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PYOGENIC COCCI
Staphylococcus, Streptococcus, Neisseria
Junqi Zhang (张俊琪), PhD
MOH&MOE Key Lab of Medical Molecular Virology
Shanghai Medical College, Fudan University
(复旦大学上海医学院分子病毒学教育部/卫生部重点实验室)
Cocci
Streptococcus
Staphylococcus
Neisseria
Smear &
Gram Stain
Staphylococcus
Pathogenic Cocci
(Pyogenic Cocci)
Streptococcus
Enterococcus
Neisseria
Gram stain
Positive
Gram stain
Negative
Staphylococcus
>40 species
Staph=grape-like clusters
coccus=round
Scanning Electron Microscope
General Characteristics of
the Staphylococci
 Include a major human pathogen
and skin commensals
 Spherical cells arranged in irregular
clusters
 Gram-positive
 Coagulase: an important indicator
of pathogenic staphylococci
 Lack spores and flagella
 May have capsules
Gram staining
Classification I: isolate character on
blood agar plate
 S.aureus
S.epidermidis
S. saprophyticus
Classification II: coagulase
 coagulase positive
staphylococcus
S. aureus
coagulase negative
staphylococcus, CNS
S. epidermidis
S. saprophyticus
Coagulase: an important indicator of pathogenic staphylococcus
S. aureus
 Morphology, culture, and
biological characteristics
 Pathogenesis: virulent
factors, diseases
 Diagnostic laboratory
tests
 Principles of controlling
staphylococcal infections
Culture Characteristics - S. aureus
 Facultative anaerobe
 Grows in large, round, opaque,
golden yellow colonies; βhemolysis on blood agar plate
 Optimum temperature of 37oC
 Tolerate high salt: 10%-15%
NaCl, high temperatures: 60oC
1h; 80oC 30min
 Withstands extremes in pH
 Drug resistance: MRSA
Virulence factors of S. aureus
 Structural components
 Enzymes
 Toxins
 Structural components:
inhibit phagocytosis
by PMN;
mediate attachment
elicit production of IL-1
and opsonic antibody by
monocytes; be a chemoattractant for PMN; have
endotoxin-like activity;
activate complement
bind to fibronectin and
mediate attachment
bind to Fc portion of IgG;
inhibit phagocytosis; anti
complement
responsible for adherence
of the organisms to
fibrinogen and fibrin –
forming clumps; induce
an immunogenic response
Staphylococcus
Protein A (SPA)
PHAGOCYTE
Fc receptor
SPA
IgG
BACTERIUM
antibody opsonization
 Binds to Fc portion of IgG(IgG1,2,4);
 Inhibits phagocytosis;
 Anti complement
Coagglutination
S.aureus
Antibody-sensitized
Staphylococci
Coagglutination
 Enzymes:
 Catalase – degrades hydrogen peroxide; differentiates
the staphylococci from streptococci
 Coagulase and Clumping factor– coagulates plasma
and blood; produced by 97% of human isolates; antiphagocytosis; invasive potential
 Uronidase
 Staphylokinase
 Proteinases
 lipases
 β-lactamase
swelling
Coagulase and Clumping factor
 responsible for adherence of the
organisms to fibrinogen and
fibrin – forming clumps;
 induce an immunogenic
response（vaccine efforts）
Clumping factor +
Clumping factor -
★ Toxins: (5)
 Hemolysins (α, β, γ, δ)– lyse red blood cells
 Leukocidin (PVL)
– lyses neutrophils and macrophages
 Exfoliative toxin
– separates the epidermis from the dermis
 Enterotoxin– 9 serotypes, induce gastrointestinal distress,
heat stable protein
 Toxic shock syndrome toxin (TSST)
– induces fever, vomiting, shock, systemic organ damage
superantigens
Enterotoxin, Exfoliative toxin, TSST-1 genes are on a
pathogenicity island
Exfoliative
toxin
Loss of superficial skin layers
Toxins 2010, 2(5), 1148-1165
Journal of the American Academy of
Dermatology Volume 48, Issue 2
2003 244 - 252
Superantigen Exotoxins
 Bind directly to MHC II
 Cytokines are released from large proportion of T cells
Virulence factors of S. aureus
Biological activities
Enzymes
Coagulase
Hyaluronidase
Staphylokinase
DNase
Lipases
Penicillinase
Catalase
Toxins
Hemolysins (α, β, γ, δ)
Leukocidin
Enterotoxin
Exfoliative toxin
Toxic shock syndrome toxin (TSST)
