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Salmonella

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Title: Salmonella


1
Salmonella
Praveen Rao, Sophia W. Riccardi, Danielle
Birrer Seminar in Nucleic Acids-Spring 2004 Prof.
Zubay
2
Salmonella
  • Overview
  • History and Epidemiology
  • Molecular Biology
  • Clinical
  • Weaponization

3
Overview
  • Salmonella is a rod-shaped, gram-negative,
    facultative anaerobe in the family
    Enterobacteriaceae

4
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5
Salmonella Taxonomy
  • The genus Salmonella is divided into two species,
    S. enterica and S. bongori (CDC).
  • Over 2000 strains are grouped into S. enterica.
    This species is further divided into six
    subgroups based on host range specificity, which
    also involves immunoreactivity of three surface
    antigens, O, H and Vi.
  • All strains that are pathogenic to humans are in
    species S. enterica, subgroup 1 (also called
    enterica).
  • For example, the correct taxonomic name for
    the organism that causes typhoid fever is
    Salmonella enterica ssp. enterica, serovar typhi.
    The simplified version Salmonella typhi.
  • Taxonomy has been revised several times, due to
    the degree of DNA similarity between genomes.
  • For example, In the U.S., another legitimate
    species name for enterica is choleraesuis.

6
Other Facts
  • Bacterium of 2501 identified strains, as of 2001.
    Many different diseases are caused by more than
    1,400 serotypes of this bacteria genus.
  • Salmonella derived from Dr. Salmon, a U.S.
    veterinary surgeon, who discovered and isolated
    the strain enterica or choleraesuis from the
    intestine of a pig in 1885.
  • They are ingested orally by contaminated food or
    water. Refrigeration prevents growth but does not
    kill bacteria. Heating at 57-60C or 134-140F
    has shown to be effective in killing the
    bacteria.
  • Optimal growth 37C or 98.6F

7
Disease-associated facts
  • Salmonellosis Any of several bacterial
    infections caused by species of Salmonella,
    ranging from mild to serious infections.
  • Two main kinds in humans enteric fever
    (typhoid and paratyphoid) and gastroenteritis
    (non-typhoidal).
  • The main feature for S. diseases is the Type III
    Secretion System, a needle-like multi-protein
    complex that is associated with transferring
    toxic proteins to host cells.

8
Principal habitats in different types of
Salmonella
  • Their principal habitat is the intestinal tracts
    and bloodstream of humans, and in the intestinal
    tracts of a wide variety of animals.
  • The WHO groups Salmonella into 3 types
  • - Typhoidal (enteric) Salmonella
  • (example S. typhi)
  • ?causes typhoid and paratyphoid fever
  • ?restricted to growth in human hosts
  • ?principal habitat is in intestinal
    tracts and the bloodstream

9
  • - Nontyphoidal Salmonella (example S.
    enteritidis, S. typhimurium)
  • ?prevalent in gastrointestinal tracts of a
    broad range of animals, including mammals,
    reptiles, birds and insects.
  • ?cause a whole range of diseases in animals and
    humans, mainly gastroenteritis.
  • ?usually transferred animal-to-person, through
    certain food products fresh meat, poultry, eggs
    and milk
  • - fruits, vegetables, seafood
  • ?house and exotic pets, contamination through
    contact with their feces

10
- Salmonella mostly restricted to certain
animals, such as cattle and pigs
infrequently in humans if these strains
do cause disease in humans, it is often
invasive and life-threatening.
11
Salmonella
  • Overview
  • History and Epidemiology
  • Molecular Biology
  • Clinical
  • Weaponization

12
History of Salmonella
  • Some historical figures are believed to have
    been killed by
  • Salmonella
  • Alexander the Great died mysteriously in 323
    B.C. In 2001, a group of doctors at the
    University of Maryland suggested that S. was the
    cause of death, based on a description of
    Alexanders symptoms written by the Greek author
    Arrian of Nicomedia.
  • Prince Albert, the consort of Queen Victoria,
    died of a Salmonella infection in 1861. During
    the Victorian era, an estimated 50,000 cases per
    year occurred in England.

13
History
  • Scholars working on the history of Jamestown,
    Virginia, believe that a typhoid outbreak was
    responsible for deaths of over 6000 settlers
    between 1607 and 1624.
  • Typhoid Epidemic in the Spanish-American War
    (1898)
  • - In all, 20,738 recruits contracted the
    disease (82 of all sick soldiers), 1,590 died
    (yielding a mortality rate of 7.7)
  • - It accounted for 87 of the total deaths
    from disease.
  • - A significant number of these deaths
    actually occurred at training areas in the
    southeastern United States.

14
History
  • Typhoid outbreak in British camps during the
    South African War (1899-1902)
  • - more soldiers suffered from typhoid fever
    than from battle wounds.
  • - British troops lost 13,000 men to
    typhoid, as compared to 8,000 battle deaths.
  • - outbreak was largely due to unsanitary towns
    and farms throughout Africa, and polluted soil
    was washed into the network of streams and rivers
    during the rainy season.
  • Epidemic potential during a war prominent because
    of the disposal problems of mens discharges.

