Salmonella

What family is Salmonella from?

Enterobacteriaceae

What morphology does Salmonella have (gram stain and shape)?

gram negative rods

How do salmonella obtain energy?

oxidation and reduction of organic sources

What type of pathogen is Salmonella?

facultative intracellular

What does it mean if a pathogen is facultative intracellular?

can survive both inside and outside eukaryotic cells

How are most Salmonella strains motile?

via peritrichous flagella (rotate to propel)

What is produced by most Salmonella strains?

hydrogen sulphide

Which media will enrich Salmonella (3)?

• tetrathionate broth

• MacConkey agar

• Xylose lysine deoxycholate

Why is MacConkey agar often used to detect enteric bacteria (2)?

• presence of bile salt (deoxycholate)

• phenol red indicator detects lactose fermentation

What colour are Salmonella colonies on MacConkey agar (2)?

• colourless (cannot metabolise lactose)

• Salmonella lac colonies are light pink

What media can be used to distinguish Salmonella from other Enterobactericeae (2)?

• xylose lysine deoxycholate (XLD)

• black colonies (due to hydrogen sulphide production)

How are Salmonella subdivided into different serovars (2)?

• Kaufmann White scheme

• based on O and H antigens

How many Salmonella species are there?

2 (S.bongori and S.enterica)

What tests can be carried out to further subtype Salmonella (5)?

• biochemical tests

• phage typing

• AMR profiling

• DNA profiling

• multi-locus sequencing

What are some methods of bacterial DNA profiling (3)?

• pulsed-field gel electrophoresis

• variation in length of restriction fragments (RFLP)

• random amplicons (RAPD)

What are the 4 main manifestations of Salmonella infection in humans and domestic animals?

• enteritis (watery diarrhoea)

• typhoid fever

• abortion (particularly in cattle and sheep)

• subacute infection (can be protracted ‘carrier state‘)

How are Salmonella serovars broadly classified (2)?

• host range

• disease presentation

When does the broad serovar classification of Salmonella based on host range and disease presentation apply (2)?

• ONLY in healthy, outbred adults orally infected

• if a young, immunocompromised animal or adult is injected infection will likely always be severe

Which Salmonella species are more likely to be systemic (based on broad serovar classification)?

host-specific serovars more likely yo be systemic than host ubiquitous

What is typhoid fever?

severe invasive disease of humans

How many cases of typhoid fever are seen a year and what is the fatality rate?

• 21.7m cases / year

• 1% fatality rate (30% if untreated)

How is typhoid fever (Salmonella) transmitted?

person-to-person via water and food (inadequate sanitation)

What are the main Salmonella serovars responsible for typhoid fever?

Typhi and Paratyphi A, B and C

What are the 4 stages observed in Typhoid fever?

1. slow rising fever

2. prostrate with high fever, delirium, tender abdomen and constipation

3. complications of intestinal haemorrhage

4. recovery or death

What may follow Typhoid fever and who is an example of this?

• sub-acute carrier state (long term)

• ‘Typhoid Mary’ Mallon (infected 51 people as a cook)

What is associated with the sub-acute carrier state of Salmonella (2)?

• colonisation of gallbladder

• secretes bile (and salmonella) into the duodenum

How can typhoid fever be controlled (2)?

• antibiotics

• e.g. ampicillin, chloramphenicol, ciprofloxacin

What resistance is exhibited by Salmonella (to antibiotics) (3)?

• many strains resistant to multiple antibiotics

• 3rd generation cephalosporins now first choice

• e.g. ceftriaxone or cefotaxime

How is AMR acquired by Salmonella?

horizontal gene transfer of plasmids or mobile elements

What vaccines are available for Salmonella (2)?

• injectable VI polysaccharide

• live oral Ty21a (aroA)

Which groups are / are not recommended to get the Salmonella vaccine (3)?

• NOT effective in children

• recommended for travellers to endemic areas

• boosters required

How many cases of non-typhoidal salmonellosis occur worldwide each year (2)?

