Download Lecture 15 and 16 Microbiology: Gram Positive Bacteria infections

Lecture 15 and 16 Microbiology: Gram Positive Bacteria infections
Pathogenic Grampositive Bacteria
•
Coccus
− Staphylococcus
− Streptococcus
•
Staphylococcus:
3 medically
important species
of Staphylococcus:
- S. aureus
- S. epidermidis
- S. saprophyticus
Bacillus
− Bacillus
− Clostridium
− Corynebacterium
− Listeria
− Mycobacterium
Characteristics (clusters of grapes)
• Facultative Anaerobes; Nonmotile; Irregular clusters
• Normal flora
• Grows best aerobically
• Found in soil, water and skin of humans
• Catalase-positive (Streptococcus are catalase-negative)
 Coagulase test: aurea is positive; you put it in a tube and see if it
clumps.
•
Three clinically important species:
− S. aureus most virulent (coagulase-positive)
− S. epidermidis (agent of opportunistic infections associated with
indwelling equipment (catheters, prosthetics, …)
− S. saprophyticus (agent of UTI)
Epidemiology
• Transmission – Coagulase-Positive (aureus)  Autoinfection (carrier),
Direct contact (person with lesion), Contaminated food (person with
lesion) or Fomite (Survive long periods of drying); Coagulase-Negative
(other 2 species)  Autoinfection (infections associated with implanted
catheters and prosthetic devices; UTI)
− About 30% of individuals carry S. aureus in the anterior nares
− Coagulase-negative species are normal flora of skin, nares and
ear canals
Pathogenesis and Virulence Factors
• Adhesions
− Teichoic acid
− MSCRAMMs (Microbial Surface Components Recognizing
Adhesive Matrix Molecules) MOST COMMON
• Protein A- binds to constant region- upside down!
• Pyrogenic exotoxins
− Enterotoxins (multiple types)
• Heat-stable, Superantigens resistant to gut enzymes
(just 25mg induces vomiting)

Superantigens: in MHC class II; they bind to T helper
cells which allows it to release cytokines!!!
− Toxic Shock Syndrome Toxin (TSST-1)
• Superantigen  toxic shock syndrome; directly
cytotoxic (?); In 20% of isolates
• Membrane damaging toxins
− Beta-, Delta-, Gamma-Hemolysins
• b-hemolysin degrades sphingomyelin lysing numerous
cells
• d-hemolysin dissociates into subunits to disrupt cell
membranes
• g-hemolysin components combine with PV proteins
(LukS/LukF) that lyse WBC (mainly neutrophils)
− Leukocidins
• Two-component toxins which lyse WBC (neutrophils)
by forming pores
Pathogenesis and Virulence Factors
• Exfoliatin (eats at skin and makes it look like it’s pealing)
− A = heat stable (resists boiling for 20min); B = heat labile
− Dissolve epidermal mucopolysaccharide matrix (desquamation);
Also function as Superantigen
• Enzymes
− Coagulase
• Initiates fibrin polymerization on surface of bacteria
(prevent phagocytosis)
• Clumping factor permits binding to fibrinogen and fibrin
− Hyaluronidase / Staphylokinase
• Permits invasion of tissues and dissolves fibrin clots
formed by coagulase, respectively (break through tissue
layers and go through tissue damage)
− *Bacteremia: when there is bacteria in blood stream; immune
system isn’t clearing it
Diagnosis
• Culture
− Pus/surface swab, blood, sputum specimen inoculation onto
Mannitol Salt agar (MSA)  Successful growth, Gram stain,
catalase test, coagulase test
− MSA contains 7.5% NaCl which inhibits growth of other normal
flora
− Catalase differentiates from Streptococcus
− Coagulase differentiates more virulent S. aureus from other
species
− Microdilution / Disk Diffusion susceptibility tests should be
done (this will come up later!)