15
History
  • Similar problems of sanitation occurred in urban
    areas. Many historic documents report about
    typhoid outbreaks in England
  • - Most outbreaks that were reported could be
    traced back to unsanitary water supplies or
    polluted milk supplies.
  • - Dr. William Budd (1811-1880) documented his
    observations, published them in the Lancet It
    was known then that polluted water can spread the
    disease. Budd urged for more disinfection and
    water treatment
  • - reports show that in the nineteenth century,
    population seemed powerless against this disease
    even though they knew it was perfectly
    preventable.
  • - with the introduction of piped and filtered
    water supplies in most urban areas, its
    prominence as a cause of death had diminished.

16
Salmonella vaccine
  • First preventive measure against Salmonella was
    discovered in 1896, as an antityphoid vaccine was
    developed by the British surgeon Almroth Wright.
  • Vaccine consisted of heat-denatured, rudimentary
    killed whole-cell bacteria said to be highly
    effective.
  • Early wars -Immunization known, but new
  • -the minimum dosage had not been clearly
    refined
  • British War Office
    authorized it on a voluntary basis only
  • most soldiers refused to be
    immunized because of
  • violent reaction
    following injection possible contraction
  • Urban outbreaks opposition to any type of
    vaccination a way around the problem of
    sanitation and cleanliness. It was seen as a
    disease of defective civilization due to
    defective sanitation.

17
Salmonella vaccine
  • Between 1904-1914, the vaccine had become
    respectable, in the scientific as well as
    military world.
  • Vaccine was successfully used during World War I
    to reduce the number of soldiers who died of
    enteric fever (S. typhi).

18
  • Bottling typhoid vaccine, 1944
  • Division of Biologic Products, U.S. Army of
    Medical Department Professional Service Schools
  • First typhoid inoculation, 1909 United States
    Army Medical School

19
History in the U.S.
  • Typhoid Mary Mallon was the first famous
    carrier of typhoid fever in the U.S.
  • Some individuals have natural immunity to
    Salmonella. Known as chronic carriers, they
    contract only mild or asymptomatic disease, but
    still carry the bacteria in their body for a long
    time. These cases serve as natural reservoir for
    the disease.
  • Approximately 3 of persons infected with S.
    typhi and 0.1 of those infected with
    non-typhoidal salmonellae become chronic carriers
    which may last for a few weeks to years.
  • One such case was Mary Mallon, who was hired as a
    cook at several private homes in the new York
    area in the early 1900s.

20
History Mary Mallon
  • Mary Mallon caused several typhoid outbreaks,
    moving from household to household, always
    disappearing before an epidemic could be traced
    back to the particular household Mary was working
    in. All together, she had worked for 7 families,
    with 22 cases of typhoid and one death.
  • She was finally overtaken by the authorities in
    1907 and committed to an isolation center on
    North Brother Island, NY. There she stayed until
    she was released in 1910, on the condition that
    she never accept employment involving food
    handling.
  • But She was found to work as a cook and to cause
    typhoid outbreaks again. She was admitted back
    to North Brother Island, where she lived until
    her death in 1938.

21
Recent outbreaks
  • More recently reported outbreaks in the U.S.
    involve different kinds of Salmonella strains,
    predominantly S. enteritidis and S. typhimurium.
  • In 1985, a salmonellosis (S. typhimurium)
    outbreak involving 16,000 confirmed cases in 6
    states by low fat milk and whole milk from one
    Chicago dairy.
  • Largest outbreak of food-borne salmonellosis
    in the U.S. Investigations discovered that raw
    and pasteurized milk had been accidentally mixed.

22
Oregon Intentional Contaminationof Restaurant
Salad Bars
In September of 1984, 10 area restaurants in The
Dalles, Oregon, were involved with outbreaks of
S. typhimurium
23
Outbreaks
  • January 2000 infant aged 1 month visited a
    clinic with fever and diarrhea. A stool specimen
    yielded Salmonella serotype Tennessee. One week
    before illness onset, the infant's family moved
    into a household that contained a bearded dragon
    (i.e., Pogona vitticeps).
  • During June 2002, a child aged 21 months was
    admitted to a hospital with fever, abdominal
    cramps, and bloody diarrhea. Blood and stool
    cultures yielded Salmonella serotype Poona (from
    pet Iguana).

24
Foodborne diseases
  • WHO in 2000 that globally about 2.1 million
    people died of foodborne illness
  • in industrialized countries, about 30 of people
    suffer from foodborne diseases each year around
    76 million cases occur each year, of which
    325,000 result in hospitalization and 5,000 in
    death.
  • (WHO, 2002)

25
Why do foodborne diseases emerge ?
  • Globalization of food supply for example,
    multistate outbreaks of S. Poona infections
    associated with eating Cantaloupe from Mexico
    (2000-2002)
  • Unavoidable introduction of pathogens into new
    geographic areas for example, vibrio cholerae
    introduced into waters off the coast of southern
    U.S. by cargo ship (1991).
  • Travelers, refugees and immigrants exposed to
    unfamiliar foodborne hazards.
  • Changes in microorganisms evolution of new
    pathogens, development of antibiotic resistance,
    changes in the ability to survive in adverse
    environmental conditions.

26
Why do foodborne diseases emerge ?
  • Changes in human population population of highly
    susceptible people is expanding more likely to
    succumb to bacterial infections.
  • Changes in lifestyle Great amount of people eat
    prepared meals. In many countries, the boom in
    food service establishments is not matched by
    effective food safety education and control.