coagulates plasma and blood; alter ingestion and destruction by
phagocytic cells; an important indicator of invasive pathogenic staphylococcus
digests connective tissue
digests blood clots
digests DNA
digest oils; enhances colonization on skin
inactivates penicillin
degrades hydrogen peroxide
lyse red blood cells
lyses neutrophils and macrophages
induces gastrointestinal distress
separates the epidermis from the dermis
induces fever, vomiting, shock, systemic organ damage
Structural components
Capsule/polysaccharide
Peptidoglycan
Teichoid acid
SPA
Clumping factor
(bound coagulase)
inhibits phagocytosis by PMN; mediate attachment
elicits production of IL-1 and opsonic antibody by monocytes; be a
chemo-attractant for PMN; has endotoxin-like activity; activates
complement
binds to fibronectin and mediates attachment
binds to Fc portion of IgG; inhibits phagocytosis; anti complement
responsible for adherence of the organisms to fibrinogen and fibrin
forming clumps; induces an immunogenic response
Staphylococcal Disease
• Pyogenic infections: Range from localized
to systemic
• Toxigenic disease:
-Food intoxication
-Staphylococcal scalded skin syndrome (SSSS)
-Toxic shock syndrome (TSS)
 Hospital spread is on the hands of medical personnel
 Outbreaks involve nasal carrier or worker with lesion
 Enterotoxin is produced in rich foods before they are ingested
Pyogenic infections:
Range from localized to systemic
 Localized cutaneous infections – invade skin through
wounds, follicles, or glands
– Folliculitis (毛囊炎)– superficial inflammation of hair follicle;
usually resolved with no complications but can progress
– Furuncle (疖)– boil; inflammation of hair follicle or
sebaceous gland progresses into abscess or pustule
– Carbuncle (痈)– larger and deeper lesion created by
aggregation and interconnection of a cluster of furuncles
– Impetigo (脓胞)– bubble-like swellings that can break and
peel away; most common in newborns
Cutaneous lesions of S. aureus
Furuncle
Carbuncle
Systemic infections
Bacteremia – primary origin is bacteria from
another infected site or medical devices;
endocarditis possible
Osteomyelitis – infection is established in the
metaphysis; abscess forms
Staphylococcal osteomyelitis in a long bone
Toxigenic disease:
 Food intoxication: ingestion of heat stable enterotoxins;
gastrointestinal distress
 Staphylococcal scalded skin syndrome (SSSS): toxin
induces bright red flush, blisters, then desquamation of the
epidermis; infants, adults with hypoimmunity
 Toxic shock syndrome (TSS) :toxemia leading to shock
and organ failure; fever, hypotension, diarrhea,
erythroderma (rash)
Staphylococcal scalded skin syndrome (SSSS):
Following controversial test
marketing in Rochester, New
York and Fort Wayne, Indiana,
in August 1978, Procter and
Gamble introduced
superabsorbent Rely tampons to
the United States market in
response to women's demands
for tampons that could contain
an entire menstrual flow without
leaking or replacement.
1. Rely used carboxymethylcellulose (CMC) and compressed beads of polyester for
absorption.
2. This tampon design could absorb nearly 20 times its own weight in fluid.
3. Further, the tampon would "blossom" into a cup shape in the vagina to hold menstrual
fluids without leakage.
Coagulase Negative Staphylococcus
• CNS - Normal flora
• 35 spp. ~ 15 potential human pathogens
– Staphylococcus saprophyticus
• Common cause of UTI in young women
• Treated as outpatients
– Staphylococcus epidermidis
• UTI, uncommon
• Bacterial endocarditis
• Biofilms – implanted medical devices related infections
Generally hospital acquired
– Distiguished by novobiocin in the lab
BACTERIAL BIOFILMS
Identification of Staphylococcus in
Samples
The identification of organisms is based on
cellular, cultural, and biochemical characteristics
• Specimens: pus, tissue exudates, sputum, urine,
spinal fluid, blood, etc.
