Download Micro: Lecture 17: Gram-Positive Bacteria Study Objectives •List

Micro: Lecture 17: Gram-Positive Bacteria
Study Objectives
•List the medically important Gram-positive bacteria.
•Discuss the classification of streptococci.
•Discuss the role of each virulence factor in disease.
•Describe the pathogenesis, epidemiology, diseases, diagnosis and treatment of each of the organisms.
•Identify the major characteristics of each organism (morphology, virulence factors, spore formation,
cell wall composition and any other unique characteristics…).
•Define PPD and discuss its significance in diagnosis of M. tuberculosis.
Pathogenic Gram-Positive Bacteria
•Coccus (round)
−Staphylococcus
−Streptococcus
•Bacillus (Rod)
−Bacillus
−Clostridium
−Corynebacterium
−Listeria
−Mycobacterium
Staphylococcus
Characteristics
•Facultative Anaerobes (can use O2); Nonmotile; Irregular clusters
•Grows best aerobically
•Found in soil, water and skin of humans
•Catalase-positive (Streptococcus are catalase-negative)
•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  Autoinfection (carrier), Direct contact (person with lesion),
Contaminated food (person with lesion) or Fomite – an inanimate object (Survive long periods of drying);
Coagulase-Negative  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
3 Medically Important Species of
Staphylococcus
•Staphylococcus aureus (Most common)
–Causes most Staph infections in humans
–Coagulase +
–Mannitol fermenter (Will turn yellow)
–Novobiocin sensitive
•Staphylococcus epidermidis
–Normal flora, an opportunisitc pathogen
–Coagulase –
–No mannitol fermentation
–Novobiocin sensitive
•Staphylococcus saprophyticus (Most opportunistic)
–Opportunistic, causes UTI
–Coagulase –Mannitol fermenter
–Novobiocin resistant
Differentiation of the Main Species of Staphylococci
Characteristic
S. aereus
S. epidermidis
S. saprophyticus
Color of Colonies
Often yellow
White
White to pale grey
Hemolysis
Most isolates
A few isolates
Non-hemolytic
Coagulase production
Yes
No
No
Mannitol fermentation
Yes
No
Yes
Novobiocin
Sensitive
Sensitive
Resistant
Staphylococcus
Pathogenesis
•Coagulase
−Initiates fibrin polymerization on surface of bacteria (protect from phagocytosis)
−Clumping factor permits binding to fibrinogen and fibrin
•Hyaluronidase / Staphylokinase (enzymes)
−Permits invasion of tissues and dissolves fibrin clots formed by coagulase, respectively
•Alpha-Toxin (a-toxin)
−Inserts into cell membranes resulting in lysis of cells (also a hemolysin)
•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
•Leukocidins (cytotoxin)
−Two-component toxins which lyse WBC by forming pores
•*Exfoliative Toxins A, B (detroy cell-cell adhesion)
−A = heat stable (resists boiling for 20min); B = heat labile
−Dissolve epidermal mucopolysaccharide matrix (desquamation); Also function as Superantigen
•*Toxic Shock Syndrome Toxin (TSST-1)
−Superantigen (can cause anaphylaxis) toxic shock syndrome; directly cytotoxic (?); In 20% of
isolates
•*Enterotoxins (multiple types – infects GI tract)
−Heat-stable Superantigens resistant to gut enzymes (just 25mg induces vomiting)
Virulence Determinants of S. aureus (Box A: use for cell adhesion; Protein A: Igs bind)
Staphylococcus aureus
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
−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)
−Antibiotics are not useful
Toxic Shock Syndrome Toxin-1 •A superantigen toxin that results in release
cytokines causing fever, capillary leakage, circulatory collapse and shock
•Superantigen toxins also prevent the development of a normal
immune response
of
Diagnosis
•Culture
−Pus/surface swab, blood, sputum specimen inoculation onto
Mannitol Salt agar (MSA)  Successful growth, Gram stain (all are
positive), 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
Prevention & Treatment
•To prevent recurrent infections remove contaminated fomites
•Use of soaps to reduce flora levels and antimicrobials to eliminate nasal carriage
•Prophylaxis prior to surgery reduces incidences of infection
•Methicillin-susceptible strain = Dicloxacillin
−Alt = A Cephalosporin, Vancomycin, Clindamycin
•MRSA = Vancomycin (+Gentamicin + Rifampin*)
−Alt = Linezolid, Daptomycin, Tigecycline, A Fluoroquinolone
−*Rifampin recommended if prosthetic valve or hardware present
Methicillin-Resistant 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
Streptococcus
Characteristics--------------------Alpha: no hemolyse; Beta: has hemolyse
•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