27
Relative Frequency of the disease in the U.S.
  • Estimate 2 to 4 million cases of salmonellosis
    occur in the U.S. annually (reported and
    unreported). Salmonella accounts for the
    majority of food poisoning cases in the U.S
  • Latest numbers
  • In 2002, a total of 32,308 cases were
    reported from health laboratories in 50 states.
  • The national rate of reported isolates was
    11.5 per 100,000 population. Shows decrease of
    7 compared to 1992, slight increase of 2 from
    2001.

28
Epidemiology
  • The most commonly reported serotypes, in history
    and present
  • - S. typhi
  • - S. enteritides and S. typhimurium
  • The top 20 serotypes accounted for 80 of all
    isolates reported in the U.S. in 2001.

29
Top 15 Salmonella Serotype list in the U.S., 2001
Country, Institution, Biological origin Total Serot ped Rank Serotype Count of Total Serotyped
U.S.A., Centers of Disease Control, Control and Prevention-FDDB Epi, 2001, Human 31,675 1 Typhimurium 6,999 22.1
    2 Enteridites 5,614 17.7
    3 Newport 3,158 10
    4 Heidelberg 1,884 5.9
    5 Javiana 1,067 3.4
    6 Montevideo 626 2
    7 Oranienburg 595 1.9
    8 Muenchen 583 1.8
    9 Thompson 514 1.6
    10 Saintpaul 469 1.5
    11 Paratyphi B tartrate positive 466 1.5
    12 Infantis 440 1.4
    13 Braenderup 388 1.2
    14 Agona 370 1.2
    15 Typhi 343 1.1
30
EpidemiologyS. typhi (typhoidal Samonella)
  • Causes enteric fever
  • Have no known hosts other than humans.
  • Transmission through close contact with infected
    or chronic carriers. While direct
    person-to-person transmission through the
    fecal-oral route is rare, most cases of disease
    result from digestion of contaminated food or
    water.
  • Since improvements in food handling, piped and
    filtered water supplies as well as water/sewage
    treatment have been made, enteric fever has
    become relatively rare in developed countries.

31
  • However, typhoid fever is still a big
    health-problem in developing countries.
  • The WHO estimates that there are worldwide about
    16 million of clinical cases annually, of which
    about 600,000 result in death. In comparison,
    about 400 cases occur each year in the U.S., and
    70 of these cases are acquired while traveling
    internationally.

32
Salmonella typhi in developing countries
  • Contaminated water is a common cause in the
    spread of typhoid fever. At the time of rain,
    the contaminated surface water further
    contaminates water supplies.
  • Severity, Morbidity and complication rate is much
    higher than in Europe and North America due to
    lack of antibiotics supply, water filtration and
    treatment, sterilization of water and sanitation.

33
S. Typhi in the U.S.
  • Almost 30 of reported cases in the U.S. are
    domestically acquired.
  • Although most cases are sporadic, large outbreaks
    do occur.
  • For example, outbreak linked to contaminated
    orange juice in N. Y.,
    caused by a previously unknown chronic carrier
    (1991).
  • Multi-drug resistance
  • recent trend toward an increased incidence of
    multi-drug resistant S. typhi in developing
    countries is reflected by increase in the
    proportion of U.S. cases 0.6 in 1985-1989 to
    1.2 in 1990-1994.

34
Epidemiology
  • S. enteriditis and typhimurium (non-typhoidal
    S.)
  • - are the 2 top serotypes in the U.S. since
    1980s
  • - cause gastroenteritis following ingestion of
    the bacteria on or in food or on fingers and
    other objects
  • - cause the majority of cases of zoonotic
    salmonellosis in many countries.

35
Salmonella Enteritidis
  • Humpty Dumpty
  • by R. Wayne Edwards January 1999
  • Humpty Dumpty lay on the groundA crushed and
    broken fella.No one wanted to put him
    together'Cause he had salmonella.
  • transmitted to humans by contaminated foods of
    animal origin, predominantly eggs. Raw eaten or
    undercooked eggs that have been infected in the
    hens ovaries can cause gastroenteritis

36
Salmonella Enteritidis Infections, United States,
19851999
  • During the 1980s, illness related to contaminated
    eggs occurred most frequently in the northeastern
    United States, but now it is increasing in other
    parts of the country as well.

37
  • CDC, 2002 In the Northeast, approximately one in
    10,000 eggs may be internally contaminated one
    in 50 average consumers could be exposed to a
    contaminated egg each year.
  • In 1995 high of 3.9 per 100,000 population,
  • In 1999 1.98 per 100,000, rate still
    decreasing due to prevention and control efforts
    by the government.

38
S. typhimurium
  • has been reported increasingly frequently as the
    cause of human and animal salmonellosis since
    1990, due to antibiotic resistance
  • Predominant multi-drug resistant strain DT 104,
    which initially emerged in cattle in England,
    1988
  • In 1997, the WHO stated that some countries in
    Europe had a staggering 20-fold increase in
    incidences between 1980 and 1997, and a 5-fold
    increase in the U.S. between 1974 and 1994, due
    to antibiotic resistant strains
  • intensive animal maintenance.

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40
Epidemic measures
  • Salmonellosis is a reportable disease.
  • An intensive search should be conducted for
    the source of an infection and for the means
    (food or water) by which the infection was
    transmitted.
  • Samples of blood can be taken immediately for
    confirmation and for testing for antibiotic
    sensitivity.
  • Samples of stool or urine may be taken after
    one week of onset for effective confirmation.
  • Food and water samples should be taken from
    suspected sources of the outbreak. It is
    recommended to organize temporary water
    purification and sanitation facilities until
    longer term measures can be implemented.