Sites of Infection
Staphylococcus
aureus (usually
skin infections)
Clinical Findings
• Furuncle (Boils) & Carbuncle
− Infected patient is often a carrier (anterior nares)
− Lesions of a hair follicle, sebaceous gland or sweat gland
− Resolves upon spontaneous drainage of pus
− Multiple boils form a carbuncle which can spread to the blood
• Impetigo
− Production of exfoliative toxins produce large blisters in
superficial skin
− Often affects young children (face and limbs)
− Macule that develops into pustules (rupture and crust)
− Can occur as wound infection following surgery
• Scalded Skin Syndrome (SSS)
− AKA Ritters Disease
− Production of exfoliative toxins that cause erythema (redness of
skin) and epidermal desquamation at remote sites from
staphylococcal infection
− Face, axilla, groin affected first then all parts of body possible
− Most common in neonates and children <5y
Clinical Findings – 1 Infections
• Toxic Shock Syndrome
− High fever, vomiting, diarrhea, sore throat and myalgia
− ~48h can progress to shock with evidence of renal and hepatic
damage
− Rash may develop followed by desquamation at a deeper level
than SSS
− Originally associated with use of tampons in 1980s…growth of
bacteria on tampon and release of TSST-1 into blood
− Also occur in men and children with staphylococcal wound
infections
− TSS-Staphylococcus found in vagina, on tampons, in wounds,
localized infections or throat (never the blood)
• Staphylococcal Food Poisoning
− Incubation 1-6h (sick immediately after bc injestion of
enterotoxin)
− Ingestion of enterotoxin-contaminated food
− Nausea, vomiting and diarrhea (no fever)…few hours duration
− Salted meats, custard pastries, ice cream, salad bar foods,…
− Toxins are heat-stable so reheating food does not inactivate
toxins (cannot taste in food); survives high heat and high salt!
− Antibiotics are not useful
MethicillinResistant
Staphylococcus
aureus
MRSA
• <10% of S. aureus strains susceptible to Penicillin but many still
susceptible to penicillin-derivatives
• Late 80s a nosocomial S. aureus strain demonstrated resistance to
penicillin-derivatives (methicillin)…ten years later a genetically distinct
community-acquired (CA) methicillin-resistant strain appeared
• In both cases, MRSA expressed a new resistance gene on a plasmid,
mecA
• CA-MRSA genetic distinction resided in the production of new types of
PV leukocidins (more virulent)
• Vancomycin employed to treat MRSA patients has led to generation of
vancomycin-resistant MRSA (VRSA)
• Several strains of VRSA have been detected since 2002 (most express a
plasmid with vanA gene cluster*)
• Development of such resistant strains appears to derive from resistance
generated in Enterococcus that is transmitted to Staphylococcus
through conjugation
• Antibiotics target cell wall; it was found that MRSA targets cell wall too.
• *The point is they keep changing so antibiotics are an issue in that way
Streptococcus
Characteristics
• Facultative Anaerobes; Nonmotile
• Blood agar is preferred because satisfies growth requirements and can
differentiate groups based upon hemolysis patterns
• Catalase-negative (Staphylococcus catalase-positive)
• Classification by Group-Specific Surface Carbohydrate:
− Group A
(S. pyogenes)
− Group B
(S. agalactiae)
− Group C
(S. dysgalactiae)
− Group D
(S. bovis, Enterococcus)
− Non-Groupable
(S. pneumoniae, Viridans)
• Classification by Hemolysis Pattern:
− Alpha
(Group D and Non-groupable)
− Beta (complete lysis)
(Groups A, B, C, F, G)
− Non-hemolytic
(Some Group D and Non-Groupable)
Epidemiology
• Transmission – Direct contact, Fomites or Contaminated respiratory
droplets (GAS); In utero or during birth (GBS); Direct contact with
nasal secretions or Contaminated respiratory droplets (S. pneumoniae
or Pneumococcus)
Streptococcus
pyogenes (GAS)
Pathogenesis *All of the below work to get around the immune system though
in different ways.
