Salmonella

1
Salmonella

• Salmonella infect all types of domestic animals
• One of the most prevalent agents causing
  food-borne disease-salmonellosis
• Classification is based on serology and phage
  susceptibility assays
• More than 2600 serovars
• S. enterica is divided into seven subspecies with
  group I causing disease in humans and other
  warm-blooded animals
• Examples Typhimurium, Enteritidis, Choleraesuis,
  Dublin, Gallinarum, and Pullorum

2
Salmonella

• Salmonella are gram (-) motile rods
• Facultative aerobes
• Salmonella grow at an optimum of 37oC
• Resistant to heat if in foods with lower pH, or
  low water activity or in food with a high fat
  content
• Viability declines during frozen storage,
  especially if near the freezing point
• Optimum pH for growth of Salmonella is 6.5-7.5
  may grow at a pH range of 4.5-9.0
• Succinic and formic acid reduce the persistence
  of Salmonella in foods

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Salmonella Prevalence

• Factors which favor the continued presence in the
  food chain
• Ubiquity of salmonella in the environment
• Intensive husbandry practices in the meat, fish,
  and shellfish industries
• Lack of microbiological control on animal feeds
  favors the continued prevalence of this pathogen
  in the food chain
• Poultry products remain the main reservoir of
  salmonella
• Eggs and egg products are also a concern since
  there is Transovarian transmission of the
  pathogen into the interior of the egg

4
Salmonella Prevalence

• Developed countries more than 80 of the
  Salmonella cases occur individually rather than
  in outbreaks
• Large Salmonella Outbreaks
• Foods linked to transmission include milk powder,
  raw milk, cheddar cheese, egg products, and liver
  pate
• Some fruits and vegetables have also been linked
  to outbreaks-cantaloupes, chocolate, mustard
  dressing
• Infectious dose will vary, as few as 1 to 10
  cells
• Foods with a high fat content may have a low
  infectious dose due to the organism being trapped
  in micelles which protect it against acidic ph

5
Salmonella Prevalence

• Food-borne infections account for 1.3 billion
  cases of acute diarrhea with 3 million deaths
  world-wide
• Salmonellosis in the U.S. is 40,000 cases
  annually
• Recent years a notable increase in cases related
  to a multi-drug resistant S. typhimurium DT104
• Case-fatality and hospitalization rates due to
  this strain are twice that of other Salmonella
  infections

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Salmonella Pathogenesis

• Disease
• Disease depends on age of the host-more severe in
  newborns, infants, the elderly
• Disease also depends on the serovar and type of
  host
• Host specific serovars typhi, paratyphi, sendai
  cause disease only in humans
• Pullorum/gallinarum in poultry, dublin in cattle
  choleraesuis serovars in pigs can also infect
  humans typhimurium and enteritidis are the major
  serovars that cause disease in humans, cattle,
  poultry, sheep, pigs, horses, and wild rodents.

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Salmonellosis

• Salmonellosis
• Diarrhea, abdominal pain, vomiting and fever
• Bacteremia has been associated with highly
  invasive serovars such as choleraesuis or dublin
• Other clinical sequels include erythrema nodosum,
  meningitis, osteomielitis, septic arthritis,
  pnuemonia, choleocydtitis, endocarditis,
  pericarditis, and cystitis
• A complication of infection is the establishment
  of a carrier state which lasts for 5-6 months.
• Carrier state is responsible for the spread of
  the bacterium to others, farms, food-handlers,
  and consumers.

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Salmonella Invasion

• Salmonella passes through the stomach and
  survives the acid pH by eliciting an acid
  tolerance response
• Colonizes the distal ileum portal of entry is
  the M-cell and it binds by means of fimbriae from
  the lpf operon or from the pcf operon
• Heritable phase variation mechanism
• Triggered by intestinal-derived signal

9
Salmonella Invasion

• Penetration and destruction of M-cell
• Localized accumulation of cytoskeletal proteins
  such as actin, vinculin, vimentin, and ezrin at
  site of entry
• Formation of membrane ruffles occurs with every
  cell type examined
• Recruitment of lymphoid cells and PMN
• Secretion of large amount of fluid
• Leads to inflammation of the area

10
Salmonella Invasion

• Colonizes intestinal epithelial cells
• In vivo
• Microvilli denuded transiently, membrane ruffling
• Bacterial penetration 1-2 h
• Cytosketetal rearrangements, 30-60 min
• Actin filament rearrangement
• No microtubule involvement
• Ca flux
• In vitro
• Distruption of tight junctions
• Causes depolarization of epithelial cells

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Salmonella Invasion

• Pathogenecity island of 40kb has been identified
  which also contains a type III secretion system
• SPI operon encoded type III secretion system
• InvA-C operon
• Functional filamentous appendages appear upon
  cell contact and require a function type III
  secretion system
• Activated by a shift from acidic to alkaline pH
  in the absence of eukaryotic cells also
  activated by environmental conditions such as
  high osmolarity, low oxygen tension, DNA
  supercoiling, and alkaline pH
• More than 15 proteins are inserted into the inner
  and outer membrane. Classes inner and outer
  membrane proteins InvA, SpaP, SpaQ, SpaR, SpaS,
  InvG, InvH, PrgH, PrgK an energizer of the
  system, the inner membrane ATPase InvC secreted
  proteins with putative targets in the host cell
  known as effector proteins SipB, SipC, SptP,
  AvrA other secreted proteins SipA cytosolic
  chaperons which prevent premature degradation of
  secreted proteins SicA, SicP transcriptional
  regulatory proteins InvF, HilA, and InvE

