Salmonellosis

clinical symptoms of salmonellosis

1. Incubation (time between ingestion and first symptoms): 12 hours to 36 hours 2. Target: Children and adults 3. Stomach cramps 4. Watery diarrhoea 5. Fever 6. Sometime vomiting Symptoms usually last for a few days.

Epidemiology of salmonellosis

1. Nature 2. Distribution 3. Causation 4. Transfer 5. Prevention 6. Control

Facultative Anaerobe test

Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycolate broth Facultative anaerobes can grow with or without oxygen because they can metabolise energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than fermentation Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest. Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest. Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube but not the very top. Aerotolerant anaerobes do not require oxygen as they use fermentation to make ATP. Unlike obligate anaerobes, they are not poisoned by oxygen. They can be found evenly spread throughout the test tube.

what are food-borne diseases?

"Foodborne diseases encompass a wide spectrum of illnesses. They are the result of ingestion of foodstuffs contaminated with microorganisms or chemicals. The contamination of food may occur at any stage in the process from food production to consumption and can result from environmental contamination, including pollution of water, soil or air."

2. Distribution of salmonellosis

Around 100.000 cases reported in Europe each year (8000 in UK) each peak cases peak in july with over 10,000 cases and dip to the lowest in january with under 6000 cases - this averages out at 8000 cases a year there is also no differences between male and female but children under 10 take up the most lab reports with people over 80 having lowest

4. Transfer of Salmonella - Reservoir

Contaminated food is the major mode of transmission for non-typhoidal salmonellae because salmonellosis is a zoonosis and has an enormous animal reservoir. The most common animal reservoirs are chickens, turkeys, pigs, and cows; dozens of other domestic and wild animals also harbor these organisms (A). Feces of production animals can contaminate irrigation water and cause their transfer to produce products (B). Wild animals as wild boars or foxes can contaminate produce products with their feces (C). Produce products contaminated with Salmonella can enter into the food chain and be consumed by humans (D). Pork meat is a source of Salmonella (E). Beef and specially lamb meat are a source of Salmonella enterica subspecies no-enterica. Also, milk and cheese can be a source of this microorganism (F). Poultry and poultry products are considered the principal source of human salmonellosis. Although the principal serotypes isolated from these kind of products belong to subspecies enterica, the no-enterica subspecies are also isolated (G). Cold-blooded animales are the principal source of non-enterica subspecies. Consumption of their meat and the use of these animals as pets are the principal routes the transference of no-enterica subspecies of Salmonella between humans and cold-blooded animals

what are zoonosis?

Derives from Greek zōon "aninal" and nosos "diseases" " A zoonosis is any disease or infection that is naturally transmissible from vertebrate animals to humans. Zoonoses may be bacterial, viral, or parasitic, or may involve unconventional agents."

5. Prevention of Salmonella

EU: integrated approach to food safety from the farm to the fork 1. keep clean - Wash your hands with soap and warm water before handling food. - Clean surfaces, such as kitchen countertops, before fixing food on them. - Wash your hands after you've had contact with animals, their food or treats or their habitats. 2. Separate raw and cooked food 3. Cook thoroughly 4. Keep food at safe temperature 5. Use safe water and raw materials

Salmonella pathogenicity island 1 (SPI-1) and 2 (SPI-2)

Systemic infection depends on the ability to invade and grow within host cells such as epithelial cells and cells of the monocyte/granulocyte lineage, including macrophages These include many 'effector' proteins that are translocated into host cells by either one or both of two type III secretion systems (T3SSs), encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). Spi-1 codes for 10 effectors with Spi-2 coding for 30 effectorrs Genes of the SPI-1 T3SS are expressed in response to signals sensed by bacteria in the intestine of the infected host. This T3SS becomes active upon contact with epithelial cells, translocating effectors across the host cell plasma membrane In S. Typhimurium, SPI-1 T3SS effectors also trigger the activation of mitogen-activated protein kinase (MAPK) pathways, leading to the production of proinflammatory cytokines such as interleukin (IL)-8, stimulating the recruitment of polymorphonuclear leukocytes (PMNs) and inducing acute intestinal inflammation SPI-2 functions by translocating effectors across the membrane of the Salmonella-containing vacuole (SCV) in infected host cells such as epithelial cells and macrophages. Delivery of SPI-2 T3SS effectors to the host cell cytosol is a precisely controlled process

activation of T3SS

The bacterium must know when the time is right to secrete effectors. Unnecessary secretion, when no host cell is in vicinity, is wasteful for the bacterium in terms of energy and resources. The bacterium is somehow able to recognize contact of the needle with the host cell. One method of recognition has been discovered in Salmonella, which relies on sensing host cell cytosolic pH through the pathogenicity island 2-encoded T3SS in order to switch on secretion of effectors