• 78 million cases

• 59 thousand deaths

Why is Salmonella rare in poultry in the UK (2)?

• seovars Enteritidis and Typhimurium controlled by vaccination

• incidence of <0.1%

What are the possible theories around how eggs are contaminated with Salmonella (4)?

• while descending from colonised ovarian tissue

• laterally as egg passes upper-oviduct tissues

• ascending from cloaca

• penetration of egg shell post lay from faeces

Why is the estimated number of UK pig and cattle Salmonella cases likely to be an underestimate (2)?

• one case and any subsequent cases on a premises within a month = 1 report

• e.g. could be just 1 pig or the whole herd

What are the symptoms of Salmonella in pigs (2)?

• severe diarrhoea, vomiting and pyrexia

• sub-acute carrier state

Which country has a problem with swine typhoid (2)?

• USA

• caused by S. Choleraesuis

Why is control of Salmonella in pigs important (2)?

• 100 fold reduction could = 60-80% reduction in human cases of pork origin

• potential for zoonosis

How are pork samples analysed for Salmonella presence?

‘meat juice’ ELISA test for antibodies to Salmonella LPS

What is the main Salmonella strain in cattle?

S. Dublin (89.4% cases)

Why are there few human cases of Salmonella in the UK of cattle origin ()?

strong controls over fate of gut, lymphatic system and CNS after BSE outbreak (mad cow disease)

What are the common features of Salmonella infections in domestic animals (5)?

• neonatal animals = more severe and spread to other organs

• untreated can be life threatening

• acute infection can be followed by sub-acute carrier state

• reactivation may occur following stress

• number of cases likely to be higher than reported

What are the stages of Salmonella pathogenesis (8)?

• ingestion

• evasion of innate defences

• attachment

• invasion

• net replication

• damage to host

• potential systemic translocation

• transmission

What are the main targets for Salmonella attachment in most mammals (2)?

• follicle-associated epithelium in distal small intestine

• enterocytes and M cells

How is Salmonella adherence mediated?

by proteinaceous fibres (fimbriae) and surface proteins

How do Salmonella move towards the mucosal surface?

flagella

What does Salmonella induce on M cells and enterocytes?

lamellipodia (membrane ‘ruffles‘)

What region of the Salmonella chromosome is required for internalisation (2)?

• Salmonella pathogenicity island (SPI)-1

• engulfed into ‘Salmonella-containing vacuoles’ (SCVs)

What is encoded on the Salmonella pathogenicity island (SPI)-1?

type III secretion system (T3SS) (molecular syringe)

What is the type III secretion system (T3SS) encoded on Salmonella pathogenicity island (SPI)-1 required for (2)?

• bacterial invasion and induction of enteritis

• injects a set of effector proteins to subvert cellular responses

How was the role of the Salmonella type III secretion system (T3SS) investigated (4)?

• male dairy cows under terminal anaesthesia

• intestine externalised and ligated into sections

• can inject different strains

• remove T3SS = no response

What 2 families of proteins are secreted by the Salmonella type III secretion system (T3SS)?

• translocon components (Sips) form the ‘needle’

• Effectors (Sops) hijack cellular processes

What are Sips and Sops secreted by the Salmonella type III secretion system (T3SS) considered to be (2)?

• novel toxins

• act in manner distinct from classical enterotoxins (like cholera toxin)

What are the roles of Sip proteins secreted by the type III secretion system (T3SS) (2)?

• form the ‘needle’

• some enter and influence invasion and enteritis

What are the roles of the SopE/SopE2 proteins secreted by the type III secretion system (T3SS) (4)?

• guanine nucleotide exchange factors for Rho family GTPases that regulate actin

• causes membrane ruffling

• structural mimic of cellular factors

• followed by SptP to restore normal cellular architecture

What is the role of the SptP proteins secreted by the type III secretion system (T3SS)?

restore normal cellular architecture after action of SopE/SopE2

What are the roles of the SopB protein secreted by the type III secretion system (T3SS) (4)?

