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

StaphylococcusStreptococcus

Neisseria

Smear &

Gram Stain

Pathogenic Cocci

(Pyogenic Cocci)

Staphylococcus

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

S.aureus S.epidermidis

S. saprophyticus

Classification I: isolate character on

blood agar plate

coagulase positive staphylococcus

S. aureus

coagulase negativestaphylococcus, CNS

S. epidermidis

S. saprophyticus

Coagulase: an important indicator of pathogenic staphylococcus

Classification II: coagulase

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; β-

hemolysis 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

inhibit phagocytosis

by PMN;

mediate attachment

elicit production of IL-1

and opsonic antibody by

monocytes; be a chemo-

attractant 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

Structural components:

SPAIgG

Fc receptor

PHAGOCYTE

BACTERIUM

Staphylococcus

Protein A (SPA)

Binds to Fc portion of IgG(IgG1,2,4);

Inhibits phagocytosis;

Anti complement

antibody opsonization

S.aureus Antibody-sensitized

StaphylococciCoagglutination

Coagglutination

Catalase – degrades hydrogen peroxide; differentiates

the staphylococci from streptococci

Coagulase and Clumping factor– coagulates plasma

and blood; produced by 97% of human isolates; anti-

phagocytosis; invasive potential

Uronidase

Staphylokinase

Proteinases

lipases

β-lactamase swelling

Enzymes:

responsible for adherence of the

organisms to fibrinogen and

fibrin – forming clumps;

induce an immunogenic

response（vaccine efforts）

Clumping factor +

Clumping factor -

Coagulase and 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

Enterotoxin, Exfoliative toxin, TSST-1 genes are on a

pathogenicity island

superantigens

Journal of the American Academy of

Dermatology Volume 48, Issue 2

2003 244 - 252

Loss of superficial skin layers

Toxins 2010, 2(5), 1148-1165

Exfoliative

toxin

Superantigen Exotoxins

Bind directly to MHC II

Cytokines are released from large proportion of T cells

Virulence factors of S. aureus Biological activities

Enzymes

Coagulasecoagulates plasma and blood; alter ingestion and destruction by

phagocytic cells; an important indicator of invasive pathogenic staphylococcus

Hyaluronidase digests connective tissue

Staphylokinase digests blood clots

DNase digests DNA

Lipases digest oils; enhances colonization on skin

Penicillinase inactivates penicillin

Catalase degrades hydrogen peroxide

Toxins

Hemolysins (α, β, γ, δ) lyse red blood cells

Leukocidin lyses neutrophils and macrophages

Enterotoxin induces gastrointestinal distress

Exfoliative toxin separates the epidermis from the dermis

Toxic shock syndrome toxin (TSST) induces fever, vomiting, shock, systemic organ damage

Structural components

Capsule/polysaccharide inhibits phagocytosis by PMN; mediate attachment

Peptidoglycan

elicits production of IL-1 and opsonic antibody by monocytes; be a

chemo-attractant for PMN; has endotoxin-like activity; activates

complement

Teichoid acid binds to fibronectin and mediates attachment

SPA binds to Fc portion of IgG; inhibits phagocytosis; anti complement

Clumping factor

(bound coagulase)

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

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

Pyogenic infections: Range from localized to systemic

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):

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.

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.

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

-On nutrient agar they tend to be white (or cream colored),

circular, entire, convex colonies.

• Cultivation

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

-On Sheep Blood Agar Staphylococcus aureus may exhibit

hemolysis of the agar in the area around the colonies.

Tests to be performed:

1. Catalase test

2. Coagulase test

3. Growth and fermentation on Mannitol Salt Agar

4. Susceptibility to the antibiotic “Novobiocin”

• Chemical and Biochemical Tests

Staphylococcus genus

Characteristics S. aureus S. epidermidis S. saprophyticus

pigment of colony Golden yellow White White

coagulase + - -

mannitol + - -

glucose + + -

heat-stable nuclease + - -

α-toxin + - -

SPA + - -

novobiocin Sensitive Sensitive Resistant

pathogenesissuppuration,

abscess, septicemia

endocarditis,

bacteremia, UTIUTI

Catalase

2 H2O2 2 H2O + O2 (g)Catalase +

S. aureus S. epidermidis S. saprophyticus

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 H2O2 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.

+_

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.

2. Coagulase Test

+

_

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.

Coagulase –Coagulase +

Staphylococcus aureus

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

InfectionsTimely 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. Colony morphology and hemolytic reaction on

blood agar

II. Lancefield classification system

III. Biochemical reactions & resistance to physical

and chemical factors

IV. Ecologic features

V. 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

Cell wall

Proteins (M protein)

Carbohydrates

(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

Streptococci

hemolysis

reaction

C-carbohydrate

A

B

C

…

V groups

M protein

1

2

3

…

100 types

Streptococci

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

protect against

lysozyme;

classifications

adherence

major, contributes to

resistance to

phagocytosis

provokes no immune

response

hinders complement and

neutrophil response

**group A streptococcus,

Streptococcus pyogenes

Adherence factors

(Surface molecules)M protein－adherence, anti phagocyte

LTA－adherence

Toxins

*Streptolysins (SLO,SLS)

Erythrogenic toxin (pyrogenic exotoxin）

Capsule-anti phagocyte

Virulence

Factors Invasive enzymes Hyaluronidase

Streptodornase

Streptokinase

Superantigens

Virulence Factors of b-Hemolytic

S. Pyogenes

Extracellular enzymes

• Streptokinase

– digests fibrin clots

• Hyaluronidase

– breaks down connective tissue

• Streptodornase

– depolymerizes DNA, decrease viscosity of

purulent exudates

Help bacteria be

easier to spread

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

61

Scope of Clinical Disease

Pyogenic infections

Skin infections

• Impetigo (pyoderma)

• Cellulitis

• Erysipelas

• Puerperal fever

Throat infections

• Streptococcal pharyngitis

– strep throat

Pharyngitis and tonsillitis

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

66

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

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

• 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.

ASO test – Serologic 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

• Bacteremia

• nasal carrier state,

nosocomial infection

Bacterial pneumonia

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 warm-blooded animals

• 2 primary human pathogens:

– Neisseria gonorrhoeae (Gonococci)

– Neisseria meningitidis (Meningococci)

Characteristics of Neisseria

• Gram-negative, bean-shaped, diplococci

• None develop flagella or spores

• Pili

• Capsules on pathogens

Pili

• In samples, can usually be

found in neutrophils

• Strict parasites, do not survive

long outside of the host

• Aerobic or microaerophilic

• Produce catalase and cytochrome oxidase

• Pathogenic species require enriched complex media (chocolate plates) and CO2

Dewdrop-like colonies of

N.meningitidis on a chocolate

plate

Neisseria gonorrhoeae:The Gonococcus

2. Lipooligosaccharide (LOS): endotoxic effects; molecular

mimicry of human glycosphingolipid, evade immune recognition

3. IgA1 protease: cleaves secretory IgA, attachment

Virulence factors:

1. Pili (Fimbriae), other

surface molecules (Rmp, Por,

Opa) for attachment; slows

phagocytosis

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 intertilityand 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 non-

pathogenic 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?