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Salmonella Praveen Rao, Sophia W. Riccardi, Danielle Birrer

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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


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 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 57-60°C or °F has shown to be effective in killing the bacteria. Optimal growth: 37°C or 98.6°F

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) ٠causes typhoid and paratyphoid fever ٠restricted to growth in human hosts ٠principal habitat is in intestinal tracts and the bloodstream

9 - Nontyphoidal Salmonella (example: S. enteritidis, S. typhimurium)
٠prevalent in gastrointestinal tracts of a broad range of animals, including mammals, reptiles, birds and insects. ٠cause a whole range of diseases in animals and humans, mainly gastroenteritis. ٠usually transferred animal-to-person, through certain food products: fresh meat, poultry, eggs and milk - fruits, vegetables, seafood ٠house 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 , a group of doctors at the University of Maryland suggested that S. was the cause of death, based on a description of Alexander’s symptoms written by the Greek author Arrian of Nicomedia. Prince Albert, the consort of Queen Victoria, died of a Salmonella infection in 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 ( ) - 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 men’s 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 ( ): 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 , 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 1900’s.

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 Contamination of 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 ( ) 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. Unhygienic preparation of food provides ample opportunities for contamination, growth, or survival of foodborne pathogens.

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 7 Oranienburg 595 1.9 8 Muenchen 583 1.8 9 Thompson 514 1.6 Saintpaul 469 1.5 11 Paratyphi B tartrate positive 466 12 Infantis 440 1.4 13 Braenderup 388 1.2 14 Agona 370 15 Typhi 343 1.1

30 Epidemiology S. 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 to 1.2% in in the past 15 years, 47 culture-proven and 24 potential cases

34 Epidemiology S. enteriditis and typhimurium (non-typhoidal S.):
- are the 2 top serotypes in the U.S. since 1980’s - 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 ground A crushed and broken fella. No one wanted to put him together 'Cause he had salmonella. Salmonella Enteritidis transmitted to humans by contaminated foods of animal origin, predominantly eggs. Raw eaten or undercooked eggs that have been infected in the hen’s ovaries can cause gastroenteritis

36 Salmonella Enteritidis Infections, United States, 1985–1999
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. Salmonella Enteritidis Infections, United States, 1985–1999

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. it is difficult to control and scientific literature suggests that some of these strains have emerged due to the use of antibiotics in intensive animal farms and maintenances.


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 Many serovars Gram-negative
Facultative anaerobes Glucose-fermenting Straight, rod 2-3 µm in length Flagellated Many serovars Typhi Typhimurium Enteriditis A serovar is a further classification of a subspecies. There is a genus, species, subspecies, and then serovar. Only considering typhi and enteriditis since they are the main serovars that infect humans. Main serovars are Typhi and Enteriditis. S. Typhi is the most host-specific, causing systemic typhoid fever in both human and non-human primates. S. Eneriditis infects a wide variety of organisms and casues limited gastrointestinal infections in humans.

45 A general picture of a salmonella bacterium. Notice the flagella
S. Typhi has an outer-capsule, while most other serovars have the lipopolysaccharide coat.

46 LPS on Surface Lipopolysaccharide Coded for by rfb locus on chromosome
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. I will go into this more later, but the antibodies and the memory immune response is directed against the LPS. So highly polymorphic nature is advantageous for bacteria – can mutate and evade memory response. Typhi has no LPS so it can evade the main memory immune response, causing a worse infection – systemic typhoid fever.

48 This is a membrane of S. typhi
This is a membrane of S. typhi. Other serovars do not have the outer capsule. Notice parts of LPS. Lipid A (conserved). Polysaccharide part is variable, especially O polysaccharide (what antibodies bind to)

49 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


51 Salmonella Entry

52 Membrane Ruffling This is probably over the span of about an our or two, maybe even less. Entrance is quick once bacteria are in the area.

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 These pathogenicity islands have a lower G-C content (about 38%) compared to rest of genome (about 50%) Lateral transfer, also called horizontal transfer, is movement between bacteria via conjugation, transduction, transformation, etc. This is as opposed to vertical transfer, which is when genetic information travels from parent to daughter as cell divides. Acquisition of PI enhances virulence of bacteria, since all genes can be transferred into host together – more effective infection. This is probably selected for evolutionarily.

