Gram +/- Cocci

Thao Nguyen, PhD

Thao.nguyen@ttu.edu.vn
 Objectives
• What is Staphylococcus? Characteristics?
Diseases?
• Streptococcus?
• Neisseria?
 Lecture Setup
• Characteristics: shapes, gram stain, growth…
• Virulence factors: what is produced to cause
disease
• Epidemiology and Pathology
• Diseases
• Diagnostic lab test
• Treatment and Prevention
Staphylococcus (TỤ CẦU KHUẨN)
 Staphylococci Morphology identification

• Spherical, 1 μm
• Irregular clusters
• Young: G+; Old: G-
• Non motile
• No spore or flagella
• May have capsule
• Common of skin and
mucous membranes
 Morphology identification-Growth

• Facultative anaerobe
• Optimum temp. at 37oC
• Form pigment best 20-25oC
• S. aureus: gray to deep golden
yellow, produces hemolysis
• S. epidermidis: gray to white
on primary isolation
• No pigment: anaerobically or in
broth
Morphology identification- Growth Characteristics
Consume: Many carbohydrates
Produce:
Catalase
b-lactamase
Lactic acid
No gas
Relatively resistant to drying, heat (alive at 50oC for 30
minutes), 9% sodium chloride
Readily inhibited by certain chemicals: 3% hexachlorophene
 Antigenic Structure

• Polysaccharides from Peptidoglycan: causes production of

interleukin-1 and opsonic antibodies by monocytes
• Teichoic acid: can be antigenic
• Polysaccharide capsules, which inhibit phagocytosis by
polymorphonuclear leukocytes unless specific antibodies
are present
• Protein A: bacterial surface protein, binds Fc portion of IgG
-> disrupt opsonization and phagocytosis
 Virulence factors of S. aureus
Enzymes
•Coagulase – coagulate plasma and blood
•Hyaluronidase – digest connective tissue
•Staphylokinase – digest blood clots
•Dnase – digest DNA
•Lipases – digest oils, enhance colonization on
skin
•Penicillinase – inactivate penicillin
 Virulence factors of S. aureus
Toxins
•Hemolysins (α,β,γ,δ): lyse blood cells
• α-Hemolysin: protein that acts on a broad spectrum of eukaryotic cell
membranes, hemolyzes blood cell

• β-toxin degrades sphingomyelin

• δ-toxin disrupts biologic membranes and may have a role in S. aureus

diarrheal diseases

• γ hemolysin lyses white blood cells

 Virulence factors of S. aureus
Toxins
•Leukocidin – lyses neutrophils and macrophages
•Enterotoxin – induce gastrointestinal distress
•Exfoliative toxin – separates the epidermis and dermis
•Toxic shock syndrome toxin (TSST) – induces fever, vomiting,
shock, systemic organ damage
 Epidemiology and Pathogenesis
• Staphylococci: normal microbiota of skin, respiratory and
gastrointestinal tracts.

• In the environment: clothing, bed linens, and other fomites

• Pathogenic capacity= extracellular factors + toxins + invasive

properties

• Poor hygiene and nutrition, tissue injury, preexisting primary

infection, diabetes, immunodeficiency
 Staphylococcal Diseases
From localized to systemic

•Localized cutaneous infections – invade skin

through wounds, follicles or glands
– Folliculitis – superficial inflammation of hair follicle

– Furuncle – boil, inflammation of hair follicle/sebaceous

gland progress into abscess or pustule

– Carbuncle – extension of furuncle, usually more than

one furuncle

– Impetigo – bubble like swelling that can break and peel

away, most common in newborns
 Staphylococcal Diseases
Systemic infections
•Osteomyelitis – infection
is established in the bone;
abscess forms
•Bacteremia
 Staphylococcal Diseases
Toxigenic disease
•Food intoxication – ingestion of
heat stable enterotoxins,
gastrointestinal distress
•Staphylococcal scalded skin
syndrome
•Toxic shock syndrome – toxemia
leading to shock and organ failure
 Diagnostic Laboratory Tests
• Specimens: from abscess,
blood, sputum, spinal fluid
• Gram stain
• Growth blood agar plates S
aureus: mannitol, 7.5% NaCl
• Catalase Test
• Coagulase Test
• β-lactamase test
• PCR
 Clinical Concerns and Treatments
• 95% have penicillinase – resistant to penicillin and ampicillin
• Nafcillin, methicillin (MRSA), oxacillin: low penicillin binding
protein (MecA)

