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

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 choleraesuisfrom 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 134-140°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 2001, 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 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 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 (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. First typhoid inoculation, 1909 United States Army Medical School Bottling typhoid vaccine, 1944 Division of Biologic Products, U.S. Army of Medical Department Professional Service Schools

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 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

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 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. 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. 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.

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.

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

46. 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.

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