41
Cost Estimates
  • The cost per reported case of human salmonellosis
    range from US 100 to 1300 in North America and
    Europe.
  • The costs associated with individual outbreaks in
    North America and Europe range from around
  • 60,000 to more than 20 Million.
  • The total annual cost in the U.S. is estimated a
    total of almost 400 Million.

42
Salmonella
  • Overview
  • History and Epidemiology
  • Molecular Biology
  • Clinical
  • Weaponization

43
Salmonella Microbiology
44
Classification
  • Enterobacteria
  • Gram-negative
  • Facultative anaerobes
  • Glucose-fermenting
  • Straight, rod
  • 2-3 µm in length
  • Flagellated
  • Many serovars
  • Typhi
  • Typhimurium
  • Enteriditis

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LPS on Surface
  • Lipopolysaccharide
  • Protective outer layer of most strains
  • (not S. typhi)
  • Coded for by rfb locus on chromosome
  • Lipid core of LPS highly conserved across
    serovars, but polysaccharide side chains are
    highly polymorphic (nature of rfb gene)

47
LPS (cont.)
  • Memory immune response and antibodies directed
    against LPS
  • Polymorphic nature of side chains is advantageous
    for bacteria
  • Since Typhi has outer capsule, this infection is
    worse.

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Infection
  • Ingestion of contaminated food or water
  • Passes through mucosa of intestine to epithelial
    cells
  • Causes membrane ruffling
  • Releases effector proteins through Type III
    Secretion system
  • Endocytosis

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Salmonella Entry
52
Membrane Ruffling
53
Virulence Factors
  • Genes for virulence factors cluster in
    pathogenicity islands (PI)
  • Genes acquired through lateral transfer
  • Bacteriophage and transposon insertion sequences
    flank PI
  • Maybe vehicles for transfer of PI to Salmonella
    at one time
  • Acquisition of PI enhances virulence of bacteria

54
Horizontal Transfer
  • Transformation
  • Uptake of naked DNA
  • Mediates exchange of any part of DNA
  • Conjugation
  • F to F-
  • Requires cell to cell contact conjugation
    bridge
  • Transduction
  • Transfer of DNA by a phage
  • New phage viral coat with bacterial DNA

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Salmonella Pathogenicity Islands
  • Salmonella Pathogenicity Island 1 (SPI-1)
  • entry into intestinal epithelium
  • Enables pathogen to exploit host intestinal
    environment
  • Salmonella Pathogenicity Island 2 (SPI-2)
  • intracellular bacterial replication and
    initiation of systemic infection
  • Do not influence enteropathogenesis to any great
    extent

57
Type III Secretion System (TTSS)
  • Main way Salmonella delivers virulence factors to
    host
  • Made up of 20 proteins
  • Assemble in step-wise order
  • PrgI is a needle structure extended by protein
    base, forms a channel to host

PrgI
58
Salmonella-host Interaction
  • Two forms of TTSS
  • One encoded on SPI-1, other on SPI-2
  • SPI-1 TTSS probably causes initial interaction
  • Starts bacteria-mediated endocytosis
  • Entry activates SPI-2 TTSS to cause thorough
    infection

59
Membrane Ruffling
  • Cytoskeleton-associated proteins relocate to site
    of bacterial entry
  • Bacterial effector proteins trigger cytoskeleton
    rearrangements
  • Apical membrane surface undergoes structural
    changes, resembling ruffling
  • This triggers endocytosis into vesicles
  • Slightly different from receptor-mediated
    endocytosis

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Salmonella Containing Vesicle
  • After ingestion, Salmonella enters a SCV through
    bacteria-mediated endocytosis
  • Lives and multiplies in SCV
  • Very little known about SCV or how bacteria exist
    inside
  • A method to avoid host immune response
  • Phagosome maturing SCV

62
SPI-1 Effector Proteins
  • SipA
  • Binds actin and stabilizes filaments
  • Allows actin to polymerize more easily
  • Maximizes efficiency of Salmonella invasion
  • SipC
  • Aides in entry of other SPI-1 effector proteins
  • Activtes G-actin to form F-actin, then polymerize
  • Aides in cytoskeleton rearrangements in membrane
    ruffling

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SopB
  • Main virulence factor
  • Encoded by SPI-5
  • An enterotoxin associated with SPI-1 TTSS
  • Induces an increase in concentration of cellular
    inositol polyphosphate
  • Increased chloride secretion into lumen
  • Na follows to balance charge
  • Water follow to balance osmolarity

diarrhea
65
SPI-2 TTSS
  • Activated once bacteria enters cell
  • Necessary for systemic infection
  • SPI-2 TTSS secretes effector proteins from
    phagosome into cytosol
  • Interfere with maturation of phagosome
  • No fusion with lysosome
  • How Salmonella avoids degredation in cell

66
Flagella
  • Another antigen
  • Host cytotoxic T-cell response directed against
    flagellar epitopes
  • N- and C- termini are highly conserved
  • Middle of flagellum is variable

67
Phase I / II Flagella
  • Operon encoding Phase I flagella also encodes for
    a protein that represses trascription of Phase II
  • The switch mediated by an enzyme that inhibits
    Phase I, allowing Phase II
  • May help Salmonella avoid cell-mediated immune
    response