• inositol phosphatase that acts on cellular messengers (hijacks messages):

  • activates RhoGTPase Cdc42 to augment invasion

  • activates Akt to delay apoptosis

  • antagonises Cl- ion channel closure = fluid secretion

What are the roles of the SipA protein secreted by the type III secretion system (T3SS) ?

promotes release of neutrophil chemoattractant hepoxilin A3 into gut lumen

Which TLR senses the Salmonella flagella and what is the response (2)?

• TLR5

• pro-inflammatory cytokine release (e.g. IL-8)

What causes Salmonella during salmonellosis (4)?

• SopB = chloride ion secretion into lumen

• osmotic flow of water

• SopB, SopE/E2 and SipA = disruption of tight junctions between enterocytes

• inflammation = increased vascular permeability and loss of epithelial integrity

• loss of surface area = loss of net absorption

How does inflammation confer a selective advantage for Salmonella in terms of metabolism (3)?

• inflammation = oxidation of thiosulphur in gut

• releases tetrathionate

• used by Salmonella as a respiratory electron acceptor during anaerobic growth

What evidence exists for the role of inflammation conveying an advantage to Salmonella?

mutants unable to use tetrathionate due to ttrA mutation are outcompeted by WT in the inflamed intestine

How does inflammation confer a selective advantage for Salmonella in terms of colonisation (3)?

• alters composition of gut microbiota (shown in mice)

• removes the colonisation resistance provided by indigenous flora

• dependent on T3SS-1 mediated colitis

What is a key strategy employed by Salmonella during cellular ninvasion?

prevent fusion of SCV with lysosomes

What mediates Salmonella intracellular net replication?

second type III secretion system (T3SS-2) on (SPI)-2

Where does the second type III secretion system (T3SS-2) inject it’s proteins?

into cytosol across the SCV membrane

What are the roles of proteins secreted by the second type III secretion system (T3SS-2) (5)?

• maintains SCV integrity and prevents lysosome fusion

• impairs assembly of NAPDH oxidase on SCVs = prevents oxidative burst

• induces macrophage death by apoptosis

• promotes migration of APCs to aid systemic spread

• inhibits MHC peptide loading and expression

What happens to a proportion of Salmonella cells following macrophage infection (2)?

• become non-replicating ‘persistors’

• reversible state where cells are refractory to antibiotics targeting growth dependent processes

What are the features of Salmonella pathogenicity islands (SPIs) (4)?

• rich in virulence genes

• low %GC

• often associated with tRNA loci

• often flanked by direct repeats / insertion sequences and contain ‘motility’ genes

What are 2 examples of motility genes?

• integrase

• transposons

What process are the 4 different SPIs involved in?

• SPI-1 = invasion

• SPI-2 = intracellular survival

• SPI-4 = adherence

• SPI-5 = enteritis

What is encoded on the via locus on S. Typhi (systemic typhoid fever in humans)?

capsular polysaccharide Vi antigen

What are the roles of the Vi antigen encoded on the via locus on S. Typhi (systemic typhoid fever in humans) (4)?

• mediates resistance to complement killing

• mediates phagocytosis resistance

• masks LPS from TLR-4

• prevents neutrophil chemotaxis

What is the Vi antigen encoded on the via locus on S. Typhi (systemic typhoid fever in humans)?

polysaccharide chain forming a capsule over the bacterium surface

What is the role of the TviA antigen encoded on the via locus on S. Typhi (systemic typhoid fever in humans)?

represses flagellin expression and thus TLR-5 activation

What are the theories behind why S. Typhi (systemic fever) is more virulent than S. Typhimurium (short-lived enteritis) ()?

• via locus containing Vi antigen

• contains deletions and broken genes conferring loss of function:

  • may be why S. Typhi is specific to humans

  • may also result in slow growth = dissemination by stealth