• Smears: Gram staining-positive, cocci in clusters;
can't distinguish saprophytic from pathogenic
organisms
• Cultivation
-On nutrient agar they tend to be white (or cream colored),
circular, entire, convex colonies.
-On Sheep Blood Agar Staphylococcus aureus may exhibit
hemolysis of the agar in the area around the colonies.
Colonies of S. aureus
(medium sized, smooth
and glossy, raised, opaque,
inaurate liposolubility
pigment)
Colonies of S. aureus
with zone of hemolysis on
a blood agar plate
White colonies of
S. epidermidis on a
blood agar plate
• Chemical and Biochemical Tests
Tests to be performed:
1.
Catalase test
2.
Coagulase test
3.
Growth and fermentation on Mannitol Salt Agar
4. Susceptibility to the antibiotic “Novobiocin”
Staphylococcus genus
S. aureus
S. epidermidis
S. saprophyticus
Catalase
Catalase +
2 H2O2
2 H2O + O2 (g)
Characteristics
S. aureus
S. epidermidis
S. saprophyticus
pigment of colony
Golden yellow
White
White
coagulase
SPA
+
+
+
+
+
+
+
-
-
novobiocin
Sensitive
Sensitive
Resistant
pathogenesis
suppuration,
abscess, septicemia
endocarditis,
bacteremia, UTI
UTI
mannitol
glucose
heat-stable nuclease
α-toxin
1. Catalase Test
•
The Catalase test determines if the organism produces the enzyme
“Catalase”, which breaks down hydrogen peroxide (H2O2) to
water and oxygen (O2).
Catalase
2 H2 O2
2 H2O + O2 (g)
_
+
A drop of 3% hydrogen peroxide solution is placed on a slide, and a small
amount of the bacterial growth is placed in the solution.
Bubbling indicates a positive test for the presence of the catalase enzyme.
2. Coagulase Test
+
Staphylococcus aureus
_
Coagulase +
Coagulase –
Citrated rabbit (or human) plasma
diluted 1:5 is mixed with an equal
volume of broth culture or growth from
colonies on agar and incubated at 37oC.
If clots form in 1-4 hours, the test is
positive.
Agglutination of the “Test” latex with
no agglutination of the “Control” latex
is considered a positive (+) test for the
presence of this enzyme.
All reactions occurring after 20
seconds should be ignored.
3. Mannitol Salt Agar
•
•
•
•
Two different characteristics of the
organism are determined with this agar.
The first is the organism’s ability to
tolerate a high salt environment. Evidence
of growth on the slant indicates the
organism can grow in a high salt
environment.
Organisms that can ferment the sugar
Mannitol produce an acid end-product that
changes the red pH indicator (Phenol red)
in the media to yellow.
red at neutral pH (~ 7.0)
yellow under acidic conditions
Any yellow in the media is considered a
positive test for Mannitol fermentation.
It is possible to have growth, but no
Mannitol fermentation.
4. Novobiocin Susceptibility
•
•
A zone of growth inhibition 16 mm or less in diameter indicates resistance (R) to
Novobiocin.
If the zone is greater than 16 mm the organism is susceptible (S) to Novobiocin.
