lect 4 oral micro b

GRAM POSITIVES

Staphylococcus, Micrococcus & Streptococcus spp.

Dr. T Chisholm-Hedge
DMD 1017

AREAS TO FOCUS ON

  • Habitat and transmission
  • Characteristics
  • Culture and Identification
  • Pathogenesis
  • Treatment and Prevention

Staphylococcus spp.

General Properties
  • Gram Positive
  • Shape: Spherical (cocci)
  • Etymology: Derived from Greek words:
    • staphyle: meaning bunch of grapes
    • coccus: meaning grain or berry
Staphylococcus Characteristics
  • Species Count: Consists of 35 species and 17 subspecies
  • Cocci Arrangement: Mainly in clusters but can occur singly, in pairs, or in short chains
  • Size: Ranges from 0.5 μm to 1.5 μm
  • Oxygen Requirement: Facultative anaerobes
  • Growth in Bile Salts: Can grow in the presence of bile salts
  • Growth in High Salinity: Grows in media with high concentrations of Sodium Chloride (NaCl)
  • Temperature Range: Grows in a temperature range from 18°C to 40°C; optimum at 37°C
  • Habitat: Many species found in humans, classified as mesophiles
Staphylococcus Species Causing Infections
  • Major Pathogens:
    • Staphylococcus aureus: Causes a variety of infections
    • Staphylococcus epidermidis: Common human colonizer and opportunistic pathogen
    • Staphylococcus saprophyticus: Common human colonizer
    • Staphylococcus lugdunensis: Common cause of human disease
    • Staphylococcus haemolyticus
  • Additional Species Listed:
    • Staphylococcus capitis
    • Staphylococcus saccharolyticus
    • Staphylococcus warneri
    • Staphylococcus hominis: Common human colonizer
    • Staphylococcus auricularis: Rare cause of human disease
    • Staphylococcus coagulans
Physiology and Structure
Cell Wall Structures
  • Capsule:
    • Composed of polysaccharides; serves as a virulence factor, protects from opsonization and phagocytosis
  • Extracellular slime:
    • Composed of monosaccharides, proteins, and small peptides; binds bacteria to tissues and foreign bodies (e.g., prostheses)
Cell Wall Components
  • Peptidoglycan:
    • Comprises 50% of the weight of the cell wall
  • Teichoic Acid:
    • Species-specific phosphate-containing polymers (30 to 50% of weight of the cell wall); important for attachment to mucosal surfaces
  • Protein A:
    • IgG binding protein that attaches to the Fc region of antibodies preventing complement activation; present in S. aureus but not in coagulase-negative staphylococci; can be utilized for specific identification of S. aureus
Additional Components
  • Coagulase:
    • A surface protein (clumping factor-bound coagulase); converts fibrinogen to insoluble fibrin; important in the identification of S. aureus
  • Cytoplasmic Membrane:
    • Acts as an osmotic barrier and anchorage for biosynthetic and respiratory enzymes
  • Other Proteins:
    • Fibrinectin, fibrinogen, elastin, and proteins in collagen; involved in various adhesion processes
Epidemiology
  • Ubiquitous Organism: Staphylococcus spp. are found everywhere
  • Coagulase Negative Staphylococcus (C.N.S): Present on skin
  • Transmission Pathways:
    • S. aureus and C.N.S found in the oropharynx, gastrointestinal tract (GIT), genitourinary tract (GUT), and nasopharynx
    • Transient colonization with S. aureus occurs at the umbilical stump, skin, and perineal area of neonates
    • Approx. 30% of healthy adults are persistent carriers of S. aureus in the nasopharynx
  • High-Risk Groups: Higher incidence in hospital patients, medical personnel, individuals with skin diseases, intravenous drug users (IVDU), and diabetics
  • Adherence Factors: Regulated by surface adhesins allowing organisms to remain on surfaces for extended periods
Survival on Various Surfaces
  • Staphylococcus aureus: 2.5 hours on hands, 7 months on surfaces
