Staphylococci and Streptococci

1
Staphylococci and Streptococci
Medical Microbiology

• BIOL 533
• Lecture 10

2
Staphylococci

• Important human pathogen
• Causes both relatively minor and serious diseases
• One of the hardiest of the non-sporeforming
  bacteria
• Can exist on dry surfaces for a long period
• Relatively heat-resistant temperature range of
  18 - 40 C

3
Staphylococci

• Morphology
• Gram grape-like cluster, but in clinical
  specimens, can be a single cocci or diplococci
• General physiological characteristics
• Nonmotile
• Facultatively anaerobic
• Catalase
• Grows in media containing 10 NaCl

4
Staphylococci

• Relationship to disease (only 3 important)
• S. aureuscauses a number of diseases
• S. epidermidispresent in normal flora (normally
  benign, except when introduced via catheters,
  etc.)
• S. saprophyticuscauses uninary tract infections

5
Staphylococci

• Microbial physiology and structure
• Capsule may not be found growing on media, but it
  is usually present in vivo
• Teichoic acids are phosphate containing
  polysaccharides bound to both peptidoglycan and
  cytoplasmic membrane
• Species specific
• Poor immunogens, but when bound to peptidoglycan,
  get an antibody response

6
Pathogenesis of S. aureus

• Features typical of staphylococci infections
• Initial lesion is normally mild and localized
• Results in a boilnormally, it is self-limiting
• Can result in systemic infection

7
Pathogenesis of S. aureus

• Stage I encounterhumans are major reservoir for
  S. aureus
• Colonize nose and are found in about 30 of
  individuals
• Transiently found on skin, oropharynx, and feces
• Transmitted via
• Hand contact
• Aerosols from pneumonia patients

8
Pathogenesis of S. aureus

• Stage I, continued
• Certain occupations are more prone to
  colonization
• Physicians, nurses, hospital workers
• Certain classes of patients are more prone to
  colonization
• Diabetics, hemodialysis patients, and drug abusers

9
Pathogenesis of S. aureus

• Stage II entrynot normally through unbroken
  skin
• Can enter if large numbers have accumulated
  through poor hygiene

10
Pathogenesis of S. aureus

• Stage III spread and multiplication
• Survival depends on
• Number of organisms
• Site involved
• Speed with which inflammatory response is mounted
• Immunological competence of host
• If inoculum is small and host immunologically
  competent infection normally defeated

11
Pathogenesis of S. aureus

• Stage IV damage
• Local infection leads to formation of abscess
  (collection of pus)
• In skin, boils or furuncles
• Interconnected abscesses are called carbuncles
• May also spread in subcutaneous or submucosal
  tissuescellulitis

12
Pathogenesis of S. aureus

• Stage IV, continued
• Developmentinvolves both host and bacterial
  factors
• Acute inflammatory reaction
• Proportion of bacteria survive and are capable of
  lysing neutrophils that engulfed them
• Outpouring of lysosomal enzymes that damage
  surrounding tissues
• Inflammatory area surrounded by fibrin clot

13
Virulence Factors of S. aureus

• Stage IV, continued
• Virulence factorsmost designed to avoid
  phagocytosis or survive once ingested
• Wall components
• Surrounded by capsule not as effective as
  pneumococcus or meningococcus
• Cell wall murein activates complement by
  alternative pathway
• Teichoic acid also activates and involved in
  adherence
• Protein A interferes with opsonization by binding
  with Fc region of Abcomplement activated primary
  pathway

14
Virulence Factors of S. aureus

• Stage IV, continued
• Secretion of enzymes
• Catalasehydrogen peroxide to water and oxygen
  (all staphylococci produce)
• Coagulasemakes fibrin clot (wbc penetrate badly
  only S. aureus)
• Hylauronidasedegrades connective tissues
  (facilitates spread 90 of S. aureus strains)
• Fibrinolysin (staphylokinase)dissolve fibrin
  clots (virtually all S. aureus)

15
Virulence Factors of S. aureus

• Stage IV, continued
• Secretion of enzymes
• Lipasesrequired for invasion into cutaneous and
  subcutaneous tissues (found in all S. aureus and
  30 of others)
• Nucleaseheat stable (role is uncertain S.
  aureus)
• Penicillinase

16
Virulence Factors of S. aureus

• Stage IV, continued
• Secretion of toxins
• Cytolytic (membrane-damaging by pores)
• Alpha, beta, (sphingomyelinase C), delta, gamma,
  leukocidin (cannot lyse red blood cells)
• Others lyse rbc and leukocytes (referred to
  previously as hemolysins)
• Cause lysis of neutrophils leading to massive
  lysosomal enzyme secretion

