence of 40% Bile
• Enterococcosel agar – selective differential medium based on the esculin
hydrolysis and is also selective by incorporation of inhibitory oxgall (bile
salts) to inhibit growth of other gram-positive organisms
• BEA and Enterococcosel with Vancomysin – Detection of
Vancomycin-Resistant Enterococcus
Laboratory Diagnosis
• Incubation Condition and Duration
• Most are facultative anaerobes, with some preferring a
CO2-enriched environment.
• In 5% to 10% carbon dioxide. This is the preferred atmosphere for
S. pneumoniae
• Blood agar plates should be inoculated by stabbing the inoculating
loop into the agar several time
• Colonies can then grow throughout the depth of the agar, producing
subsurface oxygen-sensitive hemolysins (i.e., streptolysin O). Most
organisms will grow on agar media within 48 hours of inoculation
• Colony Appearance:
• The beta-hemolytic streptococci may have a distinctive buttery
odor.
• Viridans streptococci have a butterscotch odor, especially on
 ß-hemolytic
Streptococcus

Approach to
Identification
• Pyrrolidonyl Arylamidase
(PYR) test - rapid test used for
the presumptive identification of
GABHS and Enterococci.
• All isolates of GAS and more than
99% of isolates of Enterococcus
are PYR positive.
• Bacitracin susceptibility or
using latex agglutination for the
Lancefield group A antigen –
confirms GABHS.
Bacitracin Sensitivity Test
 ß-hemolytic
Streptococcus

Approach to
Identification
• Hippurate Hydrolysis - used to detect
the ability of bacteria to hydrolyse
hippurate into glycine and benzoic acid
by action of hippuricase enzyme
present in bacteria.
• Ninhydrin reagent for reacts with
glycine = purple color
• CAMP Test - detects production of a
diffusible, extracellular protein that
enhances the hemolysis of sheep
erythrocytes by Staphylococcus aureus
Approach to
Identification
• Confirmation of presumed GBS
• From sterile body sites - serotyping
(using latex agglutination or
coagglutination tests) or
MALDI-TOF.
• For culture of vaginal/rectal swab
specimens from pregnant women -
broth enrichment be used along with
or as a replacement for agar-based
media.
• Selective broth media, including Lim
broth or selective Todd-Hewitt broth, Granada agar, on which colonies of GBS will appear yellow
can be used as enrichment media
to orange for ease of detection
Approach to
Identification
• Streptococcus pneumoniae: flat
mucoid colony with depressed center
• Optochin Test: a filter paper disk (“P”
disk) impregnated with optochin is
placed on a blood agar plate previously
streaked with a lawn of the suspect
organism. The plate is incubated at 35°
C for 18 to 24 hours and read for
inhibition
• Bile solubility – S. pneumonia is
susceptible to bile
Approach to
Identification
• PYR hydrolysis- enterococci are PYR
positive, and viridans streptococci are
negative.
• All enterococci grow in the presence of
6.5% NaCl, but viridans streptococci do
not.
• Bile Esculin - Enterococci hydrolyze
esculin in the presence of bile (causing
visible growth and blackening of the agar),
but up to 10% of viridans streptococci are
also bile–esculin positive.
Approach to
Identification
• Additional biochemical assays or
MALDI-TOF are required to identify
enterococci to the species level.
• A majority of vancomycin-resistant
enterococci (VRE) are E. faecium.
• Identification of individual species Leuconostoc and Pediococcus rarely cause disease, but it
of viridans streptococci requires is important to correctly identify them as they are
conventional biochemical testing, intrinsically resistant to vancomycin.
molecular methods, or MALDI-TOF
 Which titer increases in pyoderma?
A. Antistreptolysin O (ASO)
B. Anti-DNase B
C. Antistreptokinase
D. Antihyaluronidase
Serodiagnosis
• GABHS - the most common are
antistreptolysin O (ASO), anti-DNase B,
antistreptokinase, and
antihyaluronidase
• Pharyngitis – rise in all titers
• Pyoderma – rise in antiDNase B only
• Useful in patients with prior infection who
has not been cultured; Serum obtained as
long as 2 months after infection usually
demonstrates increased antibodies.
Antimicrobial Susceptibility Testing
• Groups A, B, C, and G streptococcus = susceptible to penicillin.
• Routine susceptibility testing is not necessary unless penicillin cannot be
used.
• Inducible resistance to clindamycin - specific guidelines for
streptococcal D–zone testing recommend that the clindamycin and
erythromycin disks be separated by 12 mm instead of the 15 to 16
mm recommended for testing S. aureus
• S. pneumoniae - A screening test using disk diffusion with a 1-μg
oxacillin disk (≥20 mm = susceptible) may be performed
• vanA, vanB, and vanC genes – confer resistance to Vancomycin
(Enterococcus)
Gemella and Aerococcus
• Other nonstreptococcal gram-positive, catalase-negative cocci
of increasing importance
• G. haemolysans - associated with endocarditis and meningitis.
• Gram stain usually demonstrates diplococci with adjacent sides
flattened that can be confused with a Neisseria sp. because cells can
easily become decolorized.
• A. urinae and A. viridans - both resemble viridans streptococci
or enterococci on agar plates; however, appear in tetrads
• Can be responsible for UTI
Merci
Beaucoup!