• Molecular typing
Polymerase chain reaction (PCR)
Pulsed-field gel electrophoresis (PFGE)
Multilocus sequence typing
ELISA - toxins
Epidemiology
 Present in most environments frequented by humans
 Readily isolated from fomites
 High carriage rate:
healthy adults – 20~50%; medical staff – ~70%
 Carriage is mostly in anterior nares, skin,
nasopharynx, intestine
 Predisposition to infection include: poor hygiene and
nutrition, tissue injury, preexisting primary infection,
diabetes, immunodeficiency
Clinical Concerns
95% have penicillinase and are resistant to
penicillin and ampicillin
Nosocomial infections
Increase in community acquired methicillin
resistance
-MRSA (methicillin-resistant S. aureus)
-Carry multiple resistance
-Some strains have resistance to all major drug groups
except vancomycin
Treatment of Staphylococcal
Infections
 Timely cleanliness, hygiene, and aseptic
management of lesions
 Abscesses have to be surgically perforated
 Prescription of appropriate antibiotics:
 Perform antimicrobial susceptibility tests to help
in the choice of drugs
 Systemic infections require intensive lengthy
therapy
Prevention of Staphylococcal Infections
• Prevent nosocomial infections
 Proper hygiene, segregation of carrier from highly
susceptible individuals
 Precautions and frequent surveillances: areas at highest
risk; high risk patients
 Protection of health care workers
 Good aseptic techniques when handling surgical
instruments
• Hygiene and cleansing
Streptococci
 Morphology, culture, and
biological characteristics
 Pathogenesis: virulent
factors, diseases
 Diagnostic laboratory
tests
 Principles of controlling
staphylococcal infections
Classification of Streptococci
I.
II.
III.
IV.
V.
Colony morphology and hemolytic reaction on
blood agar
Lancefield classification system
Biochemical reactions & resistance to physical
and chemical factors
Ecologic features
Molecular genetics
I. Classification based on colony morphology
hemolysis reaction on blood agar
In 1903 J.H. Brown grouped streptococcus by their ability to lyse RBCs
 -hemolysis: complete disruption of erythrocytes with clearing of the
blood around the bacterial growth
pathogens A, B, C, G and some D strains
 -hemolysis: incomplete lysis of erythrocytes with reduction of
hemoglobin and the formation of green pigment
S. pneumoniae and others collectively called viridans
conditional pathogens
 -hemolysis: no effect on RBCs, nonhemolytic streptococcus
Hemolysis patterns on blood agar
II. Lancefield classification system
Capsule
Proteins (M protein)
Carbohydrates
Cell wall
(C carbohydrate)
Peptidoglycan
Cell membrane
Cytoplasma
II. Lancefield classification system
In 1927 Rebecca Lancefield originated the “Lancefield groups”
of Streptococci that we still use now:
Based on surface antigens: C-carbohydrate
 17 groups (A-H, K-U)
 each group has one or more species
 Group A, B and D - most important;
Group A: ~90% human pathogenic Streptococci
Group C, G, F - rare
Lancefield later determined that Streptococcus viridans &
pneumococci did NOT possess antigens that reacted with her
antisera.
Summarize of
classification system
hemolysis 
reaction
Streptococci

1

2
A M protein
B
Streptococci C-carbohydrate C
…
V groups
3
…
100 types
Human Streptococcal Pathogens
S. pyogenes
S. agalactiae
Enterococcus faecalis
Viridans streptococci
S. pneumoniae
-hemolytic S. pyogenes
The most important member of Group A
Most serious streptococcal pathogen
Strict parasite
Inhabits throat, nasopharynx, occasionally
skin
Virulence Factors of -Hemolytic S. pyogenes
– Structural components
hinders complement and
neutrophil response
major, contributes to
resistance to
phagocytosis
protect against
lysozyme;
classifications
provokes no immune
response
adherence
**group A streptococcus,
Streptococcus pyogenes
Capsule-anti phagocyte
Adherence factors
M protein－adherence, anti phagocyte
(Surface molecules)
LTA－adherence
Virulence
