Gram-negative Facultatively-anaerobic Rods Flashcards
What are the general characteristics of enterobacteriacea?
- Gram-negative
- Straight rods
- Motile (peritrichous), or non- motile
- Non-sporulating
- Facultative anaerobes
- Production of acid from glucose
- Catalase-positive Oxidase-negative
What is the major genera of enterobacteriacae?
- Escherichia
- Salmonella
- Shigella
- Citrobacter
- Proteus
History of Salmonella
- First identified in 1885 by Daniel Elmer Salmon (a vetinary surgeon)
- Theobald Smith was his assistant
- In 1990 Joseph Leon Ligneres proposed that the pathogen discovered by Salmon’s group be called Salmonella in his honor
Nomenclature
- 2600 different serotypes of salmonella (distinct variations within a species of bacteria)
- Split into salmonella bongori and Salmonella enterica (subdivided into 6 groups)
- Subroup I (enterica) is split into typhoid salmonella and non-typhoid salmonella
Typhoidal Salmonella
Salmonella typhi (tyhoid) has an incubation period of 8-14 days
Salmonella paratyphi A, B, or C (paratyphoid) has an incubation period of 8-14 days
They both spread in the sam eway:
They organism is ingested with contaminated food or water then it directly enters the bloodstream
Sympotoms: High fever, stomach ache, loss of appetite and rash
What are the generalised Characteristics of Salmonella?
- Gram-negative
- Facultative anaerobe
- Non-sporulating
- Peritrichous flagella
- diameters of ~0.7-1.5μm
- lengths of 2-5μm
- Growth:
Temp. 5.5 – 45.6°C (37°C) pH 3.8 – 9.5 (7-7.5)
Water activity (Aw) 0.99
Burden of Salmonella In The EU
Notification Pyramid For Gastrointestinal Infections
Pathogen-specific estimates of proportion foodborne from reported outbreaks, UK 2001-2008
The changing epidemiology of Salmonella
- 1940s : Lowest number of cases during World War II
- 1953-1954: saw a rapid increase of animal slaughter, a rise in meat and egg consumption
- 1959: highlighted a phenomenal surge in Salmonella infections linked to hens’ eggs
- 1980s: Enteritidis phage type 4 (PT4) was identified as the most prevalent serotype and the leading cause of foodborne infection was linked to shelled eggs
As a result of all eggs in the UK and USA were pasteurised and washed to effectively remove faecal contamination prior to packing
The changing epidemiology of Salmonella (1990s onwards)
1990s- Edwina Currys’ statement - sales of eggs plummeted 60% overnight
2002- introduction of the vaccine Nobilis Salenvac, which was administered to laying chickens
stimulates the production of maternally derived antibodies against S.Enteritidis in the chick before it hatches
All eggs laid from vaccinated hens are labelled with a red lion stamp and a best before date
Direct vs Indirect causes of food borne diseases
DIRECT: Contact with infected animals or environments
INDIRECT: Comtaminated- utensils, hands, food, water
What are the routes and reservoirs of salmonella transmission
Major routes: Farm animas→huaman food→Man
Minor routes: Animal importation→ Slurry/sewage environmental pollution→products eaten by animals
Foodborne outbreaks recorded in England and Wales from 1992 to 2008 showing causative agent by implicated food vehicles
- 20% salmonella cases attributed to meat and poultry
- 4.4% due to fish
What are the Signs & Symptoms of salmonella?
Nausea, vomiting, fever, diarrhoea and abdominal cramps
From 4-7 days illness ranges from mild to severe; most people recover without treatment
How does salmonella progress?
- Bacteria travel to small intestine adhere to lining; begin life cycle
- In severe cases, bacteria break through intestinal wall to bloodstream; can be deadly if not properly treater
What is the treatmentment for salmonella?
Oral or injected antibiotics, usually for 2 weeks
Salmonella in the body
Salmonella enters the body via consumption of contaminated foods
→Although human stomach acid can reduce and sometimes eliminate Salmonella spp, some bacteria get through to the intestine and then attach and penetrate the cells.