68
Tumor Necrosis Factor-a
  • Flagella from S. Typhimurium induces expression
    of TNF-a through cell-mediated reponse
  • Phase II flagella are less-potent inducers
  • Switching mechanism may provide bacteria with a
    way to down-regulate inflammatory response within
    host

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Immune Response
  • White blood cells recognize trigger T cells, B
    cells
  • Two types of B cells one to attack now, one for
    memory
  • Macrophages and neutrophils attack bacteria,
    secrete interleukins, causing cell-mediated
    response by T-cells
  • Antibodies from B cells attach to bacteria,
    allowing cytotoxic T cells, macrophages, and
    neutrophils to kill the organism

71
Inside Macrophages
  • SPI-2 TTSS works in macrophages as well
  • Bacterium produces enzymes that inactivte toxic
    macrophage compounds
  • Homocysteine (Nitric Oxide antagonist)
  • Superoxide dismutase (inactivates reactive
    peroxides)
  • Salmonella must produce additional factors to
    survive limited nutrient base
  • Allows bacteria to travel throughout body,
    causing systemic infection (only in S. typhi)

72
Septicemia
  • Invasion of bloodstream
  • spv genes causes detachment of cells to ECM and
    apoptosis
  • Spreads infection
  • Bacteria can enter bloodstream and lymphatic
    system
  • Main cause of death by Salmonella

73
How do we respond?
  • Microbiological view
  • Vaccines
  • Dam
  • Antibiotics

74
Salmonella Vaccine Strategy
  • Delete chromosomal regions that code for
    independent and essential functions. This results
    in
  • - low probability of acquiring both traits
  • - both traits
  • aro genes aromatic compound biosynthesis
  • pur genes purine metabolism biosynthesis
  • Deletion strains
  • - can be grown on complete medium in lab
  • - in vivo, growth is reduced
  • - only a low level of infection is established
  • - immune system can mount a response
  • Vaccine suitable for humans and mice, chickens,
    sheep, cattle

75
DNA adenine methylase (Dam)
  • Enzyme that methylates specific adenine residues
    in Salmonella genome
  • Disrupts regulation of DNA replication and repair
  • Regulates expression of about 20 bacterial genes
    active during infection
  • Dam (-) mutants are not virulent
  • Good antimicrobial potential
  • Current hot topic of research

76
Antibiotics
  • Antibiotics are selective poisons
  • Do not harm body cells
  • Target different aspects of bacteria, such as
    ability to synthesize cell wall, or metabolism
  • MIC Minimum Inhibitory Concentration
  • the minimum amount of agent needed to inhibit the
    growth of an organism

77
Antibiotic Resistance
  • Bacteria can counteract antibiotics by
  • Preventing antibiotic from getting to target
  • Changing the target
  • Destroy the antibiotic
  • Bacteria can acquire resistance
  • Horizontal transfer from another bacteria
  • Vertical transfer due to natural selection

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Salmonella
  • Overview
  • History and Epidemiology
  • Molecular Biology
  • Clinical
  • Weaponization

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How Do You Catch Salmonella?
  • Food borne
  • Transmitted via improperly prepared, previously
    contaminated food or water
  • - Meat poultry, wild birds, pork
  • - Dairy eggs
  • Pet turtles and lizards

83
How does Salmonella affect the body?
  • Three clinical forms of salmonellosis
  • - Gastroenteritis (S. typhimurium)
  • - Septicemia (S. Choleraesius)
  • - Enteric Fevers (i.e. S. typhi Typhoid Fever)

84
Who Can Be Infected?
  • Everyone
  • Especially the elderly, infants,
    immunocompromised patients (AIDS, sickle cell
    anemia)

85
Factors Increasing Susceptibility
86
Identification I
  • Laboratory identification of genus Salmonella
    biochemical serological tests
  • HOW?
  • - stool or blood specimens are plated on agar
    media (bismuth sulfite, green agars, MacConkey)
  • Suspect colonies further analyzed by triple sugar
    iron agar/ or lysine-iron agar
  • - confirmed by antigenic analysis of O (somatic)
    and H (flagellar) antigens Test for antigens

87
Identification II
  • Use phenol red test
  • - testing for lactic acid production
  • - if negative, diagnose (presence of red
    spots surrounded by a bright red zone)

Salmonella typhimurium
88
Nontyphoidal Salmonella
  • General Incubation 6 hrs-10 days Duration 2-7
    days
  • Infective Dose usually millions to billions of
    cells
  • Transmission occurs via contaminated food and
    water
  • Reservoir
  • a) multiple animal reservoirs
  • b) mainly from poultry and eggs (80 cases from
    eggs)
  • c) fresh produce and exotic pets are also a
    source of contamination (gt 90 of reptile stool
    contain salmonella bacterium) small turtles ban.
  • General Symptoms diarrhea with fever, abdominal
    cramps, nausea and sometimes vomiting

89
Nontyphoidal Salmonella
  • Caused by S. typhimurium and S. enteritidis
  • Rainy season of tropical climates Warm season of
    temperate climates
  • Growing rapidly in the U.S. five-fold increase
    between 1974-1994
  • Centralization of food processing makes
    nontyphoidal salmonellosis particularly prevalent
    in developing countries
  • Resistance is a concern, especially with
    multi-drug resistant S. Typhimurium known as
    Definitive Type 104 (DT 104)