Factors
Streptokinase
Invasive enzymes Hyaluronidase
Streptodornase
Toxins
*Streptolysins (SLO,SLS)
Erythrogenic toxin (pyrogenic exotoxin）
Superantigens
Virulence Factors of b-Hemolytic
S. Pyogenes
Extracellular enzymes
Help bacteria be
easier to spread
• Streptokinase
– digests fibrin clots
• Hyaluronidase
– breaks down connective tissue
• Streptodornase
– depolymerizes DNA, decrease viscosity of
purulent exudates
Virulence Factors of -Hemolytic
S. pyogenes
Extracellular toxins:
Streptolysins – hemolysins; streptolysin O (SLO)
and streptolysin S (SLS) – both cause cell and
tissue injury
Erythrogenic toxin (pyrogenic exotoxin) –
induces fever and typical red rash, superantigen
Superantigens – strong monocyte and
lymphocyte stimulants; cause the release of
tissue necrotic factor
Epidemiology and Pathogenesis
 Source of infection: patients and carriers
 Transmission – contact, droplets, food, vomits
 Portal of entry generally skin or pharynx
 Children predominant group affected for
cutaneous and throat infections
 Systemic infections and progressive sequelae
possible if untreated
Scope of Clinical Disease
Pyogenic infections
Skin infections
• Impetigo (pyoderma)
• Cellulitis
• Erysipelas
• Puerperal fever
Throat infections
• Streptococcal pharyngitis
– strep throat
Pharyngitis and tonsillitis
61
Scope of Clinical Disease
Systemic infections
• Scarlet fever（猩红热）
– strain of S. pyogenes carrying a prophage
that codes for erythrogenic toxin; can lead to
sequelae
• Septicemia
• Pneumonia
• Streptococcal toxic shock syndrome
（链球菌毒素休克综合征）
Long-Term Complications of
Streptococcus Group A Infections
• Rheumatic fever
– follows overt or subclinical pharyngitis in
children; carditis with extensive valve damage
possible, arthritis, chorea, fever
• Acute glomerulonephritis
– nephritis, increased blood pressure,
occasionally heart failure; can become chronic
leading to kidney failure
-Hemolytic Streptococci:
Viridans Group
• Large complex group
– Streptococcus mutans, S. oralis, S. salivarus,
S. sanguis, S. milleri, S. mitis
• Most numerous and widespread residents of the
gums and teeth, oral cavity, and also found in
nasopharynx, genital tract, skin
• Not very invasive; dental or surgical procedures
facilitate entrance
Viridans Group
• Bacteremia, meningitis,
abdominal infection, tooth
abscesses
• Most serious infection：
subacute endocarditis –
Blood-borne bacteria settle and
grow on heart lining or valves
• Colonization of heart by
forming biofilms
• Persons with preexisting heart
disease are at high risk
• S. mutans produce slime layers (biofilms) that
adhere to teeth, basis for plaque; involved in
dental caries
66
Group B: Streptococcus Agalactiae
无乳链球菌
Regularly resides in human vagina, pharynx,
and large intestine
Can be transferred to infant during delivery
and cause severe infection
Most prevalent cause of neonatal pneumonia,
sepsis, and meningitis
Pregnant women should be screened and treated
Wound and skin infections and endocarditis in
debilitated people
Group D Streps and Enterococci
• Group D:
- Streptococcus bovis, S. equinus
• Enterococci:
- Enterococcus faecalis, E. faecium, E. durans, etc.
– Normal colonists of human large intestine
– Cause opportunistic urinary, wound, and skin
infections
Identification
• Specimens: a throat swab, pus, blood, etc.
• Smears: Gram staining-positive, often show
single cocci or pairs rather than chains
• Cultivation – hemolysis
• Rapid diagnostic tests based on monoclonal
antibodies that react with C-carbohydrates
ASO test – Serologic test
• When the body is infected with streptococci, it
produces antibodies against the various antigens that
the streptococci produce.
• Anti-streptolysin O (ASO) is the antibody produced
against an antigen produced by group A streptococci.
• A rise in the titer of ASO can be estimated by this
test.
>400 unit
Treatment and Prevention
• Groups A and B are treated with penicillin
G.