• M Protein / Lipoteichoic Acids / Protein F
− Adherence to nasopharynx and skin epithelia
− M protein and C5a (compliment) peptidase block phagocytosis
and PMN recruitment, respectively
 C refers to compliment (ex: C5a peptidase)
• Streptodornase / Streptokinase
− DNase and Fibrinolysin to break up Neutrophil Extracellular
Traps (NET) which are networks of granule protein (fibrin) and
chromatin to capture pathogens so PMN antimicrobials can kill
microbes
• Pyrogenic Exotoxin (Erythrogenic Toxin) A, B, C
− Superantigens  toxic shock syndrome
−
Pyrogenic Exotoxin A is produced by Streptococcus carrying
lysogenic phage
− Streptococcal TSS and Scarlet Fever
• Diphosphopyridine Nucleotidase
− Lyse WBC
• Streptolysin O / Streptolysin S
− (O) Anaerobic Hemolysin that is rapidly inactivated in the
presence of oxygen
− (S) Aerobic Hemolysin; induced upon bacteria exposure to
serum
− Both damage tissue cells and lyse phagocytes
Clinical Findings
• Streptococcal Pharyngitis (Strep Throat)
− Sore throat, fever, headache (tonsil, soft palate, uvula  red,
swollen, covered with yellow exudate)…1wk duration (any
age…5-15y)
 If body doesn’t clear this infection, the heart or kidneys
can be targeting causing serious problems.
• Impetigo
− Small vesicle surrounded by erythema on face or lower
extremities…enlarges over few days…develops into
pustule…breaks to form crusted lesion (2-5y)
• Erysipelas
− Spreading area of erythema/edema (face), pain, fever,
lymphadenopathy…previous history of streptococcal
pharyngitis
• Streptococcal TSS
− Involves any site of GAS infection…myalgia, chills, severe pain at
infected site
− Necrotizing fasciitis and myonecrosis, nausea/vomiting,
diarrhea…hypotension, shock, organ failure (bacteremia)
• Scarlet Fever
− Buccal mucosa, temples, cheeks are deep red (pale area around
mouth and nose)
− Tongue covered with yellow-white exudate and red papillae
(strawberry tongue)
− “Sandpaper” rash on d2 (chestextremities)
• Acute Rheumatic Fever (ARF) / Acute Glomerulonephritis (AGN)
− (ARF) Fever, carditis, chorea (involuntary movement disorder),
arthritis…3wks following “strep throat”…recurrent attacks upon
S. pyogenes encounter can result in damage to the heart (heart
failure)
− (AGN) 3-6wks after “strep throat” or skin infection…lesions of
glomeruli
Diagnosis
• Ag Detection
− Rapid detection kit for Group A Carbohydrate (must follow with
culture as only 90-95% sensitive…goal to avoid ARF)
• Culture
− Throat swab, pus, sputum, tissue specimen inoculation onto
Blood agar  Beta hemolysis
− Also test susceptibility to Bacitracin (GAS is susceptible while
others are not)
Streptococcus
agalactiae (GBS)
Characteristics
• Leading cause of sepsis and meningitis in the first few days of life
• Normal resident of the GI tract…can spread to the vagina (10-30% of
women)
• During pregnancy and delivery…GBS may gain access to the amniotic
fluid or colonize the newborn as it passes through the birth canal
• About 2 cases/1,000 births in US
• GBS exposed to mucus membranes and quickly spreads to the blood
(lungs, CNS)
• GBS capsule binds serum Factor H – another compliment protein(binds
to host cell glycosaminoglycans or GAG and degrades C3b) to prevent
alternate pathway of complement activation.
 C3 is a targeted pathway for infection of this bacteria.
• Biggest problem when baby is being born.As long as mom has the IgG
against GBS, the newborn is protected by the classical pathway of
complement. If needed, mommy gets antibiotics.
• Onset is first few days of life  Presents as respiratory distress, fever,
lethargy, irritability, hypotension…pneumonia (common) and meningitis
(5-10%)…~20% mortality (if CNS infection…20-30% permanent brain
damage)
•
•
•
Streptococcus
pneumoniae
Late-onset develops at 1-3mo
Diagnosis by detection of group B Ag in blood and culture on Blood agar
Prophylactic therapy of colonized women has dramatically reduced
transmissions
Virulence Factors and Pathogenesis
• Choline Binding Protein
− Binds phosphocholines of bacterial cell wall with carbohydrates
of nasopharynx epithelia
• Capsule
− Prevents C3b deposition on bacterial cell surface
− Blocks phagocytosis
• Pneumolysin
− Produced but not secreted
− Production of peroxides during growth induces autolysins
− Autolysins degrade peptidoglycan thereby lysing the bacteria
− Pneumolysin is released:
o Directly cytotoxic to endothelial cells…(permits
dissemination into the bloodstream)
o Impairs ciliary action (paralyzed) keeps everything in
o Directly suppresses phagocytic activity (no cleanup)
o Suppresses local inflammatory immune response (
anti-inflammatory cytokines)
o Triggers platelet activation (DIC can occur from
concurrent vascular leakage due to endothelial damage
in lungs)
o Pneumococcal pneumonia does not elicit extensive
damage to the lung tissue as in other infections such as
influenza (local immunosuppression)
*Cascade of effects!