  • Vancomycin-resistant Enterococcus (VRE): 1 hour on hands, 4 months on surfaces
  • Gram-negative bacteria: 1 hour on hands, varies on surfaces
  • Clostridium difficile (C. diff): 24 hours on hands or 5 months on surfaces
  • Pseudomonas spp.: 3 hours on hands, 6 hours-16 months on surfaces
  • Acinetobacter: >3 hours on hands, 3 days-5 months on surfaces
  • Influenza: 10-15 minutes on hands, 12-48 hours on surfaces
  • Rotavirus: >4 hours on hands, 6-60 days on surfaces
Clinical Conditions Associated with Staphylococcus spp.
Skin and Soft Tissue Infections
  • Common Conditions:
    • Impetigo
    • Furuncles (boils)
    • Carbuncles
    • Cellulitis
    • Abscesses
    • Staphylococcal scalded skin syndrome (SSSS)
Invasive/Bloodstream Infections
  • Types:
    • Bacteremia/Sepsis: Presence of bacteria in the bloodstream potentially leading to septic shock and multi-organ failure
    • Endocarditis: Infection of heart valves, particularly in IV drug users or those with prosthetic valves
    • Metastatic infections: Secondary infections seeded from bloodstream to distant sites
Bone and Joint Infections
  • Examples:
    • Osteomyelitis: Bone infection that may be acute or chronic
    • Septic arthritis: Infection within joints causing pain, swelling, and risk of joint destruction
    • Prosthetic joint infections are particularly problematic due to biofilm formation
Respiratory Infections
  • Types:
    • Pneumonia: Can be community-acquired or hospital-acquired; PVL-positive strains cause severe necrotizing pneumonia
    • Empyema: Infection of the pleural space
    • Lung abscesses: Localized pus collection in lung tissue
Toxin-Mediated Syndromes
  • Examples:
    • Toxic shock syndrome (TSS): Caused by TSST-1 or enterotoxin; associated with tampon use, surgical wounds, or skin infections
    • Food poisoning: Resulting from preformed enterotoxins in contaminated foods, rapid onset (2-6 hours), with nausea, vomiting, diarrhea
Device-Related Infections
  • Types:
    • Catheter-associated infections: Particularly common with central lines and urinary catheters (especially S. saprophyticus)
    • Prosthetic device infections: Involves heart valves, joints, pacemakers, vascular grafts
    • Biofilm-related infections: Particularly problematic with coagulase-negative staphylococci like S. epidermidis
Central Nervous System Infections
  • Examples:
    • Meningitis: Infection of meninges; often occurs post-neurosurgical or via bacteremia
    • Brain abscess: Localized infection in the brain
    • Epidural abscess: Spinal infection that can lead to neurological compromise
Staphylococcal Virulence Factors
Enzymes
  • Coagulase: Converts fibrinogen to fibrin, protecting bacteria with a fibrin coat
  • Staphylokinase: Dissolves fibrin clots to enable spread of bacteria
  • Hyaluronidase: Degrades hyaluronic acid in connective tissues, aiding invasion
  • Lipases and nucleases: Facilitate tissue destruction and nutrient acquisition
Toxins
  • Hemolysins (α, β, γ, δ): Form pores, destroying red blood cells (RBCs), white blood cells (WBCs), and other host cells
  • Panton-Valentine leukocidin (PVL): Destroys leukocytes and leads to tissue necrosis, associated with severe skin infections and necrotizing pneumonia
  • Toxic shock syndrome toxin-1 (TSST-1): A superantigen leading to toxic shock
  • Exfoliative toxins (ETA, ETB): Responsible for disrupting cell adhesion in the epidermis, causing SSSS
  • Enterotoxins (SEA-SEE and others): Superantigens that cause food poisoning and contribute to toxic shock
Biofilm Formation
  • Polysaccharide intercellular adhesin (PIA): Major component of biofilms on medical devices, protecting bacteria from antibiotics and immune responses
Immune Evasion Factors
  • Capsule: Polysaccharide coating that inhibits phagocytosis