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Nontyphoidal Salmonella Gastroenteritis
  • Incubation 8-48 hrs Duration 3-7 days for
    diarrhea 72 hrs. for fever
  • Inoculum large
  • Limited to GI tract
  • Symptoms include diarrhea, nausea, abdominal
    cramps and fevers of 100.5-102.2ºF. Also
    accompanied by loose, bloody stool
    Pseudoappendicitis (rare)
  • Stool culture will remain positive for 4-5 weeks
  • lt 1 will become carriers

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Nontyphoidal SalmonellaBacteremia and
Endovascular Infections
  • 5 develop septicemia 5-10 of septicemia
    patients develop localized infections
  • Endocarditis Salmonella often infect vascular
    sites preexisting heart valve disease risk
    factor
  • Arteritis Elderly patients with a history of
    back/chest prolonged fever or abdominal pain
    proceeding gastroenteritis are particularly at
    risk.
  • - Both are rare, but can cause complications
    that may lead
  • to death

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Septicemia
  • Serotype S. choleraesius causes septicemia
  • - prolonged state of fever, chills, anorexia,
    and anemia
  • - lesions in other tissues
  • - septic chock, death

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Incidence of S. Enteritidis
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Nontyphoidal SalmonellosisLocalized Infections
  • INTRAABDOMINAL INFECTIONS
  • Rare, usually manifested as liver or spleen
    abscesses
  • Risk factors hepatobiliary, abdominal
    abnormalities, sickle cell disease
  • Treatment surgery to correct anatomic damages
    and drain abscesses
  • CENTRAL NERVOUS SYSTEM INFECTIONS
  • Usually meningitis (in neonates, present with
    severe symptoms e.g. seizures, hydrocephalous,
    mental retardation, paralysis) or cerebral
    abscesses
  • PULMONARY INFECTIONS
  • Usually lobar pneumonia
  • Risk factors preexisting lung abnormalities,
    sickle cell disease, glucocorticoid usage

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Typhoidal Salmonellosis Enteric Fever
  • Incubation 7-14 days after ingestion Duration
    several days
  • Infective Dose 105 organisms
  • Symptoms
  • a) 1st week slowly increasing fever, headache,
    malaise, bronchitis
  • b) 2nd week Apathy, Anorexia, confusion, stupor
  • c) 3rd week rose spots (1-2 mm diameter on the
    skin) duration 2-5 days, variable GI symptoms,
    such as abdominal tenderness (majority),
    abdominal pain (20-40 of cases) and diarrhea
    enlargement of the spleen/liver, nose bleeds, and
    bradycardia
  • neuropsychiatric symptoms delirium and mental
    confusion
  • Long term effects arthritis

96
Typhoidal Salmonellosis
  • Late stage complications include intestinal
    perforation and gastrointestinal hemorrhage
  • Immediate care such as increase antibacterial
    medications or surgical resection of bowel
  • Other rare complications include inflammation of
    the pancreas, endocardium, perocardium,
    myocardium, testes, liver, meninges, kidneys,
    joints, bones, lungs and parotid gland and
    hepatic/splenic abscesses
  • In general, symptoms of paratyphoid fever are
    similar to typhoid fever, but milder with a lower
    mortality rate
  • Majority of bacteria gone from stool in 8 weeks
    However, 1-5 become asymptomatic chronic
    carriers gallbladder is the primary source of
    bacterium

97
Typhoidal Salmonella
Chest PA view shows pleural effusion, left lower
pulmonary lobe atelectasis, medial and downward
shift of bowel gas, and an increase in the
air-fluid level in the abdomen
98
Pictures
(A)
(B)
(A) In sub-acute infections, multiple white to
yellow foci occur in the liver, spleen is
enlarged, and mesenteric lymph nodes may be
enlarged (B) Histopathological examination may
reveal necrotizing splenitis and hepatitis, with
necrotic foci often accompanied by colonies of
bacteria (arrow in right photo).
99
Treatment of Typhoidal Salmonellosis
  • Third generation cephalosporins or quinolones is
    the current treatment
  • IV or IM ceftriaxone (1-2g) is also prescribed
    usually 10-14 days (5-7 days for uncomplicated
    cases)
  • Multi Drug Resistant (MDR) strains of S. typhi
    quinolones are the only effective oral treatment
  • Nalidixic acid resistant S. typhi (NARST) must be
    tested for sensitivity to determine course of
    treatment
  • Sever typhoid fever (altered consciousness,
    septic shock) dexamethasone treatment
  • Chronic carriers 6 weeks of treatment with
    either oral amoxicillin, ciprofloxacin,
    norfloxacin
  • Surgical intervention to remove damaged cells

100
Prevention
  • Typhoidal S.
  • - Generally treated with antibiotics
  • - vaccinations available the CDC currently
    recommends vaccination for persons traveling to
    developing countries
  • - Education of general public, especially in
    developing countries identification of all
    carriers and sources of contamination of water
    supplies
  • - avoid risky foods drinks
  • buy bottled water or boil water for at least
    1 minute
  • COOK and CLEAN food thoroughly, avoid raw
    vegetables and fruits
  • - WASH YOUR HANDS WITH SOAP AND WATER!!!

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  • Preventive measures for non-typhoidal S.
  • - pasteurization of milk-products Eggs from
    known infected commercial flocks will be
    pasteurized instead of being sold as grade A
    shell eggs.
  • - tracebacks, on-farm testing, quality assurance
    programs, regulations regarding refrigeration,
    educational messages for safe handling and
    cooking of eggs
  • - Cross-contamination uncooked contaminated
    foods kept separate from cooked, ready-to-eat
    foods.