→Toxins produced by the bacteria (enterotoxin and cytotoxin) can damage and kill the cells that line the intestines, which results in intestinal fluid loss (diarrhea)
→Some Salmonella can survive in cells of the immune system and can reach the bloodstream, causing blood infection (bacteremia)
→Some Salmonella spp can enter the gallbladder, leaving the affected patient a chronic carrier of the organisms. Salmonella can then be shed with the bile from the gallbladder into the feces and then may infect other people
Salmonella Pathogenicity Island encoded type III secretion system
Salmonella has 5 pathogenicity islands in total
Pathogenicity island no.1- responsable for inflammation, diarhoeaand getting the cells into the intestines
Toxins released:
- SipA
- InJ
- SptP
- AvrA
- SopABDE
- sopE2
- SsH1
Pathogenicity island no.2: Spreading across intestine to other organs (systemic spread)
- SpiC
- SspH2
- SspH1
- SseBCD
Host pathogen interactions during pathogenesis
Describe the intracellular lifestyle of salmonella typhimurium (once engulfed)
- Vacuole formed during the engulfment of the bacterium by the host cell membrane
- A different type 3 secretion system is assembled and injects a different set of salmonella effectors across the vacuolar membrane to manipulate host function to protect the bacterium from host defences
- Thin filaments extend from the vacuole coeverd in salmonella effectors e.g SifA
- Now the baterium is safe and starts to replicate
- Salmonella triggers a profound inflammatory response
Salmonella’s type 3 secretion system
- Specialised needle like organelle (type 3 secretion system) to deliver its toxin directly into the host cells
- This system functions like a molecular syringe that injects the salmonella toxins as soon as it gets in contact with the host cell surface
- Toxins (effector proteins) include Sip A and SopE2 into the intestinal cells to manipulate the functions of cellular proteins and lipids
- As a result, the epitherlial cell membrane extends outward leading to the engulfment of the invading bacterium untill it is taken up completely into the interior of the host cell
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Food types tested in the laboratory
- Storage and Preparation of Samples
- Salmonella is not able to grow at low temperatures and samples should be refrigerated if they cannot be sent for analysis immediately.
- 25g food samples are cultured in detection testing
- dried foods require a resuscitation stage for sub-lethally damaged cells in non-selective pre-enrichment media, such as buffered peptone water
What is the primary method for salmonella identification?
Gram stain
Thin layer of peptidoglycan so wont take up purple dye so they appear as pink rods in the laboratory
What is the compostion of Selenite Cystine Broth- Enrichment Media?
Primary and secondary (selective) enrichment media are used to nurture any injured or stressed cells.
- Enzymatic Digest of Casein & Enzymatic Digest of Animal Tissue are sources of nitrogen & vitamin.
- Lactose is the carbohydrate
- Disodium Phosphate is the buffer.
- Sodium Selenite is the selective agent against Gram-positive bacteria and most enteric Gram-negative bacilli.
- L-Cystine is a reducing agent.
Agar media in the laboratory: Blood agar
Enriched medium
- Incubated in 5-10% CO2 at 35–37°C for 18-24hr
- Colonies are moist and 2-3mm in diameter.
Lots of organisms can grow on this media, won’t specifically identify salmonella
Agar media in the laboratory: Cystine-lactose-electrolyte deficient (CLED) agar
- Incubated in air at 35–37°C for 18- 24hr.
- Flat blue colonies of Salmonella
E.Coli appears yellow
Agar media in the laboratory: Desoxycholate citrate (DCA) agar
- Incubated in air at 35–37°C for 18-24hr.
- Colonies are colourless, and usually with a black centre (some serotypes eg Salmonella Paratyphi A)
Salmonella Typhi may not produce a black centre
Agar based methods: Xylose-Lysine-Desoxycholate (XLD) agar
Selective for salmonella alone
Colonies are red, and usually with a black centre (some serotypes eg S.Paratyphi A and S. Typhi may not produce a black centre).