17
Virulence Factors of S. aureus

• Stage IV, continued
• Secretion of toxins
• Exfoliative toxin (scalded skin syndrome)
  extrachromosomal
• Toxic Shock Syndrome toxin-1 (enterotoxin
  F)exotoxin secreted during growth
• Some produce enterotoxin B instead (role not
  clear)

18
Virulence Factors of S. aureus

• Stage IV, continued
• Secretion of toxins
• Enterotoxins (A-E)found in both S. aureus and
  S. epidermidis
• Resistant to hydrolysis by gastric and jejunal
  enzymes
• Stable to heating at 100C for 30 minutes
• Mechanism of toxin activity not understood no
  satisfactory animal model
• Stimulate intestinal peristalsis and have CNS
  effect intense vomiting

19
Pathogenesis of S. aureus

• Treatment
• Antibiotics
• Types
• Methicillin, oxacillin, nafcillin, and
  dicloxacillin (semisynthetic penicillins
  resistant to ß-lactam hydrolysis)
• Majority of patients can be treated, but 10-15
  S. aureus and 40 coagulase-negative
  staphylococci are resistant treat with
  vancomycin

20
Pathogenesis of S. aureus

• Treatment
• Antibiotics
• Resistance
• Plasmid-borne (hydrolysis of ß-lactam ring)
• Chromosomalchange in structure of
  penicillin-binding proteins

21
Streptococci

• Fermentative (oxygen tolerant) Gram cocci in
  chains
• Sensitive to penicillins
• Human reservoirpassed from person to person

22
Streptococci

• Properties of Lancefield Groups (CHO antigens
  on wallsee handout)
• Group A cross-reaction can lead to
• Rheumatic fever
• Glomerulonephritis

23
Streptococci

• Recent Group A Streptococcus virulence factors
• M-like proteinsbind IgM IgG (protease inhibitor)
  and alpha2 macroglobulin
• F proteinadherence to epithelial cells
• C5a peptidasedegrades C5A pyrogenic exotoxins
  previously called erythrogenic toxins

24
Staph and Strep Toxins

• S. aureus toxic shock TSST-1
• S. pyogenes toxic shock TSSL-1
• S. pyogenes scarlet fever SPE-1
  (children, not adults immunity)

25
Staph and Strep Toxins

• S. aureus Toxic Shock Syndrome
• Fever, diffuse rash
• Exfoliation of skin on palms and soles of feet
• Normally doesnt compete well in relatively
  anaerobic vaginal area

26
Staph and Strep Toxins

• S. aureus Toxic Shock Syndrome
• Super-absorbent tampon
• Created aerobic pockets
• Removed Mg?producing toxin
• After removed tampon, cases declined did not
  disappear
• Still associated with wounds, rare nasal surgery

27
Staph and Strep Toxins

• S. pyogenes Toxic Shock-Like Syndrome
• Skin or wound infection ---gt bloodstream
• Death rate 30 over 10-fold higher than TSST
• Seen in immunocompromised people
• Also other infections occurred soft tissue
  infection with influenza symptoms
• High fatality rate because rapid development of
  shock and multiple organ failure

28
Staph and Strep Toxins

• S. pyogenes Toxic Shock-Like Syndrome
• Features in common with scarlet fever
• Occur in healthy people
• Both associated with high fatality rate
• Produce same exotoxin streptococcal pyrogenic
  exotoxin (Spe)
• Similar in mechanism to TSST-1

29
Staph and Strep Toxins

• Comparing TSLS-1 and TSST-1
• Rash, fever, shock, multiple organ failure
  resemble endotoxin septic shock
• Both toxins superantigens
• Same mechanisms of action
• Limited similarity at amino acid sequence level

30
Staph and Strep Toxins

• TSLS-1 related to erythrogenic toxin (scarlet
  fever SpeA)
• Serotypes
• Spe ATSLS or invasive S. progenes
• Spe B
• Spe C
• Some strains dont produce Spe A, so Spe B or Spe
  C also has role

31
Staph and Strep Toxins

• How do TSST-1 and SPE cause shock and multiple
  organ failure?
• Hypotheses not mutually exclusive

32
Shock and Organ Failure

• First Hypothesis same as LPS triggering release
  cytokines IL1,TNF?
• Consistent with role as superantigen
• Promote association between macrophage and helper
  T cells?proliferation of T cells producing high
  IL2 level
• Secondarily produce IL1 TNF?
• Inject TSST-1 into rabbits elevated levels IL1
  TNF?