• Plasmid-mediated resistance to tetracycline, erythromycin,

aminoglycosides

• Vancomycin: Inhibitory: MIC 2 μg/mL or less

• Systemic infections require intensive lengthy therapy
• Abscess: properly drain and clean
 Control
• Hand hygiene

• Antiseptics to skin lesions

• Sterility in newborn nurseries, intensive care

units, operating rooms, and cancer
chemotherapy wards

• Contact precautions to patients who tested +

STREPTOCOCCi (liên cầu khuẩn)
Morphology

• G+, Spherical, chains/pairs

• Non spore, nonmotile

• Catalase negative

• Capsule: polysaccharides or hyaluronic acid

• Cell wall: antigenic proteins (M, T, R); carbohydrate,

peptidoglycan. Streptococci group A: pili

 Morphology identification - Growth

• Aerobic, microaerophilic, strictly anaerobic

• 37oC, Group D (enterococci): 15oC-45oC
• Only grow rich media: BHI, blood agar… Significant
growth in CO2, glutamine, riboflavin, pantothenic acid,
pyridoxin, biotin. Enterococci: can grow in NaCl 6.5% or
methylene blue 0.1%
• Temperature: most are killed at 50oC, 30-60min
• Antibiotics, chemicals: easily killed by aseptic agents
• Streptococci β-hemolytic group A are very sensitive to
low concentration of penicillin and bacitracin
 Classification
Recently based on

•Cell wall Antigen – 17 groups (A, B, C…)

•Hemolytic reactions on blood agar

– α hemolysis – S. pneumoniae and viridans

– β hemolysis – A, B, C, G and some D strains

•Biochemical reactions and resistance to

physical and chemical factors
 Morphology identification - Growth
• α-Hemolysis: partial lysis
or oxidizing RBC to green
methemoglobin
• β-Hemolysis: complete
lysis of red blood cells
(RBC)
• γ-Hemolysis: non-
hemolysis
Human Streptococcal Pathogens
 β hemolytic S. pyogenes
• Strict parasite in human
• Inhabits throat nasopharynx, occasionally skin
• Most serious streptococcal pathogen
 Virulence factors of S. pyogenes
Protein surface antigens
•C-carbohydrates – protect against lysozyme
•Fimbriae – adherence
•M-protein – resistant to phagocytosis
•Hyaluronic acid capsule – provokes no immune
response
•C5a protease hinders complement & neutrophil
response
 Toxins and Enzymes
A. Streptokinase (Fibrinolysin)

• Group A, C, G β-hemolysis
• Transforms plasminogen into plasmin-> digest
fibrin and other proteins
• Interfered by nonspecific serum inhibitors and
by a specific antibody, antistreptokinase
• Given intravenously for treatment of venous
thrombosis (huyết khối)
 Toxins and Enzymes
B. Streptodornase

• DNAse
• Facilitate the spread of streptococci in tissue by
liquefying pus (like streptokinase)
• Streptodornase+Streptokinase: liquefy exudates and
facilitate removal of pus and necrotic tissue
 Toxins and Enzymes
C. Hyaluronidase

• Hydrolyze hyaluronic acid -> spreading factor

• Antigenic

D. Streptococcal Pyrogenic Exotoxin (Spe)

(Erythrogenic Toxin)
• Group A
• Protein
• Associated with streptococcal toxic shock syndrome and
scarlet fever
 Toxins and Enzymes
D. Hemolysin
β-hemolytic group A S pyogenes
Two hemolysins (streptolysins):
•Streptolysin O: hemolytically active in the reduced state
o AntiStreptolysin O (ASO) combines SO -> block
hemolysis
•Streptolysin S: responsible for the hemolytic zones
around streptococcal colonies growing on the surface of
blood agar plates
o not antigenic
 Epidemiology and Pathogenesis
• Human reservoir only; Inapparent carriers
• Transmission – contact, droplets, food, fomites
• Portal of entry: skin; nasopharynx
• Children – predominant group affected for cutaneous and
throat infections
• Produce disease only when established in parts of the body
where they do not normally occur (eg, heart valves)
• In temporary bacteremia, antimicrobial agents are often
administered to persons with known heart valve deformity
 Clinical Diseases
Skin infections