Diarrhoea

The gastric innate immune response to Salmonella. (A) Following the invasion of the mucosa, the presence of Salmonella is detected by pattern recognition receptors. Extracellular Salmonella are detected by Toll-like receptors inducing a transcriptional response leading to the expression of pro-inflammatory cytokines such as IL-23. Intracellular Salmonella activate NOD-like receptors that can induce IL-23 expression, as well as the assembly of NLRC4/NLRP3 inflammasomes that activate Caspase-1, promoting the secretion of mature IL-1β and IL-18. SPI-1 mediated activation of Caspase-1 in epithelial cells might contribute to IL-18 secretion. (B) IL-18 and IL-23 amplify the inflammatory response by paracrine signaling. IL-18 induces the release of IFNγ from T cells, while IL-23 induces the release of IL-22 and IL-17. These cytokines induce the increased production of mucins and antimicrobial peptides, and promote the release of CXC chemokines leading to an influx of neutrophils into the mucosa. (C) Infiltrating neutrophils are crucial for the killing of extracellular Salmonellae. Although considered an intracellular pathogen, Salmonella can be found extracellularly following transcytosis through M cells or after pyroptosis induced host cell lysis. Besides clearing the pathogen, neutrophil influx can also lead to damage to intestinal tissue, resulting in the loss of epithelial cell barrier function and promoting diarrhea.

Pathogenesis model of Salmonella in humans

The infectious dose around 10^3 bacteria 1) Salmonella strains that resist the low pH-environment of the stomach reach the bowel, traverse the intestinal mucus layer and adhere to intestinal epithelium by adhesins as those encoded in SP-3 and SP4. Once attached, Salmonella express the multiprotein complex T3SS located in SPI-1 and the microorganism is engulfed into the epithelial cell 2) The cecal mucose convert the H2S produced by the microbiota in Thiosulphate as a protective response. During Salmonella invasion, neutrophiles are released to the intestinal lumen and convert the thiosulphate in tetrathionate, that can be used as a respiratory electron acceptor by Salmonella and confers to them confers the ability of grow more than the fermenting commensal competitors 3) Inside the cytoplasm, Salmonella is located in Salmonella Containing Vacuoles (SCV) and express a second T3SS located in SPI-2, of key importance to survive and replicate inside host cells (epithelial cells and macrophages). Mature SCV migrate near to the Golgi apparatus and Salmonella cells replicate. 4) When Salmonella crosses the epithelium is engulfed by phagocytes as macrophages, and survive and replicate in SCV with a similar response than in epithelial cells. Migration of the phagocytes facilitates dissemination in the host via the bloodstream.

structure of the T3SS

Three membranes separate the two cytoplasms: the double membrane (inner and outer membranes) of the Gram-negative bacterium and the eukaryotic membrane. The needle provides a smooth passage through those highly selective and almost impermeable membranes The needle complex starts at the cytoplasm of the bacterium, crosses the two membranes and protrudes from the cell. The part anchored in the membrane is the base (or basal body) of the T3SS. the extracellular part is the needle. A so-called inner rod connects the needle to the base. The needle itself, although the biggest and most prominent part of the T3SS, is made out of many units of a single protein.

S. Typhimurium virulence factors

adhesins secretion system - T6SS and T3SS immune mimicry Extracellular polymeric substances Mobility Nutrient acquisition Exotoxin Endotoxin

what is the Type 3 Secretion System (T3SS)?

are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm, bypassing the extracellular milieu Salmonella-induced diarrhea is dependent on disruption of the intestinal epithelial barrier via the T3SS Salmonella enterica serovar Typhimurium SPI1 T3SS effectors SipA and SopA, -B, -D, and -E2 were shown together to induce fluid accumulation in intestinal bovine loops and diarrhea in vitro and in vivo in calve

3. Causation: Salmonella spp.

is a genus of rod-shaped (bacillus) Gram-negative bacteria of the family Enterobacteriaceae The two species of Salmonella are Salmonella enterica and Salmonella bongori. S. enterica is the type species and is further divided into six subspecies: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), indica (VI) these subtypes are further divided up inot 153 serotypes the top five serotypes responsible for human disease are S. Enteritidis, S. Typhimurium, S. Infantis, S. Stanley and S. Newport. (ATTENTION: S. Typhi and S. Paratyphi)

Salmonella enterica

is a rod-shaped, flagellate, facultative aerobic, Gram-negative bacterium and motile facultative aerobic = is an organism that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation or anaerobic respiration if oxygen is absent. Xylose Lysine Deoxycholate agar (XLD agar) is a selective growth medium used in the isolation of Salmonella and Shigella species from clinical samples and from food Salmonella species: red colonies, some with black centers. The agar itself will turn red due to the presence of Salmonella type colonies. Brilliant Green Agar is a selective and differential medium for the isolation of Salmonella species typical Salmonella colonies appear as pinkish-white or red colonies surrounded by a red halo in the medium.