33
Shock and Organ Failure

• Second hypothesis increase bodys sensitivity to
  LPS consistent with
• Acts synergistically with LPS to amplify toxic
  effects in vitro and in animals
• Conceivablelow levels leaching into blood due to
  lysis of resident microflora
• Normally no effect
• Presence of Spe or TSST-1 causes an effect

34
Shock and Organ Failure

• Evidence to support role for LPS in TSST and TSLS
• Injecting TSST-1 or Spe is lethal to rabbits
• Injecting exfolatin and concanavalin A not lethal
  to rabbits
• Both elicit T cell proliferation, but dont
  enhance sensitivity to LPS

35
Shock and Organ Failure

• Evidence to support role for LPS in TSST and TSLS
• TSST-1 not lethal to gnotobiotic animals
• Wouldnt expect leakage, but still T cell
  response
• Therefore, both suggest T cell proliferation not
  as important as synergy of LPS
• Not conclusive difficult to prove same level T
  cell stimulation, proliferation occurred in all
  cases

36
Shock and Organ Failure

• Third hypothesis
• TSST-1 can act directly on endothelial cells
• Damage causes malfunction in circulatory system,
  which creates hypotension
• Data swelling associated with massive leakage of
  fluid from capillaries is marked symptom of both
  TSST and TSLS
• Could also be result of action of blood vessels
  by cytokines, coagulation, or complement cascade

37
Staph and Strep Toxins

• Mortality of S. pyogenes vs. S. aureus
• TSLS higher than TSS
• TSLS strains enter bloodstream
• TSS, only the toxin circulates
• S. pyogenes known to be invasive killed by PMNs
  and macrophage if ingested

38
S. pyogenes Invasiveness

• Strategies for evading phagocytosis (1)
• M protein binds H factor better than factor B
• Leads to degradation of C3b
• Therefore, prevents opsonization by C3b and
  formation of C3 convertase

39
S. pyogenes Invasiveness

• Strategies for evading phagocytosis
• Data supporting
• M mutants yield more susceptible to
  phagocytosis less virulent than wild type
• Ab against M protective
• 80 serotypes of M possibly evades host
  antibodies by changing serotype however, no data
  to support this hypothesis

40
S. pyogenes Invasiveness

• Strategies for evading phagocytosis (2)
• Protease cleaves C5a
• Chemoattractant stimulates oxidative burst
• Some activation of complement could occur in
  spite of M protein because
• Lysis releases wall components that activate
  complement
• Streptococci could protect themselves- C5a
  peptidase
• Data supporting
• C5a mutants less virulent that wild type in
  animals

41
S. pyogenes Invasiveness

• Strategies for evading phagocytosis (3)
• M like proteins
• Sequence and structural similarity to M
• COOH embedded NH2 exposed
• Most similar to M and each other at carboxy end
• These proteins bind Fc portion of IgG and IgA

42
S. pyogenes Invasiveness

• Strategies for evading phagocytosis
• M like proteins possible roles
• Coat with host proteinless likely detected as
  invader by complement and immune system
• Adherence for body cells that contain Ab on
  surface
• Also can bind host protease inhibitor such as ?2
  macroglogulin
• Host uses protease inhibitor to protect against
  proteases released by phagocytes

43
S. pyogenes Invasiveness

• Strategies for evading phagocytosis (4)
• F proteinbinds fibronectin
• Adherence of bacteria to tissues
• Evasion of immune system
• Summary of invasiveness
• No direct evidence M-like proteins involved in
  virulence
• Found in impetigo strains, not always in severe
  invasive strains
• Need mutant studies to answer questions

44
S. pyogenes Virulence

• Regulation of S. pyogenes virulence genes
• Expression M, C5a peptidase, and some M-like
  proteins regulated at transcriptional level
• Responds to CO2 levels
• Increased CO2 causes increased production

45
S. pyogenes Virulence

• Regulation of S. pyogenes virulence genes
• Regulatory gene
• mry transcriptional activator sequence analysis
  shows it is part of two-component system
• Sensornot found
• Activator
• Also known that speA gene on temperate phage

46
S. pyogenes Pathogenesis

• Treatment and prevention
• TSST, TSLS are medical emergencies
• Surgical debridement of wounds prevents further
  production of toxin
• Antibiotics penicillin
• Toxic effects TSST-1 countered by intravenous
  rehydration counter hypotension

47
Streptococcal Treatment

• Prevention
• Vaccine possible
• Target against M
• Possible problems
• serotypes, but severe invasive disease caused
  by few
• AB against M cross-reacts with heart