54 Horizontal Transfer Transformation Conjugation Transduction
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


56 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 SPI-1 encodes for genes necessary for entry into intestinal epithelium SPI-2 encodes for genes necessary for intracellular bacterial replication and initiation of systemic infection in the host, which leads to the enteric fever symptoms seen with S. typhimurium Both of these pathogenicity islands code for protein complexes that are part of the Type III Secretion System (TTSS) Mutations in these pathogenicity islands cause the bacteria to no longer be virulent.

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 This system is not unique to Salmonella. It is also present in E. coli and Y. pestis, which is the causative agent of the plague. InvJ is another protein inside bacteria that regulates which proteins get injected into host at the right time in the infection cycle and in the right place.

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 This process is slightly different from receptor mediated-endocytosis, because some of these vesicles travel to basolateral surface to allow apical surface to resume non-ruffled appearance.


61 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 A phagosome is analogous to an endosome, prior to fusion with a lysosome.

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 Different strains of Salmonella secrete different effector proteins, which is what causes the variance in virulence and epidemiology. Usually the concentration of G-actin is kept below the amount required for polymerization, thus making synthesis of more G-actin a requirement for filament formation. These effector proteins stabilize the actin filaments, thus decreasing the minimum cellular concentration of G-actin necessary for actin polymerization.


64 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 An enterotoxin is something that is produced by microorganisms an is involved in gastrointestinal infections SopB can increase cellular inositol phosphate becase it has two domains that are homologous to eukaryotic inositol polyphosphate 4-phosphatases 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 Both S. enteriditis and S. typhimurium infections begin in the epithelium. S. typhimurium has this additional SPI-2 TTSS system that allows it to leave epithelium and invade macrophages, causing a more widespread, systemic infection.

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 An epitope is the region on the antigen that is responsible for elliciting the immune response. What makes it an “antigen.” The middle of the flagellum is highly variable among different serovars. Influences virulence.

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 Salmonella genome encodes for two types of flagella, Phase I and Phase II.

68 Tumor Necrosis Factor-α
Flagella from S. Typhimurium induces expression of TNF-α 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 Tumor Necrosis Factor-alpha is a cytokine involved in the inflammatory response. A cytokine is just a chemical substance that delivers a message between different cells in the body. Switch to Phase II causes less of an inflammatory response.

69 The scenario on the left is what happens with the switch to a Phase II flagellum. The inflammatory response is not triggered On the right, there is no switch, so Tumor Necrosis Factor-alpha is expressed, cell busts, releasing Salmonella into bloodstream and lymphatic system This activates the standard inflammatory immune response

70 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 This is a standard immune response. Most people with a healthy immune system can rid themselves of a usual Salmonella infection without the use of antibiotics.

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) S. Enteriditis cannot survive inside macrophages, but S. typhi can, allowing it to cause a more severe, systemic infection – typhoid fever.

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 Bacteria can travel through lymphatic system and infect the liver and spleen, as well as causing septicemia in the bloodstream.

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 Dam turns all these 20 genes ‘on’ that are normally only active during infection. If this is used as a live-attenuated vaccine, then we can produce antibodies for all the expressions of these genes, giving us immunity to nearly all Salmonella strains. Also, if we can block Dam, then we can make Salmonella not virulent.

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



80 Salmonella Overview History and Epidemiology Molecular Biology
Clinical Weaponization


82 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 Salmonella typhimurium 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 (> 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 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)

90 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 ºF. Also accompanied by loose, bloody stool; Pseudoappendicitis (rare) Stool culture will remain positive for 4-5 weeks < 1% will become carriers

91 Nontyphoidal Salmonella: Bacteremia 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

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

93 Incidence of S. Enteritidis

94 Nontyphoidal Salmonellosis: Localized 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

95 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 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!!!


102 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: Megan™Vac 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.

105 Salmonella Overview History and Epidemiology Molecular Biology
Clinical Weaponization

106 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 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.

109 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

110 First Use of Salmonella as a Bioterrorist Weapon
From , 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

112 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

113 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.

114 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. Problem protecting ourselves from bioterrorism is that the U.S. does not do well detecting and reporting its own naturally occurring food-borne outbreaks

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 aren’t 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 don’t even have to be inside the United States to do damage For example: It would not be difficult for a terrorist to pose as an employee and, using a sprayer attached to a garden hose, wet down several hundred tons of lettuce with salmonella.

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

117 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.

118 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

119 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.

120 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. Even if a bioterrorism attack never occurs, this type of health surveillance will spot disease outbreaks or associations quickly and will in turn reduce the national disease burden for humans, plants, and animals. With the ease of electronic communication, there should be daily monitoring of disease patterns across the country at the local, regional, and national levels.

121 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

122 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.

123 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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