5Salmonella TaxonomyThe 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.
6Other FactsBacterium 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
7Disease-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.
8Principal 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 fever٠restricted to growth in human hosts٠principal 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, seafood٠house 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.
11Salmonella Overview History and Epidemiology Molecular Biology ClinicalWeaponization
12History of SalmonellaSome historical figures are believed to have been killed bySalmonella: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.
13HistoryScholars 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.
14HistoryTyphoid 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.
15HistorySimilar 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.
16Salmonella vaccineFirst 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 ofviolent reaction following injection; possible contractionUrban 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”.
17Salmonella vaccineBetween , 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).
18Bottling typhoid vaccine, 1944 Division of Biologic Products, U.S. Army of Medical Department Professional Service SchoolsFirst typhoid inoculation, 1909 United States Army Medical School
19History 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.
20History: Mary MallonMary 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.
21Recent outbreaksMore 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.
22Oregon: Intentional Contamination of Restaurant Salad Bars In September of 1984, 10 area restaurants in The Dalles, Oregon, were involved with outbreaks of S. typhimurium
23OutbreaksJanuary 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).
24Foodborne diseasesWHO: in 2000 that globally about 2.1 million people died of foodborne illnessin 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)
25Why 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.
26Why 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.
27Relative 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.SLatest 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.
28EpidemiologyThe most commonly reported serotypes, in history and present:- S. typhi- S. enteritides and S. typhimuriumThe “top 20” serotypes accounted for 80% of all isolates reported in the U.S. in 2001.
29Top 15 Salmonella Serotype list in the U.S., 2001 Country, Institution, Biological originTotalSerotpedRankSerotypeCount% of TotalSerotypedU.S.A., Centers of Disease Control, Control and Prevention-FDDB Epi, 2001, Human31,6751Typhimurium6,99922.12Enteridites5,61417.73Newport3,158104Heidelberg1,8845.95Javiana1,0673.46Montevideo6267Oranienburg5951.98Muenchen5831.89Thompson5141.6Saintpaul4691.511Paratyphi Btartrate positive46612Infantis4401.413Braenderup3881.214Agona37015Typhi3431.1
30Epidemiology S. typhi (typhoidal Samonella) Causes enteric feverHave 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.
31However, 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.
32Salmonella 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.
33S. 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% inin the past 15 years, 47 culture-proven and 24 potential cases
34Epidemiology 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.
35Salmonella Enteritidis Humpty Dumptyby R. Wayne Edwards January 1999Humpty Dumpty lay on the ground A crushed and broken fella. No one wanted to put him together 'Cause he had salmonella.Salmonella Enteritidistransmitted 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
36Salmonella 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
37CDC, 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.
38S. typhimuriumhas been reported increasingly frequently as the cause of human and animal salmonellosis since 1990, due to antibiotic resistancePredominant multi-drug resistant strain DT 104, which initially emerged in cattle in England, 1988In 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 strainsintensive 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.
40Epidemic 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.
41Cost EstimatesThe 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.
42Salmonella Overview History and Epidemiology Molecular Biology ClinicalWeaponization
44Classification Enterobacteria Many serovars Gram-negative Facultative anaerobesGlucose-fermentingStraight, rod2-3 µm in lengthFlagellatedMany serovarsTyphiTyphimuriumEnteriditisA 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.
45A general picture of a salmonella bacterium. Notice the flagella S. Typhi has an outer-capsule, while most other serovars have the lipopolysaccharide coat.
46LPS 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 chromosomeLipid core of LPS highly conserved across serovars, but polysaccharide side chains are highly polymorphic (nature of rfb gene)
47LPS (cont.) Memory immune response and antibodies directed against LPS Polymorphic nature of side chains is advantageous for bacteriaSince 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.
48This 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)
49Infection Ingestion of contaminated food or water Passes through mucosa of intestine to epithelial cellsCauses membrane rufflingReleases effector proteins through Type III Secretion systemEndocytosis
52Membrane RufflingThis is probably over the span of about an our or two, maybe even less. Entrance is quick once bacteria are in the area.
53Virulence FactorsGenes for virulence factors cluster in pathogenicity islands (PI)Genes acquired through lateral transferBacteriophage and transposon insertion sequences flank PIMaybe vehicles for transfer of PI to Salmonella at one timeAcquisition of PI enhances virulence of bacteriaThese 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.