48
Streptococcal Sequellae Hypotheses

• First Autoimmune theory
• Epitopes that cross react with epitopes on
  cardiac myosin and sarcolemmal membrane proteins
• Thus, T cells or antibodies could attack tissue
• Inflammatory response damages heart valves

49
Streptococcal Sequellae Hypotheses

• Glomerulonephritis
• High levels Ab to streptococcal Ag circulating in
  blood stream causes AgAb complexes to accumulate
  in kidney
• Inflammatory response attacks kidney interfering
  with kidney function

50
Streptococcal Sequellae Hypotheses

• Data supporting
• Ag-Ab complexes visible in people with
  glomerulonepheritisglomeruli
• Decrease in C3 and other complement components
  also seen supports hypothesis that inflammatory
  response is occurring
• Second Toxins cause sequellae

51
Streptococcal Sequellae Hypotheses

• Main argument against
• Time lag between initial infection and
  development of rheumatic fever (RF several
  weeks) or glomerulonephritis (10 days)
• Normally, if due to toxin, within a week
• Candidates for toxin most likely to cause
  glomerulonephritis streptococcal O,
  streptokinase, or Spe

52
Glomerulonephritis Hypotheses

• Streptococcal O cytotoxin
• Mechanism and aa sequence similarity to
  pneumolysin
• Pore-forming toxin
• Injected into lab animals damages heart
• Therefore, may have role in RF
• Also, very immunogenic maybe Ab damage

53
Glomerulonephritis Hypotheses

• Streptokinase
• Plasminogen?plasmin
• Therefore causes symptoms similar to
  glomerulonephritis in animals
• Interesting, but not proven

54
Rheumatic Fever Hypotheses

• Spe
• RF strains produce Spe others dont
• Enhances cardiotoxicity caused by Streptococcal O
  in animals
• Havent explained long time lag

55
Rheumatic Fever Hypotheses

• Mysterious feature of RF unexplained
• Treated with antibiotics for as late as 9 days
  after symptoms, still protected against RF
• After 9 days, toxic products should be
  circulating and immune response underway
• Recurrence of disease
• Normally, infection results from different strain
• Some result from same strain
• RF symptoms take as long to develop as in original

56
Rheumatic Fever Hypotheses

• Mysterious feature of RF unexplained
• If caused by autoimmune response, would expect
  faster response
• Possible explanation previously exposed produces
  primed immune system

57
Streptococcal Sequellae

• Treatment and prevention
• Strep throat self-limiting
• Treat with antibiotics and prevent RF and
  glomerulonephritis
• Only ¼ S. pyogenes strains cause RF or G
• Not all people colonized actually contract RF or
  G
• Because of the seriousness, treat any strain with
  antibiotics

58
Streptococcal Sequellae

• Tests
• Blood agar
• Hemolysis
• Bacitracin sensitivity
• Rapid test and culture test both yield high
  number of false negatives
• For patients who are allergic to penicillin, use
  erythromycin

59
Streptococcal Sequellae

• Previous damage
• Even heart murmur
• Dentists recommend prophylactic penicillin before
  dental work
• Kill bacteria escaping into blood stream from
  mouth (oral bacteria are susceptible to
  penicillin)
• Reduces chance of colonization

60
Staphylococcal Enterotoxins

• Normal enterotoxins cause diarrhea
• Water loss from small intestine mucosa
• Cause c-GMP or c-AMP levels in mucosal cells to
  rise

61
Staphylococcal Enterotoxins

• Staph toxins operate in a different mode
  hypotheses include
• 1) Stimulates vagus nerve endings in stomach
  lining that control emetic (vomiting) response
• If hypothesis correct, its a neurotoxin, not
  enterotoxin
• 2) Superantigen?IL2
• Administering IL2 to human volunteers produces
  many of the same symptoms (nausea, vomiting,
  fever, malaise)

62
Staphylococcal Enterotoxins

• Neither hypothesis conclusively proven
• Could be a combination of both
• Seven serotypes SEA, SEB, SEC1, SEC2, SEC3, SED,
  SEE
• 30 kDa proteins share considerable aa similarity
  and single internal S-S

63
Staphylococcal Enterotoxins

• SE closely related to Spe
• Genes coding for SEC1 and Spe A have 60
  nucleotide identity
• SE produced in different amounts by different
  strains
• entA produces much less toxin than entB
• Differences in promoter strength

64
Staphylococcal Enterotoxins

• SEB on integrated plasmid
• SEA on lysogenized phage
• One strain entB, entC, plasmid-borne
• SED entD on 27.6 kb plasmid

65
Lecture 10

• Questions?
• Comments?
• Assignments...