103
Salmonella Vaccines I
  • Poultry vaccine MeganVac 1
  • - applied to baby chicks via drinking water and
    cattle. It stimulates immunity in the chickens,
    preventing Salmonella infection during the
    growing period which may result in contamination
    and subsequent food borne infection of humans
  • - targets S. Enteritidis
  • - Salmonella infection is stopped at lower
    levels of the food chain will mean increased
    productivity for the farmer and a break in the
    cycle of Salmonella transmission from animals à
    humans

104
Salmonella Vaccines II
  • Today, three types of Typhoid Vaccines are
    available
  • (1) inactivated whole-cell vaccine 2 doses/
    4wks. Apart single booster dose recommended
    every 3 years
  • (2) Ty21a a live, attenuated S. typhi vaccine.
    Administered orally (4 doses). Efficacy 7 years
  • (3) Vi polysaccharide vaccine from purified Vi
    polysaccharide from S. typhi. Administered
    subcutaneously or intramuscularly. To maintain
    protection, revaccination recommended every 3
    years.
  • These vaccines have been shown be 70-90
    effective.

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Salmonella
  • Overview
  • History and Epidemiology
  • Molecular Biology
  • Clinical
  • Weaponization

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Salmonella as A Bioweapon?
107
CDC classification
  • Category B agent includes microorganisms that
    are moderately easy to disseminate, have moderate
    morbidity (i.e., ability to cause disease) and
    low mortality, but require enhanced disease
    surveillance.
  • Biosafety Level 2
  • Risk Level 2 associated with human disease that
    is rarely serious and prophylactic intervention
    is often available.
  • 9 different species Salmonella arizonae,
    cholerasuis, enteritidis, gallinarum-pullorum,
    meleagridis, paratyphi (Type A,B,C), spp., typhi,
    and typhimurium
  • Salmonella typhi is the only species that
    requires import and/or export permit from CDC
    and/or Department of Commerce has high droplet
    or aerosol production potential

108
WHO Global Salm Surv (GSS)
  • GSS is an international Salmonella surveillance
    program initiated in January 2002. It collects
    annual summary data from member institutions all
    over the world.
  • The goal is to enhance the quality of Salmonella
    surveillance, serotyping and antimicrobial
    resistance testing and leading local
    interventions that reduce the human health burden
    of Salmonella.
  • A total of 138 laboratories were enrolled in the
    GSS in September 2003.

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Salmonella as a Bioterrorist Weapon What states
are most at risk?
  • The states most vulnerable to terrorist attack on
    the agricultural sector are those with several or
    most of the following attributes
  • High density, large agricultural area
  • heavy reliance on monoculture of a restricted
    range of genotypes
  • major agricultural exporter, or heavily dependent
    on a few domestic agricultural products
  • suffering serious domestic unrest, or the target
    of international terrorism, or unfriendly
    neighbor of states likely to be developing BW
    programs

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First Use of Salmonella as a Bioterrorist Weapon
  • From 1932-1945, Japan conducted biological
    warfare experiments in Manchuria
  • At Unit 731, a biological warfare research
    facility, prisoners were infected with Salmonella
    typhosa among other biological agents
  • Additionally, a number of Chinese cities were
    attacked. The Japanese contaminated water
    supplies and food items with Salmonella. Cultures
    were also tossed into homes and sprayed from
    aircraft
  • Due to inadequate preparation, training, and/or
    lack of proper equipment, the Chekiang Campaign
    in 1942 led to about 10,000 biological casualties
    and 1,700 deaths among the Japanese troops.

111
  • Oregon 1984 a religious cult known as the
    Rajneeshees, a Buddhist cult sought to run
  • the whole country by wining the local election in
    1984 using salmonella bacteria. They
  • brewed a "salsa" of salmonella and sprinkled it
    on the town's restaurant salad bars. Ten
  • restaurants were hit and more than 700 people got
    sick.
  • First large scale bioterrorism attack on
    American soil
  • A communitywide outbreak of salmonellosis
    resulted at least 751 cases were
  • documented in a county that typically reports
    fewer than five cases per year.
  • Health officials soon pinned down salmonella as
    the cause of the sudden outbreak, but
  • put the blame on food handlers. In 1984, who
    could have imagined bioterrorism?  
  • caused by S. typhimurium as this type

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Salmonella as a Bioterrorist Weapon
  • Wide distribution of food contaminated food
    produced in one country can cause illness in
    other countries
  • Traceability
  • Antimicrobial resistance strains of
  • Salmonella are being found that have
  • multiple drug resistance
  • Capacity building Salm-gene project
  • used to enhance outbreak detection by
  • routinely sub-typing certain salmonellas
  • using molecular methods

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Salmonella as a Bioterrorist Weapon
  • Contaminating unguarded food supplies
  • Some terrorist acts may be designed purely to
    spread panic contaminating the food supply could
    bring economic and agricultural production to a
    standstill
  • EX. If numerous food-borne outbreaks occurred
    across the country, people would soon fear their
    meals
  • Unfortunately, people have reason to worry all
    these contaminations have occurred naturally
    every year. If Mother Nature can do this
    repeatedly, then a terrorist should have no
    problem recreating these outbreaks over and over
    in any number of American cities.