• Long-term penicillin prophylaxis for people
with a history of rheumatic fever or
recurrent strep throat
• Enterococcal treatment usually requires
combined therapy
Streptococcus Pneumoniae
• Causes 60-70% of all bacterial
pneumonias
• Small, lancet-shaped cells
arranged in pairs and short
chains
• Culture requires blood or
chocolate agar
• Growth improved by 5-10%
CO2
• Lack catalase and peroxidases
– cultures die in O2
Virulence factors
• Capsule: 90 different capsular types
major virulence factor, anti-phagocytosis,
Smooth(S)/Rough(R)
• Pneumolysin O: hemolysis
• LTA: attachment
• Neuraminidase:
digest N-Acetylneuraminic Acid
colonization, proliferation, spreading
Diseases
• Main cause of
bacterial pneumonia,
chronic bronchitis, and
meningitis
• Otitis
Bacterial pneumonia
• Bacteremia
• nasal carrier state,
nosocomial infection
Otitis
Cultivation and Diagnosis
• Gram stain of specimen – presumptive identification
Gram +
Capsule +
Diplococcus
• Bile solubility – the most reliable method
• Quellung test or
capsular swelling reaction
• α-hemolytic;
optochin sensitivity
• Animal model: intraperitoneal injection,
dead – S. pneumoniae
Treatment and Prevention
• Traditionally treated with penicillin G or V
Increased drug resistance;
Vancomycin
• Two vaccines available for high risk
individuals:
– Capsular antigen vaccine for older adults and
other high risk individuals – effective 5 years
– Conjugate vaccine for children 2 to 23 months
The Neisseria
• Gram-negative cocci
• Residents of mucous membranes of warmblooded animals
• 2 primary human pathogens:
– Neisseria gonorrhoeae (Gonococci)
– Neisseria meningitidis (Meningococci)
Characteristics of Neisseria
Pili
•
•
•
•
•
•
•
•
•
Gram-negative, bean-shaped, diplococci
None develop flagella or spores
Pili
Capsules on pathogens
In samples, can usually be
found in neutrophils
Strict parasites, do not survive
Dewdrop-like colonies of
N.meningitidis on a chocolate
long outside of the host
plate
Aerobic or microaerophilic
Produce catalase and cytochrome oxidase
Pathogenic species require enriched complex media
(chocolate plates) and CO2
Neisseria gonorrhoeae:The Gonococcus
Virulence factors:
1. Pili (Fimbriae), other
surface molecules (Rmp, Por,
Opa) for attachment; slows
phagocytosis
2. Lipooligosaccharide (LOS): endotoxic effects; molecular
mimicry of human glycosphingolipid, evade immune recognition
3. IgA1 protease: cleaves secretory IgA, attachment
Causes gonorrhea, in top 5 STDs
Gonorrheal damage to the male and female
reproductive tracts
Males – urethritis, yellowish
discharge, scarring, and infertility
Females – vaginitis, urethritis,
salpingitis mixed anaerobic abdominal
infection, common cause of intertility
and ectopic （tubal ）pregnancies
Gonorrhea in Newborns
• Infected as they pass through birth canal
• Eye inflammation, blindness
• Prevented by prophylaxis immediately after
birth
Neisseria Meningitidis: The Meningococcus
• >13 serogroups; serotypes A, B, C, X, Y
and W-135 cause most cases
• Virulence factors:
–
–
–
–
Capsule
Adhesive pili (fimbriae)
IgA protease
Lipooligosaccharide (LOS): endotoxic effects
Epidemiology and Pathogenesis
• Prevalent cause of meningitis; sporadic or epidemic
• Human reservoir – nasopharynx; 5-30% of adult
population may harbor meningococci; 70% carrier
rate during epidemics
• High risk individuals are those living in close
quarters, children 6 months-3 years
• Disease begins when bacteria enter bloodstream,
cross the blood-brain barrier, permeate the meninges,
and grow in the cerebrospinal fluid
• Very rapid onset; neurological symptoms; endotoxin
causes hemorrhage and shock; can be fatal
Dissemination of the meningococcus from a
nasopharyngeal infection
One clinical sign of meningococcemia
Thrombosis
petechia
Immunity, Vaccination and treatment
• humoral immunity: IgG, IgM, IgA
• Vaccines: against capsular polysaccharides
types: A, C ; A, C,Y, W-135
• Treatment: penicillin G, cephalosporin
Key points
• The virulence factors of staphylococcus and
streptococcus
• Diseases caused by staphylococcus and
streptococcus
• Differences between pathogenic and nonpathogenic staphylococcus
• Classification of streptococci
Questions
1. Chapter 13: 2,3,4,5,6,7,10,11
2. Chapter 14: 1,2,3,4,5,6,7,9,10
3. Chapter 20: 1,4,6,7,9
4. How to distinguish pathogenic staphylococcus species from
non-pathogenic ones ?
5. Describe the main virulence factors of staph, strep and
Neisseriae genus.
6. Please definition SPA.
7. “Strep” vs. “staph”, how do they differ?