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Salmonella Invasion

• Other Invasion Factors
• Motility is needed
• Intact LPS is also required for invasion
• Fimbriae are also needed
• Host Factors for Invasions
• Subversion of host cell essential functions
• Actin cytoskeleton reorganization
• Membrane ruffling
• Signal transduciton pathways
• SopE (substances of the SpI-1 encoded system)
  stimultes actin reorganization by increasing
  GDP/GTP nucleotide exchange in several GTPases
• Stimulates production of proinflammatory
  cytokines as IL-8, and activates transcriptional
  factors

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Proliferation within Nonprofessional Phagocytes

• Salmonella proliferates in membrane-bound
  vacuoles
• Salmonella containing vacuole fuses with host
  compartments containing lysosomal-membrane
  glycoproteins and bypasses compartments of the
  endocytic route
• SPI-2 is required for intracellular proliferation
  within epithelial cells
• Recent findings suggests that macrophages and
  specialized epithelial cells allow Salmonella to
  proliferate intracellularly

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Proliferation within Nonprofessional Phagocytes

• Although non-growing, remain viable for periods
  of 10-14 days
• Salmonella have been shown to reduce their
  metabolic rate to perpetuate with the host cell
• The PhoPQ system is involved in preventing growth
  of intracellualr Salmonella phoP mutants grow
  actively in non-permissive cells
• First example of a bacterial-mediated mechanism
  that prevents its own proliferation in a specific
  host cell
• Long persistance of Salmonella within
  non-phagocytic cells could be linked to
  Salmonella derived reactive arthirits, chronic
  infections, or carrier asymptomatic

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Proliferation within Professional Phagocytes

• Active membrane ruffling and macropinocytosis
  occurs upon ingestion of Salmonella by phagocytic
  ells
• Trafficking route used by Salmonella within a
  cultured macrophage is different than within
  epithelial cells
• Fusion of the endosome with mature lysosomes
  seems to occur
• Nevertheless that compartment does not behave as
  a conventional mature lysosomal since there is no
  processing of lysosomal enzymes contained within
• Acidification triggers bacterial response for
  survival and replication within these cells
• Salmonella may exist within two populations one
  activity growing and the other static but not
  viable

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Proliferation within Professional Phagocytes

• Salmonella is also able to trigger apoptosis of
  the infected macrophage
• Products involved in triggering apoptosis are
  SPI-1 oxygen regulated protein (OrgA), OmpR/EnvZ,
  SPI-1 secreted proteins such as InvJ, SpaO, SipB,
  SipC, and SipD
• Induced apoptosis may destroy phagocytic cells
  before they process antigen and activate T-cells
• Macrophage may react to infection with Salmonella
  and program its own death to avoid excessive
  release of immune mediators

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Survival within Phagocytic cells

• Proteins involved in DNA repair are needed such
  as RecA and RecBC for survival
• Increased levels of SodA (Mn dependent) results
  in increase survival within macrophages in vitro
  but not for survival in the mouse model
• Increased levels ot SodC (Zn2-Cu2) results in
  increased survival in the mouse model but not in
  macrophages in vitro
• SlyA is a transcriptional regulator involved in
  Salmonella survival by giving it resistance to
  toxic oxidative products
• Sap proteins are required for antimicrobial
  peptide resistance

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Salmonellosis

• PhoP-PhoQ regulatory System
• Two component systempair of proteins, one of
  which, the sensor, underoges a change in response
  to a change in the environment and communicates
  this change, usually in the form of a phosphate
  to another protien, response regulator, which
  then causes the appropriate cellular response
• Sensing extracellular signals such as limiting
  amounts of Mg2 and mild acidic pH

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Salmonellosis

• PhoQ is a inner membrane integral protein capable
  of binding Mg2 and Ca2
• Upon binding PhoQ auto-phosphorylates and
  transfers the phosphate to PhoP, a cytosolic
  protein
• Phosphorylated PhoP acts then as a
  transcriptional regulator or at least 40 genes
• Some virulence genes are activated as pag and
  others repressed such as prg
• Virulence traits linked to this regulatory system
  include(1) bacterial survival within
  macrophages, epithelial cell (2) resistance to
  cationic antimicrobial peptides, (3) invasion of
  epithelial cells, (4) control of Ag presentation
  by bacteria-infected macrophage, (5) resistance
  to bile acids

20
Salmonellosis

• PmrAB regulatory system
• Two component system
• Controls bacterial resistance to polymixin and
  other LPS-binding antimicrobial peptides
• Auto-regulated
• Expression of these genes is dependent on low pH
  or Mg2 limitation

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Salmonellosis

• OmpR/EnvZ regulatory system
• Two component system
• Senses changes in external medium osmolarity
• OmpR is the transcriptional regulator
• EnvZ is its cognate membrane sensor
• Controls expression of major outer membrane
  proteinsOmpC and OmpF

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Salmonellosis

• High osmolarity favors expression of OmpC while
  low osmolarity shifts the expression to PmpF
• OmpF mutants are avirulent in the mouse typhoid
  model
• OmpCF mutants are attenuated in the in vivo model
  when administered orally but not
  interperiotoneally

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Salmonellosis

• RpoS (katF) Sigma factor
• Important regulatory factor in starved bacteria
• High levels of this protein in cells that are
  reaching stationary phase
• Needed for survival in stationary phase
• Needed also for nutrient stresses in Salmonella
  such as carbon, nitrogen, or phosphate
  starvations
• Increased in intracellular bacteria
• Required for virulence

24
Salmonellosis

• Roles of a Salmonella virulence plasmid (60-80
  kb)
• Needed for the production of systemic disease
  cured strains are unable to produce systemic
  disease in the mouse typhoid system
• Mutants retain capacity for colonization the
  intestinal tract and spreading to target tissues
  and organs
• Spv operon
• Under control of the RpoS sigma factor
• Protein play a role in signaling between
  intracellular Salmonella and the host cell
• Only synthesized in intracellular bacteria