• Impetigo – superficial lesions, epidemics in school

children, poor hygiene, crowded living condition,
mosquito bites
• Erysipelas – pathogen enters through skin breaks,
spreads to the dermis and subcutaneous tissues, can
remain superficial or become systemic
 Clinical Disease
Necrotizing Fasciitis (Flesh eating
disease) – typically by S. pyogenes but
also other bacteria
•Bacteria enter through breaks in the
skin
•Soft tissue of the patient destroyed
by bacteria – necrotic
•Diabetics, immune compromised
most at risk
•Proper wound care and health
immune systems – very low risk
 Clinical Disease
Streptococcal sore throat: most common
infection caused by β -hemolytic S pyogenes
 Clinical Disease
Systemic infections:
•Scarlet fever – by S.
pyogenes producing
erythrogenic toxin – body
rash, strawberry tongue
•Septicemia
•Pneumonia
•Streptococcal toxic shock
syndrome
 Long term complications of Group A
infections
• Rheumatic fever
– Follows subclinical/overt pharyngitis in children;
damage to heart muscle and valves; arthritis
– The first attack of rheumatic fever usually
produces only slight cardiac damage, which,
however, increases with each subsequent attack
 Long term complications of Group A
infections
• Acute Glomerulonephritis
– Group A type 12, 4, 2, 49, 59, 60, 61
– Nephritis, increase blood pressure, occasionally
heart failure, can become chronic leading to
kidney failure
 Group B – S. agalactiae
• Regularly resides in human vagina, pharynx,
large intestine
• Can be transferred to infant during delivery
and cause severe infection
– Neonatal pneumonia, sepsis, meningitis
– Pregnant women should be screened and treated
• Wound and skin infections; endocarditis in
debilitated pp
 Group C, D and G
• Group D
– Enterococcus faecalis, E. faecium, E. durans
– Normal colonists of human large intestine
– Cause opportunistic urinary, wound, skin
infections (particularly in debilitated persons)
• Group C and G
– Common animal flora, frequently isolated from
upper respiratory; pharyngitis,
glomerulonephritis, bacteremia
 Diagnostic Laboratory Tests

• A. Specimens: throat swab, pus, or blood,

serum
• B. Smears: G+, often show single cocci or
pairs
• C. Culture: blood agar, 37oC, 5-10% CO2
 Diagnostic Laboratory Tests
• D. Tests
– Catalase test: negative
– Bacitracin: Group A positive
– CAMP test
– Antigen Detection Tests: enzymatic immunoassay
(EIA)
– Serologic Tests: detect ASO, antiDNase,
antihyaluronidase, antistreptokinase; anti-M type-specific
antibodies
 Treatment
• Pencillin G
• Erythromycin
• Luôn phải xác định độ nhạy cảm với kháng
sinh
 Prevention, and Control
• Detection and early antimicrobial therapy of
respiratory and skin infections
• Persons who have suffered an attack of
rheumatic fever: penicillin
• Eradication of S pyogenes from carriers
α hemolytic
Streptococci:
Viridans group
• Large complex group
– S. mutans, S. oralis, S. salivarus, S. sanguis, S.
milleri, S. mitis
• Residents of gums, teeth, oral cavity. Also
found in nasopharynx, genital tract, skin
• Not very invasive
• Dental/surgical procedures facility entrance
 Disease caused by Viridans Group