Salmonella Brilliance Agar- chromogenic agar
Reduced time to result: 2 days compared with up to 5 days for standard culture
Slide Agglutination Test
- Used for serotypng (refers to O and H antigen)
- Antigen: isolated salmonella in suspension
- Antibody: specific antisera against salmonella
- Place test Salmonella in a drop of saline on a slide
- Add a drop of antiserum, mix and rock slide for approx 1 minute
- Examine for aggulation (positive)
Agglutination test for Salmonella species
Name Biochemical Tests for salmonella
- Urease test→Salmonella species do not produce urease
- Oxidase test→ Salmonella are oxidase negative
- Indole test→ Salmonella species are indole negative
Describe Matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS)
- Analyses the protein composition of a bacterial cell
- Reproducible, fast and sensitive analysis
- Results available within a few hours rather than several days.
- Minimal consumable costs
- Utilised to aid in both the detection and species-level identification of Salmonella
- Discriminating S. enterica serovar Typhi from other Salmonella serovars
Need to first grow the bacteria on an agar plate and use the colony for MOLDI-TOF
What are the treaments for salmonella?
- Rest
- Hydration
- Antibiotics
How can salmonella be prevented?
o Avoid risk factors
o Contact with animals
o Undercooked meat
What are the generalised Characteristics of E. coli?
- Gram-negative
- Facultative anaerobe
- Non-sporulating
- Peritrichous flagella
- Growth:
Temp. 10 - 55°C (37°C)
pH 4.5 - 9 (7-7.5)
Aw 0.96 (7% NaCl)
Describe the habitat of E.Coli
- Commensal:
Human & warm blooded animals
- Rapid colonisation:
1st day of life (first to colonise the body)
Humans normally from mother
- Indicator (of disease) organism
What are the main groups of pathogenic E. coli?
- Enteropathogenic E. coli (Infant diarrhoea)
- Enterotoxigenic E. coli (Travellers diarrhoea)
- Enteroinvasive E. coli (Fresh blood- dysentery- breaks through wall of gut)
- Enterohemorrhagic E. coli (EHEC) subdivided into →Verocytotoxin/Shiga toxin-producing E. coli (VTEC / STEC) →O157 “Model strain”(Blood in faeces) mimic the sverity of disease
Enterohemorraghic E. coli (EHEC)
First isolated 1974 in Canada
- Non invasive like EIEC
- Not produce enterotoxins like ETEC
- Clinically different from EPEC Recognised as foodborne pathogen 1982
- Main serogroups =O157, O26
What makes the strain of Enterohemorraghic E. coli (EHEC) O157?
- Serotyping (Kauffmann/White)
- O antigen on the LPS membrane (173)
- H (55)
O antigen can be 1-157
H antigen can be 1-55
173 different possibilites to type the salmonella
O157:H7
O157:H-
O157 and VTEC
- VTEC= produces vero cytotoxins
- 0157= Composition (O and H antigens)
Risk Factors
- Reservoir
GI tract of cattle
Other domesticated animals Vehicles
- Food
Beefburgers, milk, vegetables
- Person - Person
Faecal - Oral
- Contact
Animals
What is the incubation period and symptoms of Enterohemorraghic E. coli (EHEC)?
Symptoms
Incubation 3 - 9 days (mean = 4 days)
- Haemorragic Colitis (HC)-gastroenteritis
- Haemolytic Ureamic Syndrome (HUS)- blood disorder
- Thrombotic Thrombocytopaenic Purpura (TTP)- blood clots
Duration 2 - 9 days
I.D. = <100 organisms (Possibly 1 organism!)
E. coli O157 Pathogenicity
Acid and desiccation resistance
Intimate Binding of EHEC: Intimin/Tir
EHEC privides the receptor to the target cell, thus allowing the two to bind
Laboratory Detection of EHEC: What is the Typical Composition (g/L) of Sorbitol MacConkey Agar SMaC Agar white colonies?
Specific for E.Coli
- Peptone →20g/L
- NaCl →5g/L
- Bile salts →1.5g/L
- Sorbitol →10g/L
- Crystal violet →0.001g/L
- Neutral red →0.03g/L
- Agar →15g/L
