Human Pathogens in Plant Rhizosphere, Phyllosphere, and Vascular Tissues

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Human Pathogens in Plant Rhizosphere,
Phyllosphere, and Vascular Tissues
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Bugs in vegetables

• Greens, sprouts, vegetables, fruits, nuts were
  linked to Salmonella and E. coli O157H7
  outbreaks
• In 1990-2003, 28,315 cases of gastroenteritis
  were from produce (the second most common vehicle
  of foodborne illnesses)
• Early outbreaks were suspected to have resulted
  from cross-contamination with meats by consumers.
  Likely not the case
• Since 1995, 19 E.coli O157H7 outbreaks from
  spinach or lettuce, 9 linked to Salinas valley
• Easy removal from the surface, not from the inner
  parts
• Where are the bugs coming from?
• field/pre-harvest
• immediately post-harvest
• warm tomatoes constrict in cool water, suck up
  bugs thru the stem scar
• CDC wash picked vegetables in warm chlorinated
  water (200 ppm)
• dicing pooling problem
• shipment
• storage (flies, fruit flies transfer bacteria)
• gamma irradiation

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Bugs in vegetables

• E.coli and Salmonella can persist in manure for
  weeks-years
• Bugs from manure colonize veg. surface and
  persist for up to 2 mo

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Most common bacteria causing produce-related
outbreaks

• Salmonella
• caused 48 of all produce-related outbreaks in
  1973-1997
• E.coli O157H7 and Shigella
• E.coli O157H7 was linked to 21 of
  produce-related outbreaks in 1982-2002
• Others
• Campylobacter
• Listeria monocytogenes
• Staphylococcus aureus
• Yersinia spp
• Bacillus cereus

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Factors involved in the emergence of
produce-linked outbreaks

• Changes in the produce industry
• Intensification, centralization of production
• Wider distribution
• Introduction of minimally processed
• Increased importation of produce
• Changes in consumer habits
• Increased consumption of meals outside the home
• Salad bars
• Increased consumption of produce
• Increased size of at-risk population
• Enhanced epidemiological surveillance
• Improved methods for tracking/detection
• Emerging pathogens with low infectious dose

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Soil
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• Rhizosphere. Root zone. A very narrow zone (a
  few mm) around the root. Contains root exudates
  and microbes.
• Phyllosphere. Leaf surface as a habitat for
  microbes
• rhizosphere is a relatively stable, nutrient-rich
  environment. Phyllosphere is an extreme
  environment
• Epiphytes. Microbes that reside in the
  phyllosphere
• Endophytes. Live within plant tissues
  intracellularly
• typically commensals or symbionts
• many strains of Klebsiella are also co-evolved
  N-fixing endophytic symbionts of grasses. Same
  Klebsiella strains are fully virulent in animals
• Salmonella, E.coli are now recognized as
  commensal endophytes
• Plants may have evolved to promote colonization
  of edible plant parts with endo- and epiphytes as
  a defense against herbivory

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Enterics in plants

• Klebsiella, Enterobacter, Salmonella, E.coli are
  often isolated from plants in pristine
  environments.
• Therefore they have not necessarily originated
  from animal discharge

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Rhizosphere
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Human pathogens colonize rhizosphere and/or
phyllosphere
Growth of S. enterica serovar Newport (open
symbols) and E. coli O157H7 (shaded symbols) on
A. thaliana shoots (triangles) and roots
(circles).
Colonization of A. thaliana leaves and roots by
GFP-labeled E. coli O157H7 localized in small
depressions (A) and over veins (B). Chloroplast
autofluorescence is colored red. Initial
colonization of roots occurs at root tips (C) and
the branch points of lateral roots (D). Red
indicates either vascular autofluorescence or
TO-PRO-3 staining of plant cells
From Cooley et al., 2003
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Steps in plant colonization by human pathogens

• Attachment
• Aggregative fimbriae are involved. Similarly to
  their role in attachment to animal cells
• Adhesin proteins from plant pathogenic
  enterobacteria Erwinia are evolutionarily related
  to hemaglutinins in animal pathogen
• Growth
• Enterics can utilize plant sugars. Yersinia,
  Klebsiella can also use pectin, a plant polymer.
  E. coli and Salmonella cant use pectin or
  sucrose (major plant carbohydrates).
  Campylobacter mostly grows off organic and amino
  acids, not a big problem in produce
• Temperature and surface wetness may prevent human
  pathogens from efficiently competing with other
  epiphytes
• Colonize plants at sites where more nutrients are
  available (stomata on leaves, root tip and
  cracks)
• Survival on surfaces
• Internalization

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Steps in plant colonization by human pathogens

• Attachment
• Growth
• Survival on surfaces
• In the field, E. coli O157H7 and Salmonella can
  persist on/in lettuce leaves for at least 6 mo
• Water availability. Especially in phyllosphere
  and dry seeds. Salmonella has been isolated from
  dried nuts
• Aggregation (biofilms) is the key to survival
• Internalization
• The goal is to get into edible plant parts to
  find a new animal host
• Because Salmonella, E. coli cant degrade pectin,
  cellulose they can only enter through stomata,
  wounds or rots caused by plant pathogens.
  Salmonella can infect tomatoes through flowers.
• Other pathogens (those that can degrade plant
  cell walls) may be able to infect plants
  directly. Not clear.

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Abiotic pressures in phyllosphere

• Frequent and extreme changes in
• Dryness/wetness
• strong UV irradiation
• Not many nutrients
• However, nutrient-rich oases
• exist on leaf surfaces
• Compare these conditions to intestines!

Bacterial microcolonies on leaf surface (from
Leveau and Lindow, 2001)
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Biotic interactions in rhizo-, phyllosphere

• Interactions with native microflora
• Competition pathogens compete with other
  microbes for nutrients, space, Fe, antibiotics.
• Disinfection of plant surfaces may leave a void
  for pathogens to colonize
• Co-colonization with plant pathogens was shown to
  promote growth of human pathogens in/on plants
• Leakage of nutrients
• Changes in pH
• Integrity of plant cells is compromised
• Conspecifics
• Conjugal plasmid transfer
• Seems to be a correlation between survival of
  pathogens and type of a crop
• Not clear whether its plant chemicals that
  directly affect pathogens or whether it is
  microflora that is specific to certain plants

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Endophytes under pressure

• Inside the plants, endophytes have to deal or
  avoid plant defenses
• Plants appear to be able to recognize the same
  antigens (O, H, K) that animals would
• The inability of Salmonella, E.coli to degrade
  cell wall polymers may be beneficial to avoid
  elicitation of plant defenses

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Summary

• Produce-associated outbreaks have been
  increasing.
• Salmonella, E. coli O157H7 cause the greatest
  number of produce-associated outbreaks
• Surveillance indicates presence of enteric
  pathogens in fresh produce, fruits, nuts
• Not all enteric pathogens are ecological
  generalists
• Salmonella, E. coli are less competitive in
  phyllo-, rhizosphere. Still are capable of
  colonizing plants, internalizing and
  persistance.
• Interactions with native microflora are crucial
  to successful colonization and persistence