• Bacteremia, meningitis,
abdominal infection,
tooth abscesses
• Most serious is subacute
endocarditis
• Colonization of heart by
forming biofilms
 Viridans Group
• S. mutans produce slime
layers that adhere to teeth,
basis for plaque
• Involve in dental caries
• Person with preexisting heart
conditions should receive
prophylactic antibiotics before
surgery or dental procedures
 S. pneumoniae – the pneumococcus
• Cause 60-70% of all bacterial pneumonias
• Small, lancet shaped cells, pairs, short chains
• Growth induced by 5-10% CO2
• Culture dies in O2
• Catalase and peroxidases negative
 S. pneumoniae – the pneumococcus
• All pathogenic strains form large capsules –
major virulence factor
• 90 capsular subtypes
• Cause pneumonia
• Otitis media in kids
 S. pneumoniae – Epidemiology and
Pathology
• 5-50% of all people carry it in nasopharynx.
Infections are usually endogenous
• Very delicate, not survive long outside its
habitat
• Kids, elderly, immune compromised, those
with lung diseases/viral infections –
predisposed to pneumonia
Pneumonocci gain access to middle ear by
way of eustachian tube
 S. pneumoniae – Cultivation and
Diagnosis
• Gram stain of specimen – presumptive
identification
• Quellung test or capsular swelling reaction
• a-hemolytic; optochin sensitivity, bile
solubility, inulin fermentation

Bile Optochin
test
 S. pneumoniae – Treatment and
Prevention
• Traditionally use Penicillin G or V
• Increase drug resistance
• Two vaccines available for high risk individual
– Capsular antigen vaccine: older adults and high
risk individuals, 5 year effective
– Conjugate vaccine for children 2-23 months
What is the major virulence factor
of Streptococcus pneumoniae?
A) M protein
B) Pyogenic exotoxins
C) Capsule
D) DNase
E) Hyaluronidase
NEISSERIACEAE
 central
nervous
system sexually transmitted
disease (STD)
irritation- gonorrhea
meningitis
 Family Neisseriaceae
• G- cocci
• Residents of mucous membranes of warm
blooded animals
• Genera include Nesseria, Branhamella,
Moraxella
• 2 primary human pathogens
– Neisseria gonorrhoeae
– Neisseria meningitidis
 Genus Neisseria
• G-, bean shaped, diplococci
• No flagella or spores
• Capsules on pathogens
• Pili
• Strict parasites, do not survive outside
hosts
• Aerobic/microaerophilic
• Catalase and cytochrome oxidase
positive
• Pathogenic spiecies require rich
complex media
 NEISSERIA MENINGITIDIS
Não mô cầu (meningococcus)
• Epidemic cerebrospinal meningitis
• Causes life-threatening sepsis (meningococcemia)
and/or meningitis
 Virulence factors
1. Capsule
• Polysaccharide
• Antiphagocytic
• Classified into 13 serogroups based on
different antigenic capsular polysaccharides:
A-E, H, I, K, L, X, Y Z
• Meningitis: usually by serogroups A, B, and C
 Virulence factors
2. Endotoxin
• Lipopolysacharides (LPS)
• Causes blood vessel destruction (hemorrhage)
and sepsis
• Hemorrage on the skin (petechiae-petechial
rash): tiny, round, red dots
 Virulence factors
3. IgA1 protease: cleaves IgA in half
4. Neisseria meningitidis can extract iron from
human transferrin via a non-energy requiring
mechanism
5. Pili: Attachment to nasopharyngeal cells;
Undergo antigenic variation to avoid attack by
the immune system
 Epidemiology and Pathogenesis
• Prevalent cause of meningitis: sporadic or epidemic
• 5% of the population: normal flora of the nasopharynx;
asymptomatically => develop natural immunization
• Spreads via respiratory secretions
• High risk groups: infants (6m-3y); army recruits, college
freshman
• Disease happens when bacteria invade the
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
 Meningococcal Disease
1) Meningococcemia
• Spiking fevers, chills, arthralgia (joint pains),
• Muscle pains, petechial rash
• Meningococci rapidly disseminate throughout
the body => meningitis and/or Fulminant
meningococcemia
 Meningococcal Disease
2) Fulminant meningococcemia (Waterhouse
Friderichsen syndrome, septic shock)
• Bilateral hemorrhage => adrenal insufficiency
• Hypotension, tachycardia
• Enlarging petechial skin lesions
• Disseminated intravascular coagulation (DIC)
• Coma
• Death can occur rapidly (6-8 hours)
 Meningococcal Disease
3) Meningitis
• Most common
• Infants < 1 y/o:
chill, fever,
vomitting,
iritabilility,
lethargy, stiff neck
• Petechial skin
Diagnostic Laboratory Tests