  • Protein A: Also interferes with complement activation
  • Chemotaxis inhibitory protein (CHIPS): Blocks recruitment of neutrophils
  • Superantigen-like proteins: Disrupt immune cell functions without inducing massive cytokine release
Toxic Shock Syndrome
  • Occurrence: TSST-1 producing strains of S. aureus thrive in tampons; toxin released into the bloodstream
  • Symptoms: Abrupt onset of fever, hypotension, rash, multi-organ system involvement
  • Mortality Rate: Initially high but now approximately 5%
Toxin-Mediated Diseases
Food Poisoning
  • Mechanism: Caused by consumption of food contaminated with enterotoxin, presenting symptoms such as vomiting, abdominal cramps, and diarrhea a few hours after consumption
  • Common Sources: Dairy products, meat, pastries
  • Infection Process: Carriers transmit organism to food
  • Incubation Period: 3-7 hours
  • Symptoms: Abrupt onset of severe vomiting, diarrhea, headache, abdominal pain; typically lasts less than 24 hours
  • Toxin Heat Stability: These toxins are resistant to heat
  • Treatment: Focus on fluid replacement and symptomatic treatment
Staphylococcal Scalded Skin Syndrome (SSS)
  • Characteristics:
    • Disseminated desquamation of epithelial cells mainly in infants
    • Localized bullous impetigo serves as a less severe manifestation
Clinical Presentation of SSS
  • Patient Profile: Primarily neonates and young children
  • Onset: Rapid, beginning with perioral erythema that spreads to the entire body
  • Symptoms: Development of large bullae or cutaneous blisters, leading to desquamation; blisters contain clear fluid but few or no white blood cells (indicating toxin, not bacteria)
  • Diagnostic Sign: Positive Nikolsky’s sign; suggests superficial separation of the skin layers
  • Outcome: Generally no scarring, as only the top layer of the epidermis is involved
Other Pyogenic Diseases
  • Conditions:
    • Impetigo
    • Folliculitis
    • Furuncles
    • Carbuncles
    • Ecthyma
    • Wound infections
Laboratory Identification of Staphylococcus spp.
Tests Used
  • Gram Stain: Displays Gram-positive cocci in grape-like clusters
  • Catalase Test: Positive for S. aureus
  • Coagulase Test: Used to differentiate S. aureus
  • Mannitol Salt Agar Test:
    • Staphylococcus aureus ferments mannitol, changing the color of the medium to yellow; S. epidermidis and S. saprophyticus will grow but not ferment mannitol
  • Deoxyribonuclease (DNAse) Test:
    • Differentiates S. aureus (produces DNAse) from other Staphylococcus species (do not produce DNAse)
Coagulase Test Details
  • Principle: Coagulase is a prothrombin-like substance activating fibrinogen to form fibrin clots, leading to identification
  • Reactions:
    • S. aureus: Positive
    • Coagulase-negative staphylococci: Negative
Treatment of Staphylococcal Infections
  • Antibiotic Susceptibility: Less than 10% of Staphylococcus is susceptible to Penicillin
  • Resistance Mechanism: Primarily due to the production of Penicillinase
  • Alternative Treatments:
    • Use Penicillin derivatives resistant to B-lactamase hydrolysis, e.g., Methicillin, Nafcillin, Oxacillin, Dicloxacillin
  • Resistance Strains:
    • MRSA (methicillin-resistant S. aureus): 30-50% incidence
    • MRSE (methicillin-resistant S. epidermidis): >50% incidence; reported rate in Jamaica is <10%
  • Vancomycin: Remains the drug of choice for MRSA and MRSE treatment despite emerging resistance reports
Prevention and Control Measures
  • Methods:
    • Proper cleansing of wounds
    • Appropriate use of disinfectants
    • Proper hand washing and covering of exposed skin infections
  • Chemoprophylaxis:
    • Consisting of Vancomycin, rifampin, mupirocin, and chlorhexidine baths aimed at preventing MRSA and MRSE spread

