Legionella

1
Legionella

• Acute pneumonia, first epidemic in 1976 (when an
  elderly group of men were attending an American
  Legion convention, many of them smoker or
  drinkers or both),1997 cases of Legionella
  infection in Houston area
• L. pneumophila (fever, disorientation, lethargy,
  lung damage cytoplamic contents of lysed
  phagocytic cells contribute to fluid seen in the
  air sacs), L. micdadei (mild form, called Pontiac
  fever after the city in Michigan symptoms of
  viral flu)
• Facultative intracellular parasite,
  gram-negative, require high levels of cysteine
  and iron, multiply freely in alveolar macrophages
• Prevents phagosomal lysosomal fusion
• Coiling phagocytosis

2
Legionella

• Incidence of Legionella pneumonia increased
  dramatically recently because of the installation
  of central air-conditioning systems in hotels,
  buildings, hospitals
• The organism is found in water and soil and
  particularly adapted to grow in air-conditioning
  cooling towers
• Aerosols produced by the air-conditioning
  machinery are inhaled introduce bacteria into
  the lungs
• Devices used in hospitals for inhalation therapy
  (water is used to humidify the air that is pumped
  into lungs orgms escape upper airway defenses
  and many patients are immunocompromised) , dust
  liquid aerosols from construction sites also lead
  to Legionella outbreaks
• People with impaired defenses (heavy alcohol use,
  smoking, old age) are prone of infection
• 25,000 30,000 cases/yr (CDC) 85 infections due
  to L.. pneumophila

3
Who gets legionellosis

• People of any age, however, most often
  middle-aged and older persons, particularly who
  smoke or have chronic lung disease
• People with suppressed immune system cancer,
  kidney failure requiring dialysis, diabetes, AIDS
• Drugs that suppress the immune system are also at
  higher risk

4
Legionella Pathogenesis

• Pathogenesis of Legionella infections begins with
  contaminated water supply
• Alveolar mfs engulf the bacteria, the organism
  being facultative intracellular parasite,
  multiplies freely in mfs
• Eventually, the mfs are destroyed, releasing new
  generation of microbes to infect other cells
• Bacterial growth death of mfs produce powerful
  chemotactic factors that elicit an influx of
  monocytes neutrophils
• Leaky capillaries allow transduction of serum
  deposition of fibrin in the alveoli resulting in
  destructive pneumonia

5
Legionella Pathogenesis

• The area of damage in lungs is not localized as
  in other pneumonias
• The damage spreads throughout the lung to give
  diffuse patchy densities in lung radiograms
• Compared to other pneumonias that spread from
  person to person, legionnaires disease spreads
  only through water

6
Animal models of legionnaires

• No good animal model as it causes disease in
  humans, particularly those with
  immuno-suppression
• Guinea pigs given aerosols of bacteria produce
  similar lung lesions but these animals are not
  immunocompromised
• A/J mice lacking neuronal apoptosis inhibitory
  protein, that inhibits apoptosis, are more
  susceptible to infection
• Macrophages, CHO cells are used in vitro

7
Extracellular Products of L. pneumophila

• Protease
• Hemolytic activity
• LPS

8
Legionella Virulence Factors

• 40 kDa metalloprotease with hemolytic activity
• Inactivates enzymatically, IL-2 CD4 receptors
  on T cells, thus inhibits T cell activation
• Antibodies to metalloprotease are protective in
  animals (e.g, guinea pigs) this enzyme when
  given intranasanally or intratracheally into
  lungs produces similar lesions as seen with
  Legionella
• Protease exhibits homology with elastase of
  Pseudomonas involved in CF

9
Legionella Virulence Factors

• Phospholipase A and C have destructive effects on
  lungs, the latter could hydrolyze phospatidyl
  choline in eukaryotic membranes
• These enzymes could destroy lung surfactant, the
  lipid that reduces surface tension experienced by
  vacuolar membranes, making it easier to refill
  emptied air sacs
• Destruction of surfactant causes the alveoli to
  collapse when the air is released, and can not be
  filled again