54Horizontal Transfer Transformation Conjugation Transduction Uptake of naked DNAMediates exchange of any part of DNAConjugationF+ to F-Requires cell to cell contact – conjugation bridgeTransductionTransfer of DNA by a phageNew phage: viral coat with bacterial DNA
56Salmonella Pathogenicity Islands Salmonella Pathogenicity Island 1 (SPI-1)entry into intestinal epitheliumEnables pathogen to exploit host intestinal environmentSalmonella Pathogenicity Island 2 (SPI-2)intracellular bacterial replication and initiation of systemic infectionDo not influence enteropathogenesis to any great extentSPI-1 encodes for genes necessary for entry into intestinal epitheliumSPI-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. typhimuriumBoth 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.
57Type III Secretion System (TTSS) Main way Salmonella delivers virulence factors to hostMade up of 20 proteinsAssemble in step-wise orderPrgI is a needle structure extended by protein base, forms a channel to hostPrgIThis 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.
58Salmonella-host Interaction Two forms of TTSSOne encoded on SPI-1, other on SPI-2SPI-1 TTSS probably causes initial interactionStarts bacteria-mediated endocytosisEntry activates SPI-2 TTSS to cause thorough infection
59Membrane RufflingCytoskeleton-associated proteins relocate to site of bacterial entryBacterial effector proteins trigger cytoskeleton rearrangementsApical membrane surface undergoes structural changes, resembling rufflingThis triggers endocytosis into vesiclesSlightly different from receptor-mediated endocytosisThis 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.
61Salmonella Containing Vesicle After ingestion, Salmonella enters a SCV through bacteria-mediated endocytosisLives and multiplies in SCVVery little known about SCV or how bacteria exist insideA method to avoid host immune responsePhagosome: maturing SCVA phagosome is analogous to an endosome, prior to fusion with a lysosome.
62SPI-1 Effector Proteins SipABinds actin and stabilizes filamentsAllows actin to polymerize more easilyMaximizes efficiency of Salmonella invasionSipCAides in entry of other SPI-1 effector proteinsActivtes G-actin to form F-actin, then polymerizeAides in cytoskeleton rearrangements in membrane rufflingDifferent 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.
64SopB Main virulence factor Encoded by SPI-5 An enterotoxin associated with SPI-1 TTSSInduces an increase in concentration of cellular inositol polyphosphateIncreased chloride secretion into lumenNa+ follows to balance chargeWater follow to balance osmolarityAn enterotoxin is something that is produced by microorganisms an is involved in gastrointestinal infectionsSopB can increase cellular inositol phosphate becase it has two domains that are homologous to eukaryotic inositol polyphosphate 4-phosphatasesdiarrhea
65SPI-2 TTSS Activated once bacteria enters cell Necessary for systemic infectionSPI-2 TTSS secretes effector proteins from phagosome into cytosolInterfere with maturation of phagosomeNo fusion with lysosomeHow Salmonella avoids degredation in cellBoth 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.
66Flagella Another antigen Host cytotoxic T-cell response directed against flagellar epitopesN- and C- termini are highly conservedMiddle of flagellum is variableAn 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.
67Phase I / II FlagellaOperon encoding Phase I flagella also encodes for a protein that represses trascription of Phase IIThe switch mediated by an enzyme that inhibits Phase I, allowing Phase IIMay help Salmonella avoid cell-mediated immune responseSalmonella genome encodes for two types of flagella, Phase I and Phase II.
68Tumor Necrosis Factor-α Flagella from S. Typhimurium induces expression of TNF-α through cell-mediated reponsePhase II flagella are less-potent inducersSwitching mechanism may provide bacteria with a way to down-regulate inflammatory response within hostTumor 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.
69The scenario on the left is what happens with the switch to a Phase II flagellum. The inflammatory response is not triggeredOn the right, there is no switch, so Tumor Necrosis Factor-alpha is expressed, cell busts, releasing Salmonella into bloodstream and lymphatic systemThis activates the standard inflammatory immune response
70Immune Response White blood cells recognize – trigger T cells, B cells Two types of B cells: one to attack now, one for memoryMacrophages and neutrophils attack bacteria, secrete interleukins, causing cell-mediated response by T-cellsAntibodies from B cells attach to bacteria, allowing cytotoxic T cells, macrophages, and neutrophils to kill the organismThis 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.