MICROCOCCI

General Characteristics
  • Catalase: Positive
  • Coagulase: Negative
  • Blood Agar Observation: White colonies (some brightly pigmented—pink, orange, or yellow)
  • Stomatococcus mucilagenosus: Previously classified as Micrococcus, now found on the lingual surface; produces extracellular slime corresponding with its habitat; role in disease remains unclear

Streptococci

General Characteristics
  • Gram Positive: Cocci approximately 1 µm in diameter
  • Arrangement: Typically in chains or pairs
  • Capsule: Usually capsulated
  • Motility: Non-motile
  • Spore Formation: Non-spore forming
  • Oxygen Requirement: Facultative anaerobes
  • Culturing Requirements: Fastidious
  • Catalase Reaction: Catalase negative (in contrast to Staphylococci which are catalase positive)
Classification of Streptococci
  • Oxygen Requirements:
    • Anaerobic: Peptostreptococcus
    • Aerobic or Facultative Anaerobic: Streptococcus
  • Serological Grouping: (Lancefield Classification)
  • Hemolysis Observations on Blood Agar:
  • Groups Listed:
    • Group A: S. pyogenes
    • Group B: S. agalactiae
    • Group C: S. equisimitis
    • Group D: Enterococcus and others (Groups E-U)
Medically Important Streptococci
  • Type Species:
    • Streptococcus pyogenes: Lancefield Group A
    • Streptococcus agalactiae: Lancefield Group B
    • S. equisimilis: Lancefield Group C
    • Enterococcus faecalis: Lancefield Group D
    • S. bovis (non-Enterococcus): Lancefield Group D
    • S. anginosus: Lancefield Group F
    • S. sanguinis: Lancefield Group G
    • S. salivarius: Lancefield Group K
    • S. suis: Lancefield Group H
Hemolysis Classification on Blood Agar
  • α-hemolysis: Partial hemolysis; green discoloration observed, e.g., non-groupable streptococci such as S. pneumoniae and S. viridans
  • β-hemolysis: Complete hemolysis; clear zone observed around colonies, e.g., Group A S. pyogenes and Group B S. agalactiae
  • γ-hemolysis: No lysis observed, e.g., Group D (Enterococcus species)
Group A Streptococci
  • Species: Only S. pyogenes present
  • Age Distribution: Affects all ages, peak incidence between 5-15 years
  • Associated Conditions: Accounts for 90% of pharyngitis cases
Pathogenesis and Virulence Factors of S. pyogenes
Structural Components
  • M Protein:
    • Interferes with opsonization and lysis
  • Lipoteichoic Acid & F Protein:
    • Involved in adhesion
  • Hyaluronic Acid Capsule:
    • Camouflages the bacteria from immune detection
Enzymes Produced
  • Streptokinases
  • Deoxyribonucleases
  • C5a Peptidase
  • Pyrogenic Toxins: Stimulate macrophages and helper T cells to release cytokines
Streptolysins
  • Streptolysin O: Lyse red blood cells, white blood cells, and platelets
  • Streptolysin S: Facilitates tissue spread for streptococci
Diseases Caused by S. pyogenes

  • Superficial Infections:

    • Pharyngitis
    • Scarlet Fever
    • Impetigo
    • Pyoderma
    • Ecthyma
    • Necrotizing fasciitis
    • Cellulitis
    • Postpartum Sepsis

  • Invasive Infections:

    • Streptococcal toxic shock syndrome
    • Myositis
    • Bacteremia
    • Pneumonia
Autoimmune Sequelae
  • Post-Infectious Conditions:
    • Acute rheumatic fever
    • Post-streptococcal glomerulonephritis
Differentiation Between β-hemolytic Streptococci
  • Tests Utilized:
    • Lanciefield Classification
    • Bacitracin Susceptibility Test: Specifically used for S. pyogenes
    • CAMP Test: Specific for S. agalactiae
Bacitracin Sensitivity Test
  • Principle: Used for identifying Group A; S. pyogenes is susceptible while Group B is resistant
  • Procedure: Inoculate blood agar plate (BAP) with heavy suspension of the organism; place Bacitracin disk (0.04 U) on the culture and observe the zone of inhibition
CAMP Test
  • Principle: S. agalactiae produces CAMP factor, enhancing lysis of RBCs in conjunction with Staphylococcus aureus b-lysin
  • Procedure: Streak Streptococcus and S. aureus perpendicular and observe for an arrowhead-shaped zone of complete hemolysis
  • Outcome: Positive for S. agalactiae; negative for non-Group B
Differentiation Between α-hemolytic Streptococci
Tests Utilized
  • Optochin Test: Test for the identification of S. pneumoniae
  • Bile Solubility Test: Tests self-lysing abilities of S. pneumoniae compared to S. viridans
  • Inulin Fermentation
  • Quellung Reaction/Test: Tests for capsular swelling
Optochin Susceptibility Test Details
  • Principle: Identifies S. pneumoniae as it is inhibited by optochin
  • Procedure: Inoculate BAP with organism, place optochin disk, and measure inhibition zone
  • Results: Zone ≥ 14mm indicates positive for S. pneumoniae; zones ≤ 13mm negative
Bile Solubility Test Details
  • Principle: S. pneumoniae has a self-lysing enzyme enhanced by bile
  • Procedure: Mix 10 parts broth culture with 1 part bile, record turbidity after incubation
  • Results: Positive test shows clearing with bile, negative shows turbidity; S. pneumoniae is soluble in bile while S. viridans is not
Summary of Differentiation Tests
  • Bacitracin Sensitivity:
    • Positive in S. pyogenes (susceptible)
    • Negative in S. agalactiae (resistant)
  • CAMP Test:
    • Positive for S. agalactiae; negative in non-group B
  • Optochin Sensitivity:
    • Positive in S. pneumoniae (soluble)
    • Negative in S. viridans (insoluble)
Conclusion and Clinical Relevance
  • Staphylococcus and Streptococcus spp. are significant in clinical settings due to their ability to cause a wide range of infections, their resistance mechanisms, and the necessity for proper identification and treatment.