10
Legionella Virulence Factors

• L. micdadei PK II kinase is dependent on cAMP
  and stimulated with the eukayotic Ca2-binding
  protein calmodulin and by phosphatidyl inositol
  (a second messenger molecule in eukaryotes)
• PK I is not stimulated by these eukaryotic
  factors but uses ATP to phosphorylate tubulin in
  PMNs and phosphatidyl inositol
• These kinases could affect regulation of the
  phagocytic functions

11
Phagocytosis by Legionella

• Coiling phagocytosis
• A long pseudopod is formed by mFs, and the
  bacterium is engulfed in a coiled vesicle
• The vesicle does not acidify, phagolysosome
  fusion is inhibited, and bacteria divide within
  vesicles
• Eventually the vesicles surrounded by the ER are
  studded with ribosomes
• LAMP-1 and 2 (required for phagosomal-lysosomal
  fusion) are missing from late-stage phagosomes
  containing virulent bacteria. Present when
  phagosomes contain dead or avirulent bacteria.

12
Life cycle of L. pneumophila in macrophages
13
Invasion by Legionella

• L. pneumophila has a 24-kDa OMP Mip (macrophage
  invasion protein) that helps in invasion in the
  absence of opsonization
• Mutation in gene encoding Mip reduces invasion
  and also increases LD50 in guinea pigs
• Mip does not play any role in intracellular
  survival of bacteria once invaded
• Mip is a peptidyl prolyl isomerase enzyme found
  in eukaryotes and probably targets some host
  proteins

14
Genes for intracellular survival of Legionella

• Mutants that do not prevent phago-lysosomal
  fusion but elicit ribosome recruitment
• Mutants that do not inhibit phagolysosomal fusion
  and also do not recruit ribosomes have been
  isolated
• A 8-kb region on the chromosome (dot defect in
  organelle trafficking) complements these mutants
• Genes involved in intracellular survival ((icm
  intracellular multiplication) are localized in
  this region some of the genes appear to encode
  type IV secretion system

15
Type IV system in L. pneumophila
16
Legionella Virulence Factors required for
infection

• Some mutations affect virulence in amoebae but
  not in mammalian cells and vice versa
• Afimbrial adhesin involved in attachment and
  invasion of amoebae, not required in human
  phagocytic cells
• Mip required for both
• Mil (macrophage infectivity locus) in human and
  animal macrophages but not in parasites
• Association between bacteria and protozoa could
  indicate that bacterial virulence factors could
  be designed to kill protozoa

17
Virulence Factors required to infect amoebae or
macrophages
18
Regulation of the virulence genes

• At 30C, pili, flagella, and type IV secretion
  system preferentially expressed, could be a heat
  shock stress response
• In vacuoles concentration of thymine and
  thymidine low, the stringent response system
  senses starvation for amino acids or other
  compounds and responds by making ppGpp
• ppGpp signals to activate expression of virulence
  genes

19
Regulation of the virulence genes
20
Legionella (survival in the environment)

• Organism parasitizes amoebae Acanthamoeba spp.
  Naegleria spp., Hartmanella which are main
  reservoir of Legionella orgm uses protozoa as
  food
• Organism in amoebae more resistant than
  free-living bacteria to biocides (Kathaon CG
  chlorine) and are more virulent thus preparing
  Legionella to infect human macrophages
• Inside amoebae, organism undergoes changes in
  membrane lipid content, changes in protein
  profile, which give resistance to bacteria
• Acanthamoeba form cysts
• Organisms in the cysts are protected from
  biocides, heat, other environment

21
Legionella (treatment prevention)

• Erythromycin effective in vitro patients also
  given rifampin
• Does this antibiotic penetrate phagocytic cells
  effectively?
• Eliminate organisms from water supplies such as
  cooling towers that produce aerosols
• Resistance of bacteria inside amoebae biofilms
  to biocides
• Hot water (60C) flushed through pipes UV light
  kill Legionella
• Expensive
• No vaccine
• Because bacteria grow in mfs, a protective
  response would involve activation of mfs
  recruitment of cytotoxic T cells that will kill
  bacterium-infected mfs