*Most common case of pneumonia from streptococcus pneumonae*
- Know how it binds and what it’s receptor is.
Clinical Findings
• Infections most common in young (<2y) and old (>60y)
• Pneumococcal Pneumonia
− A leading cause of pneumonia in world (500,000 cases/y in US)
− 5 million children die each year worldwide
− Shaking chills, high fever, cough (tinged with blood), chest
pain…5-10d duration
− Higher incidence in those over 50y
• Pneumococcal Meningitis
− One of the 3 leading causes of bacterial meningitis (N.
meningitidis, H. influenzae)
− Meningitis may develop solely or follow pneumonia or otitis
media
− Headache, stiff neck, fever, photophobia, irritability
• Other Infections
− Otitis Media
o Most frequent cause with millions of cases/y
− Sinusitis
− Bacteremia  Endocarditis, Arthritis, Peritonitis
Diagnosis
• Microscopy
− Gram stain of sputum (bronchial lavage), CSF, blood  Grampositive Diplococci
• Culture
− Sputum, CSF, blood specimen inoculation onto Blood agar 
Gram stain, susceptibility to Optochin (ethylhydrocupreine…S.
pneumoniae is susceptible while others are not)
Prevention & Treatment
• Pneumococcal Conjugate Vaccine (PCV13, Prevnar 13®)
− Children/Adults <18y…4 doses (2, 4, 6, 12-15mo) or 1-2 doses if
>2y
− 13 capsule Ags conjugated to CRM197 mutant diphtheria toxin
• Pneumococcal Polysaccharide Vaccine (PPSV23, Pneumovax®)
− All adults >65y
− 2y-64y with health condition (heart disease, AIDS,…)
− 88% of serotypes (23 capsule Ags)
Bacillus anthracis
Characteristics
• Aerobic or Facultative Anaerobes; Nonmotile; Spore-forming
• All other species are low-virulence saprophytes found in air, soil, water
• Protein capsule
• Koch used this bacteria to work out Koch’s postulates; Pasteur
developed vaccine for sheep, goats and cows using attenuation
methods
Epidemiology
• Anthrax primarily a disease of horses, sheep and cattle who acquire it
from spores of B. anthracis contaminating their pastures
• Ideal biowarfare agent since spores of B. anthracis have long-life,
stability and require few spores to produce infections (respiratory is
most severe)
• Transmission – Direct contact with spores (through skin), Ingestion of
spores (rare) or Inhalation of spores
• B. cereus and B. subtilis cause infections of the eye, soft tissues and lung
associated with immunosuppression, trauma, indwelling catheters or
contaminated medical equipment
• B. cereus also can produce an entertoxin (cAMP) and cause diarrhea
Virulence Factors and Pathogenesis
• Protein Capsule
− Adherence to tissues (?)