serotyping for Salmonella

serotyping is based on the concept that microorganisms from the same species can differ in the antigenic determinants expressed on the cell surface the "O" antigen type is determined based on oligosaccharides associated with lipopolysaccharide. the "H" antigen is determined based on flagellar proteins Scientists determine the serotype based on the distinct combination of O and H antigens. it involves the combination of an insoluble particulate antigen with its soluble antibody - if they are complementary it will form an antigen-antibody complex this will be seen as clumps and agglutinate depending on the antibodies exposed to the H and O antigens on the bacteria - this can determine the serotype of the Salmonella a = shows a positive result b, c, d = shows a negative result

6. Control of Salmonella

Rehydration: Drink plenty of fluids Manage fever: Paracetamol Antibiotics: No except if bacteria reach the blood streams or compromised immune system Loperamide: No as it prolongs the diarrhoea

Cell invasion

Salmonella typhimurium colonizes the intestinal epithelium by injecting an array of effector proteins into host cells that induces phagocytic uptake of attached bacteria. Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules Antigen-processing cells use macropinocytosis, as well as phagocytosis, to sample potential antigens for presentation to T lymphocytes Pathogenic bacteria, such as Salmonella, can also induce macropinocytosis in order to invade macrophages, dendritic cells and B cells Virulence proteins secreted into host cells stimulate extensive membrane ruffling, which is a form of macropinocytosis Formation of macropinocytic membrane extension loosely attached to the bacteria which eventually lead to bacterial internalisation

1. Nature of salmonellosis

Salmonellosis is an acute diarrhoeal infection caused by ingestion of food contaminated with the bacterium belonging to the genus Salmonella.

what are serotypes?

Serotypes are groups within a single species of microorganisms, such as bacteria or viruses, which share distinctive surface structures. The O antigens are distinguished by their different chemical make-up. The H antigens are distinguished by the protein content of the flagella. Each O and H antigen has a unique code number.

Bacterial proliferation

Invasive Salmonella use T3SS1 to translocate effector proteins into host cells. Several of these effectors drive actin-mediated ruffling and internalization of the bacteria into a modified phagosome or SCV T3SS1 effectors are also present on the SCV membrane and are important for rapid remodeling of the membranes as well as more sustained effects. The early SCV has many characteristics of early endosomes, including the phospholipid PI(3)P and proteins that interact with it such as Rab5 and SNX1. During this initial phase of infection the majority of Salmonella down-regulate T3SS1 and induce T3SS2, which is required for subsequent steps in SCV biogenesis. The majority of SCVs relocate to a juxtanuclear location within 1-2 h and become enriched in proteins, such as Lamp1, Rab7, and vacuolar ATPase, that are normally found in late endosomes and lysosomes. However, some SCVs do not undergo this maturation process and instead either lyse and release the bacteria into the cytosol or are targeted by the autophagy system. In the mature SCV replication is initiated 4-6 h post invasion and is accompanied by the formation of a dynamic tubular network that extends from the surface of the SCV. Tubules enriched in Lamp1 are known as Sifs although another population of Lamp1-ve tubules (SISTs) has recently been described. In epithelial cells cytosolic Salmonella replicate to high numbers, compared to bacteria inside SCVs, and become re-induced for T3SS1 and flagella. Salmonella can also invade cells via T3SS1-independent mechanisms (right side), although biogenesis of the SCV under these conditions has not been well studied.

Xylose-lysine-deoxycholate agar

It has a pH of approximately 7.4, leaving it with a bright pink or red appearance due to the indicator phenol red Sugar fermentation lowers the pH and the phenol red indicator registers this by changing to yellow. After exhausting the xylose supply Salmonella colonies will decarboxylate lysine, increasing the pH once again to alkaline and mimicking the red Shigella colonies Salmonellae metabolise thiosulfate to produce hydrogen sulfide, which leads to the formation of colonies with black centers and allows them to be differentiated from the similarly coloured Shigella colonies. C = Salmonella and E.coli ferment xylose (acidification) so media become yellow B (white) = BUT after xylose been consumed, Salmonella decarboxylate lysine (alkalization): media turn red B (black) = Salmonella metabolise thiosulfate: production of hydrogen sulphide (black colony

biochemical tests for Salmonella

urease test = identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide Christensen's urea agar is used to detect urease activity in a variety of microorganisms urease production is indicated by a bright pink (fuchsia) color on the slant that may extend into the butt after 1-6 hours of incubation The culture medium will remain a yellowish color if the organism is urease negative. Salmonella are urease-negative oxidase = used in microbiology to determine if a bacterium produces certain cytochrome c oxidases It uses disks impregnated with a reagent such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark-blue to maroon color when oxidized, and colorless when reduced. Salmonella are oxidase-negative indole test = to determine the ability of the organism to convert tryptophan into indole. Pure bacterial culture must be grown in sterile tryptophan or peptone broth for 24-48 hours before performing the test. Following incubation, five drops of Kovac's reagent are added to the culture broth. A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. Salmonella are indole-negative