22
Prevention of legionnaires Disease

• Chlorine dioxide (ClO2)
• Biofilm in the piping can protect Legionella from
  most disinfectant, ClO2 removes biofilm
• The bactericidal efficiency is unaffected by pH
  values between 4-10
• The required contact time is lower
• ClO2 has better solubility
• No distinct smell

23
Haemophilus

• Type b H. influenzae
• Meningitis
• 6 months 2 years
• Uncapsulated strains
• Otitis media in babies young children
• Bronchitis pneumonia in adults
• H. ducreyi
• Chancroid, a venereal disease
• Requirement for hemin (X) NAD (V) factor
• Capsulated strains penetrate nasoparyngeal
  epithelium capillary endothelium, reaching
  meninges either directly via lymphatic drainage
  or indirectly by causing bacteremia

24
Haemophilus

• Other effects of type b infection
• Deafness
• Learning disabilities
• Day care centers important sources of
  dissemination
• Organism multiplies rapidly in blood, 2 x 107
  108/ml in 24-48 hours
• Antibiotics can lead to septic shock due to
  LPS-induced cytokine response. Corticosteroids
  are given with antibiotics to reduce inflammatory
  response
• Many type b strains penicillin ampicillin
  resistant
• Ceftriaxone effective in controlling type b H.
  influenzae

25
Hemophilus Infections

• Found in the nasal secretions of 90 of healthy
  individuals infections characterized by acute
  inflammation with a discharge
• Any organ system could be affected but most
  common infection in the respiratory tract
• Person-to-person contact, contact with nasal
  discharge and other body fluids, late winter or
  early spring in USA

26
Hemophilus Infections

• Before vaccine was developed, 50 of children
  would acquire infection before reaching one yr of
  age almost all children would develop one before
  3 yr of age
• H. influenzae penumomia common in adults
  (nosocomial infection)
• Rate of Hemophilus infections in adults increased
  over the past 40 yr
• Due to overuse of antibiotics resulting in
  drug-resistant strains

27
Hemophilus infection

• Organism spreads from the nasal tissues and upper
  airway to the chest, throat, or middle ear
• Meningitis, infectious arthritis, conjunctivitis,
  cellulitis, epiglottis, or inflammation of the
  membrane surrounding the heart could occur
• Before vaccination Hib was the most common cause
  of meningitis and most cases of acquired mental
  retardation in US because of Hib

28
Haemophilus Influenzae InfectionsClinical
Manifestations
29
Hemophilus infection

• Bacterial sepsis fatal illness in newborn infants
• Disease acquired through mothers birth canal, or
  from the hospital environment
• Inflammation of the eye in newborn
• Signs fever, crankiness, feeding problem,
  breathing difficulties, pale skin
• Premature birth is the most significant risk
  factor in newborns

30
People at risk to develop Hemophilus infection

• Smoking
• Alcoholism
• Chronic lung disease
• Old age
• Living in a city with a large group of people
• Poor nutrition and hygiene
• HIV infection, or other immune system disorder

31
Causative Agents of Meningitis and the Frequency
with Which They are Associated with Disease
Frequencya (percent)
Agent

• Hemophilus influenzae
• Neisseria meningitidis
• Streptococcus pneumoniae
• Escherichia coli
• Other species

• 40 50
• 20 25
• 20 25
• 4 5
• 3 - 5

a The values are approximate and vary from year
to year as well as from one community to
another. b In children under 4 years of age H.
influenzae accounts for over 65 percent of the
cases of meningitis.
32
Haemophilus Influenzae

• Vaccine to capsular material conjugated with
  diphtheria toxoid
• Immunization at the age of 2, 4, 6, and 15 months
• Detection of the capsular material in the
  cerebrospinal fluid
• Growth on Mueller-Hinton 5 sheep blood, growth
  in CO2

33
Risk of Hemophilus vaccine

• Relationship between vaccination and development
  of insulin dependent diabetes
• In Finnish children, followed for 10 yrs
  children receiving 4 doses of vaccine, rate of
  diabetes elevated by 26
• This is equivalent to 2300 cases of diabetes a yr
  in US, which has an annual birth rate of 4
  million children
• Immunization starting in the first month of life
  has a decreased risk of diabetes and is one
  method being considered to make immunization safer