− Effective blockade of phagocytic uptake
• Anthrax Toxin
− Protective Antigen (PA)
o Binds to ATR on host cell surface (first)
o bind either EF or LF and are endocytosed into host cell
− Edema Factor (EF)
o EF functions as an adenylate cyclase to  cAMP; causes
edema (swelling)
− Lethal Factor (LF)
o Protease that cleaves MAPKK pathway proteins and
induces TNF secretion…induces cell death of
macrophages and endothelial cells (causes necrosiscell death or hypoxia)
Pathogenesis
Corynebacterium
diphtheria
Clinical Findings
• Cutaneous Anthrax
− Incubation 2-5d after spore exposure
− Erythematous papule  Vesicular lesion  Ulcerative lesion 
Scab (Black Eschar)…7-10d transition
− Accompanied by mild systemic symptoms; rare cases, local
edema is precursor to bacteremia and death
• Pulmonary Anthrax
− Historically – inhaled spores upon work in confined space with
contaminated hides, hair, wool,…(Wool Sorter’s Disease)
− Incubation 1-2d after spore exposure
− Mild fever, nonproductive cough quickly progress to respiratory
distress and cyanosis (alveoli are destroyed)
− Bacteremia ensues to “seed” every organ system…95%
mortality (untreated)
Diagnosis
• Culture
− Skin lesion, Sputum, CSF, blood specimen inoculation onto
Blood agar  Non-hemolytic growth, Gram stain (Grampositive bacillus)
− Saprophytic species are beta-hemolytic and motile (swarming)
Prevention
• Anthrax Vaccine Absorbed (AVA, Biothrax®)
− Filtrate of B. anthracis culture with added aluminum hydroxide
(adjuvant)
− Contains PA and other immunogenic proteins
− Recommended for those who may be exposed to large doses of
endospores (some military, lab workers, some animal handlers)
Characteristics / Epidemiology / Pathogenesis
• Facultative Anaerobe; Pleomorphic (club-shaped bacillus)
• 60 species…found in plants and animals; colonize skin, respiratory tract,
GI tract and urogenital tract (can be asymptomatic carriers)
• Transmission – Contaminated respiratory droplets (remain viable for
hours)
• Diphtheria Toxin (DTX)
− Encoded by a lysogenic phage
− B subunit serves as ligand for (binds to cell surface host
receptor (heparin-binding epidermal growth factor)
− A subunit inactivates elongation factor-2 (EF-2)
− One DTX molecule can inactivate all EF-2 within the cell
(interferes with cell translation)
− Cells die following uptake of DTX
Listeria
monocytogenes
(unique because
it’s an intercellular
pathogen)
Clinical Findings
• Diphtheria
− Begins as an “exudative pharyngitis”
o Sore throat, low-grade fever, malaise
− Exudate on tonsils, pharynx and larynx evolves into a grayish
“pseudomembrane” (necrotic epithelia embedded in fibrin, RBC
and WBC…active bacteria)
− Removal results in capillary damage and bleeding (removes
dead cells)
− Resolves within 1wk
− Complications:
o Breathing obstruction (dyspnea)
o Cardiac arrythmia
Coma (5-10% mortality)
Diagnosis
• Culture
− Swabs of nose and/or throat inoculated onto Blood agar
(eliminate Streptococcus) and Cystin-Tellurite agar  No
hemolysis on Blood agar and growth on Tellurite agar
− Tellurite inhibits growth of most URT microbes; tellurite is
reduced by C. diphtheriae producing black colony coloration
− PCR performed to confirm presence of DTX
Prevention & Treatment
• DTaP vaccine
Characteristics / Epidemiology / Pathogenesis
• Facultative Anaerobe; Motile; Psychrophile (growth at 4C)
• Found in intestinal tract of many animals (poultry, cattle,
sheep)
• 5-10% of humans harbor without symptoms
• Transmission – Contaminated food (unpasteurized milk,
cheeses, contaminated ice cream, raw vegetables, raw meat,
cold cuts) or Congenital to newborn (during labor/delivery)
• Internalin (protein used to get in)
− Adherence to enterocytes, M cells, phagocytes,
hepatocytes, fibroblasts (in GI tract)
− Induces uptake by cells (endosome or phagosome)
− Actin reorganization to move into other cells acquiring a
double membrane vacuole (unique)
•
Listeriolysin O
− Degrades endosome/phagosome/vacuole membranes to
escape into cytosol and avoid lysosomal fusion (prevents phagocytosis)
Clinical Findings
• Listeriosis
− Nausea, abdominal pain, watery diarrhea, fever
(self-limiting)
− Bacteria may disseminate to other sites (cellular
bacteremia)…sepsis
− Tropism for CNS – meningitis
− Fecal contamination at birth can transmit Listeria to
newborn…neonatal sepsis
Diagnosis
• Culture
− Blood, CSF specimen inoculated onto Mueller-Hinton agar with
added sheep RBC  Gram stain, Catalase test (positive),
evident small zone of hemolysis
− Can also inoculate PALCAM agar with food source