• Specimens:
blood, cerebrospinal fluid, petechial scrapings
• Neisseria culture
– best on chocolate agar (heated blood agar)
– Thayer-Martin VCN (chocolate agar+Vancomycin,
Colistin+Nystatin)
– High CO2 concentration improves the growth
 Diagnostic Laboratory Tests
• Oxidase test : positive
• Sugars fermentation
• Neisseria meningitidis can grow on maltose
while Neisseria gonorrhoeae cannot
• Agglutination with type-specific or polyvalent
serum
Treatment, Epidemiology and Control
• Penicillin G or Ceftriaxone: disseminated
meningococcemia
• Rifampin or Minocyclin: contacts with infected
patients
• Immunization with purified capsular
polysaccharides from certain strains (groups A, C,
Y, and W135)
• Duration of immunitiy in adults: 2-4 ys; in
children: does not induce immunity
 NEISSERIA GONORRHOEAE
(Lậu cầu khuẩn-gonococcus)
• Second most common STD, gonorrhea
 Virulence factors
1) Pili
• Complex genes coding for their pili, multiple
recombinations, hyper varitable amino acid
• changing antigens protect the bacteria from
our antibodies
• prevent phagocytosis, probably by holding the
bacteria so close to host cells
 Virulence factors
2) Lipopolysaccharides
3) Protein I (Outer membrane protein porins,
PorA, PorB)
•form pores in the surface through which some
nutrients enter the cell
•preventing phagosome – lysosome fusion in
neutrophils
Phagocytosis
 Virulence factors
4) Protein II (Opa Proteins)
• Membrane protein (rooted from the outer
membrane)
• Promote adhesion of gonococci within
colonies and to host cells
• Each strain has 11 or 12 genes for different
Opas, but expressions are occasionally
different (0, 1, 2..)
 Virulence factors
5) Protein III (Rmp)
• Associates with Por in the formation of pores
in the cell surface
6) Other proteins (poorly defined roles)
• Lip (H8) is a surface exposed protein
• Fbp (ferric-binding protein), is expressed when
the available iron supply is limited
7) IgA1 protease
Gonococcal Disease in Men

• Unprotected sex with an infected person

• Bacteria penetrates the mucous membranes of the
urethra => inflammation
• Asymptomatic (3-10%)
• Painful urination, purulent urethral discharge =>
Complications: epididymitis, prostatitis, and urethral
strictures
• Rectal gonococcal infection: anal pruritis, tenesmus,
and/or rectal bleeding and purulent discharge
 Gonococcal Disease in Women
• Infected urethra: Painful burning on urination,
minimal purulent discharge
• Infect cervix: Lower abdominal discomfort,
fever, purulent vaginal discharge
• Pelvic inflammatory disease (PID)
=>Infertility (20%)
=>Ectopic pregnancy
Gonococcal Disease in Both
Men and Women
1) Gonococcal bacteremia (Diseminate
gonococcal infection)
• Rarely, Neisseria gonorrhoeae can invade the
bloodstream.
• Fever, joint pains, and skin lesions (which
usually erupt on the extremities)
• Endocarditis and meningitis are rare
Gonococcal Disease
2) Septic arthritis
• Fever, pain and swelling of 1 or 2 joints
• Progressive destruction of the joint

Gonococcal Disease in Infants

• During delivery
• Causes ophthalmia neonatorum => blindness
• Erythromycin eye drops are given to all
newborn.
 Diagnostic Lab Test
• Specimens: Pus and secretions from the
urethra, cervix, rectum, conjunctiva, throat,
synovial fluid
• Gram Stain, Culture on Thayer-Martin VCN,
Oxidase, sugar fermentation
• Serological tests: detect antibody (IgA, IgG
against pili, outermembrane proteins, LPS) by
ELISA
 Immunity

• Repeated gonococcal infections are common

• Protective immunity to reinfection does not

develop due to the antigenic variety of
gonococci

• IgA, IgG: have little protective ability

 Treatment

• In the past, simple use of sulfonamides and

penicillin G, Tetracycline. Resistance increased.

• Recently, Ceftriaxone + azithromycin