71Inside Macrophages SPI-2 TTSS works in macrophages as well Bacterium produces enzymes that inactivte toxic macrophage compoundsHomocysteine (Nitric Oxide antagonist)Superoxide dismutase (inactivates reactive peroxides)Salmonella must produce additional factors to survive limited nutrient baseAllows 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.
72Septicemia Invasion of bloodstream spv genes causes detachment of cells to ECM and apoptosisSpreads infectionBacteria can enter bloodstream and lymphatic systemMain cause of death by SalmonellaBacteria can travel through lymphatic system and infect the liver and spleen, as well as causing septicemia in the bloodstream.
73How do we respond?Microbiological viewVaccinesDamAntibiotics
74Salmonella 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 biosynthesisDeletion 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 responseVaccine suitable for humans and mice, chickens, sheep, cattle
75DNA adenine methylase (Dam) Enzyme that methylates specific adenine residues in Salmonella genomeDisrupts regulation of DNA replication and repairRegulates expression of about 20 bacterial genes active during infectionDam (-) mutants are not virulentGood antimicrobial potentialCurrent “hot topic” of researchDam 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.
76Antibiotics Antibiotics are selective poisons Do not harm body cellsTarget different aspects of bacteria, such as ability to synthesize cell wall, or metabolismMIC: Minimum Inhibitory Concentrationthe minimum amount of agent needed to inhibit the growth of an organism
77Antibiotic Resistance Bacteria can counteract antibiotics by:Preventing antibiotic from getting to targetChanging the targetDestroy the antibioticBacteria can acquire resistanceHorizontal transfer from another bacteriaVertical transfer due to natural selection
86Identification ILaboratory identification of genus Salmonella: biochemical + serological testsHOW?- 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:
87Identification 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
88Nontyphoidal Salmonella General Incubation: 6 hrs-10 days; Duration: 2-7 daysInfective Dose = usually millions to billions of cellsTransmission occurs via contaminated food and waterReservoir:a) multiple animal reservoirsb) 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
89Nontyphoidal Salmonella Caused by S. typhimurium and S. enteritidisRainy season of tropical climates; Warm season of temperate climatesGrowing rapidly in the U.S.: five-fold increase betweenCentralization of food processing makes nontyphoidal salmonellosis particularly prevalent in developing countriesResistance is a concern, especially with multi-drug resistant S. Typhimurium known as Definitive Type 104 (DT 104)
90Nontyphoidal Salmonella: Gastroenteritis Incubation: 8-48 hrs ; Duration: 3-7 days for diarrhea & 72 hrs. for feverInoculum: largeLimited to GI tractSymptoms 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
91Nontyphoidal Salmonella: Bacteremia and Endovascular Infections 5% develop septicemia; 5-10% of septicemia patients develop localized infectionsEndocarditis: Salmonella often infect vascular sites; preexisting heart valve disease risk factorArteritis: 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 leadto death
92Septicemia Serotype S. choleraesius causes septicemia: - prolonged state of fever, chills, anorexia, and anemia- lesions in other tissues- septic chock, death
94Nontyphoidal Salmonellosis: Localized Infections INTRAABDOMINAL INFECTIONS:Rare, usually manifested as liver or spleen abscessesRisk factors: hepatobiliary, abdominal abnormalities, sickle cell diseaseTreatment: surgery to correct anatomic damages and drain abscessesCENTRAL NERVOUS SYSTEM INFECTIONS:Usually meningitis (in neonates, present with severe symptoms e.g. seizures, hydrocephalous, mental retardation, paralysis) or cerebral abscessesPULMONARY INFECTIONS:Usually lobar pneumoniaRisk factors: preexisting lung abnormalities, sickle cell disease, glucocorticoid usage
95Typhoidal Salmonellosis: Enteric Fever Incubation: 7-14 days after ingestion; Duration: several daysInfective Dose = 105 organismsSymptoms:a) 1st week: slowly increasing fever, headache, malaise, bronchitisb) 2nd week: Apathy, Anorexia, confusion, stuporc) 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 bradycardianeuropsychiatric symptoms: delirium and mental confusionLong term effects: arthritis
96Typhoidal Salmonellosis Late stage complications include intestinal perforation and gastrointestinal hemorrhageImmediate care such as increase antibacterial medications or surgical resection of bowelOther rare complications include inflammation of the pancreas, endocardium, perocardium, myocardium, testes, liver, meninges, kidneys, joints, bones, lungs and parotid gland and hepatic/splenic abscessesIn general, symptoms of paratyphoid fever are similar to typhoid fever, but milder with a lower mortality rateMajority of bacteria gone from stool in 8 weeks; However, 1-5% become asymptomatic chronic carriers: gallbladder is the primary source of bacterium
97Typhoidal SalmonellaChest PA view shows pleural effusion, left lower pulmonary lobe atelectasis, medial and downward shiftof bowel gas, and an increase in the air-fluid level in the abdomen
98Pictures(A)(B)(A) In sub-acute infections, multiple white to yellow foci occur in the liver, spleen is enlarged, andmesenteric lymph nodes may be enlarged(B) Histopathological examination may reveal necrotizing splenitis and hepatitis, with necroticfoci often accompanied by colonies of bacteria (arrow in right photo).