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Salmonella as a Bioterrorist Weapon
  • readily accessible and easy to grow or make
  • Centralized food production largely unmonitored
    food supply food that is tampered with can be
    widely quickly distributed
  • Terrorist groups could use infectious disease
    agents to confuse public health officials into
    believing that outbreaks are naturally occurring
    it is estimated that 1.4 million salmonella
    infections occur each year, but the CDC gets
    reports of only about 38,000 annually
  • According to the Centers for Disease Control
    (CDC), only 32 of the reported outbreaks have a
    known etiology.

115
Salmonella as a Bioterrorist Weapon
  • No food product is safe vegetables and fruits
    are the easiest to contaminate. Fresh-produce
    wholesalers and distributors are notorious for
    employing illegal immigrants and not checking
    their background information.
  • Even processed foods arent safe Terrorists
    could use heat-stable toxins that would survive
    the packaging process.
  • As more of our food becomes imported, especially
    hard-to-clean off-season fruits and vegetables,
    bioterrorists dont even have to be inside the
    United States to do damage

116
Salmonella as a Bioterrorist Weapon Who might be
tempted to initiate an attack on the agricultural
sector?
  • Terrorist groups might be interested in
    agricultural bioweapons for a variety of reasons
  • 1. international terrorist organizations cause
    harm/injury to enemy states or peoples
  • - in an ideologically-motivated terrorist
    attack there would be willing assumption of
    responsibility by the perpetrator OR an attempt
    to disguise the outbreak as natural.
  • 2. Extreme activist groups
  • - EX. anti-GMO groups for their potential value
    in deterring farmers from the use of genetically
    engineered crops or animals

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Salmonella as a Bioterrorist Weapon What goals
might an attack on the agricultural sector serve?
  • Food attack by a terrorist group initiate
    point-source epidemics using available Salmonella
    strains
  • Destabilize a government by initiating food
    shortages/unemployment the potential for immense
    economic damage due to contamination of the food
    supply
  • Alter supply and demand patterns for a commodity
    an outbreak can trigger the imposition of trade
    restrictions. This is turn would open up or close
    markets for others.

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Salmonella as a Bioterrorist Weapon What are the
special features of an attack on the agricultural
sector?
  • Salmonella is not hazardous to perpetrators
    easy to produce, stockpile, and disseminate
  • Few technical obstacles to weaponization it
    would not be difficult to obtain Salmonella
    strains on the open market.
  • Low security of vulnerable targets Fields,
    supermarkets, restaurants have essentially no
    security at all.
  • Point source to mimic natural introduction
    Because of the high incidence of
    naturally-occurring diseases, a deliberately
    instigated outbreak could be mistaken for a
    natural one
  • Multiple point source outbreaks can be initiated
    by contaminating imported feed or fertilizer,
    without even entering the country allows the
    possibility of initiating multiple outbreaks over
    a large geographic area, in a way that mimics a
    natural event

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Salmonella Dilemma
  • Dissemination of genomic knowledge of salmonella
    can facilitate bio-weapons development
  • Alternative 1 Restrict dissemination of genomic
    knowledge
  • - short term hinders development of a
    super-Salmonella terror weapon
  • - long run leaves us at the mercy of multi-drug
    resistant salmonella strains ranging from
    incapacitating to lethal
  • Alternative 2 Disseminate genomic knowledge, but
    support development of salmonella specific-drugs
  • - knowledge may provide a terrorist org. with
    the ability to develop super-Salmonella terror
    weapons, but it provides us with the opportunity
    to defend against all salmonella infection.

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Combating Salmonella Bioterrorism
  • Establish a national disease surveillance system
    that could not only help uncover a terrorist
    attack but also recognize naturally occurring
    outbreaks that now go undetected
  • New technology creating a diagnostic gene chip
    covering all major diseases could give the health
    care provider instant diagnoses. Similar gene
    chips could monitor the health of livestock,
    poultry, and crops. Chips could be used during
    various steps of food processing to ensure
    quality control and food safety.

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Lines of Defense
  • Food processors should limit access to their
    production, storage and packaging areas
    rerouting traffic, installing locks
  • Randomized safety checkpoints will increase fear
    of detection
  • COSTS
  • Increase work force
  • Sampling and test costs
  • Record keeping

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Government Action
  • CDC monitors the frequency of Salmonella
    infections in the country and assists the local
    and State Health Departments to investigate
    outbreaks and devise control measures
  • FDA inspects imported foods, milk pasteurization
    plants, promotes better food preparation
    techniques in restaurants and food processing
    plants, and regulates the sale of turtles and it
    also regulates the use of specific antibiotics as
    growth promotants in food animals
  • USDA monitors the health of food animals,
    inspects egg pasteurization plants, and is
    responsible for the quality of slaughtered and
    processed meat.
  • EPA regulates and monitors the safety of our
    drinking water supplies.

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Biological Weapon Prevention
  • BTWC (Biological and Toxin Weapons Convention)
    drafted in 1972
  • - intended to prevent the development,
    production and stockpiling of biological weapons
  • - pathogens or toxins in quantities that have no
    justification for protective or peaceful services
    are to be eliminated
  • - today, 159 countries have signed the
    convention and 141 have ratified it
  • - however, more can be done Factories in the
    former Eastern Europe supply viruses that cause
    fatal diseases, such as E-Coli and Salmonella,
    without checking the identities of the
    purchasers (from the trials of the largest
    fundamentalist org. in Egypt, Abu-al-Dahab)

124
Acknowledgements
  • Dr. Geoffrey Zubay
  • Salwa Touma
  • Kathleen Kehoe

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