(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)
Main Pathogenic Groups of Streptococci
Characteristics Used to Differentiate Major Groups of Streptococci
Streptococcus pyogenes (GAS)
Pathogenesis
•M Protein / Lipoteichoic Acids / Protein F (on outside of bacteria)
−Adherence to nasopharynx and skin epithelia
−M protein and C5a peptidase (will degrade) block phagocytosis and PMN recruitment, respectively
•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
Group A Streptococcal Virulence
Factors
Exotoxins
Streptococcus pyogenes (GAS)
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)
•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)
Prevention & Treatment
•Penicillin G or V
−Alt = (if allergic) Erythromycin, A Cephalosporin, Clindamycin
•Treatment within 10d of symptoms onset prevents ARF (although does not prevent AGN)…goal is to
remove the Ag so as not to develop crossreactive Igs
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 (binds to host cell glycosaminoglycans or GAG and degrades C3b) to
prevent alternate pathway of complement activation
•As long as mom has the IgG against GBS, the newborn is protected by the classical pathway of
complement
•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)
•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
•Penicillin G
−Alt = Ampicillin, A Cephalosporin, Vancomycin
Streptococcus pneumoniae
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:
oDirectly cytotoxic to endothelial cells…(permits dissemination into the bloodstream)
oImpairs ciliary action (paralyzed)
oDirectly suppresses phagocytic activity
oSuppresses local inflammatory immune response ( anti-inflammatory cytokines)
oTriggers platelet activation (DIC can occur from concurrent vascular leakage due to endothelial damage
in lungs)
oPneumococcal pneumonia does not elicit extensive damage to the lung tissue as in other infections
such as influenza (local immunosuppression)
Streptococcus pneumoniae
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
oMost frequent cause with millions of cases/y
−Sinusitis
−Bacteremia  Endocarditis, Arthritis, Peritonitis
Diagnosis
•Microscopy
−Gram stain of sputum (bronchial lavage), CSF, blood  Gram-positive 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)
•Penicillin G or V (if susceptible)
−Alt = (if allergic) Erythromycin, A Cephalosporin
•Ceftriaxone (if intermediate resistance to penicillin G)
•Vancomycin (if high level of resistance to penicillin G)
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
Pathogenesis
•Protein Capsule
−Adherence to tissues (?)
−Effective blockade of phagocytic uptake
•Anthrax Toxin
−Protective Antigen (PA)
oBinds to ATR on host cell surface
oHost furin (membrane protease) cleaves PA into monomer …heptamerization of PA induces clustering
of ATR on lipid rafts…bind either EF or LF and are endocytosed into host cell
−Edema Factor (EF)
oEF functions as an adenylate cyclase to  cAMP
−Lethal Factor (LF)
oProtease that cleaves MAPKK pathway proteins and induces TNF secretion…induces cell death of
macrophages and endothelial cells
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 (Gram-positive bacillus)
−Saprophytic species are beta-hemolytic and motile (swarming)
Prevention & Treatment
•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)
−3 initial doses (2wks apart)…then 3 booster doses (6, 12, 18mo)
•Ciprofloxacin
−Alt = A Tetracycline, Penicillin G, Amoxicillin
Corynebacterium diphtheriae
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 – works when Iron is low! Then will produce the toxin!
−B subunit serves as ligand for
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
−Cells die following uptake of
DTX
Clinical Findings
•Diphtheria
−Begins as an “exudative pharyngitis”
oSore 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
−Resolves within 1wk
−Complications:
oBreathing obstruction (dyspnea)
oCardiac arrythmia
oComa (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
•Antimicrobials and Diphtheria Antitoxin
−Antitoxin given on day of diagnosis
•Erythromycin
−Alt = Penicillin G
Listeria monocytogenes
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
−Adherence to enterocytes, M cells, phagocytes,
hepatocytes, fibroblasts
−Induces uptake by cells (endosome or phagosome)
−Actin reorganization to move into other cells acquiring a
double membrane vacuole
•Listeriolysin O
−Degrades endosome/phagosome/vacuole membranes to
escape into cytosol and avoid lysosomal fusion
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
Prevention & Treatment
•Ampicillin