99Treatment of Typhoidal Salmonellosis Third generation cephalosporins or quinolones is the current treatmentIV 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 treatmentNalidixic acid resistant S. typhi (NARST) must be tested for sensitivity to determine course of treatmentSever typhoid fever (altered consciousness, septic shock): dexamethasone treatmentChronic carriers: 6 weeks of treatment with either oral amoxicillin, ciprofloxacin, norfloxacinSurgical intervention to remove damaged cells
100Prevention 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!!!
102Preventive 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.
103Salmonella 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
104Salmonella 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.
105Salmonella Overview History and Epidemiology Molecular Biology ClinicalWeaponization
107CDC classificationCategory 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 2Risk 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 typhimuriumSalmonella 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
108WHO 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.
109Salmonella 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 areaheavy reliance on monoculture of a restricted range of genotypesmajor agricultural exporter, or heavily dependent on a few domestic agricultural productssuffering serious domestic unrest, or the target of international terrorism, or unfriendly neighbor of states likely to be developing BW programs
110First Use of Salmonella as a Bioterrorist Weapon From , Japan conducted biological warfare experiments in ManchuriaAt Unit 731, a biological warfare research facility, prisoners were infected with Salmonella typhosa among other biological agentsAdditionally, 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 aircraftDue 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.
111Oregon 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. Theybrewed a "salsa" of salmonella and sprinkled it on the town's restaurant salad bars. Tenrestaurants were hit and more than 700 people got sick.First large scale bioterrorism attack on American soilA communitywide outbreak of salmonellosis resulted; at least 751 cases weredocumented 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, butput the blame on food handlers. In 1984, who could have imagined bioterrorism? caused by S. typhimurium as this type
112Salmonella as a Bioterrorist Weapon Wide distribution of food: contaminated food produced in one country can cause illness in other countriesTraceabilityAntimicrobial resistance: strains ofSalmonella are being found that havemultiple drug resistanceCapacity building: Salm-gene projectused to enhance outbreak detection byroutinely sub-typing certain salmonellasusing molecular methods
113Salmonella as a Bioterrorist Weapon Contaminating unguarded food suppliesSome terrorist acts may be designed purely to spread panic: contaminating the food supply could bring economic and agricultural production to a standstillEX. If numerous food-borne outbreaks occurred across the country, people would soon fear their mealsUnfortunately, 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.
114Salmonella as a Bioterrorist Weapon readily accessible and easy to grow or makeCentralized food production: largely unmonitored food supply; food that is tampered with can be widely + quickly distributedTerrorist 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 annuallyAccording 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
115Salmonella 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 damageFor 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.
116Salmonella 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
117Salmonella 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 strainsDestabilize a government by initiating food shortages/unemployment: the potential for immense economic damage due to contamination of the food supplyAlter 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.
118Salmonella 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 disseminateFew 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 oneMultiple 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
119Salmonella DilemmaDissemination 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 lethalAlternative 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.
120Combating 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 undetectedNew 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.
121Lines of DefenseFood processors should limit access to their production, storage and packaging areas: rerouting traffic, installing locksRandomized safety checkpoints: will increase fear of detectionCOSTS:Increase work forceSampling and test costsRecord keeping
122Government ActionCDC monitors the frequency of Salmonella infections in the country and assists the local and State Health Departments to investigate outbreaks and devise control measuresFDA 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 animalsUSDA 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.
123Biological 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)