Genus Salmonella:
- Salmonella is an enterobacteria (fermentative, facultative anaerobes, oxidase -ve, gram -ve rods, catalase +ve).
- Motile (generally), aerogenic, non-lactose fermenting urease -ve, citrate +ve, Acetyl methyl carsinol -ve, KCN- sensitive.
- The genus Salmonella includes bacilli which parasitize the intestine of several vertebrates and infect human beings, causing enteric fever, gastroenteritis, septicemia with or without focal suppression and carrier state.
- The most important member of this genera is Salmonella Typhi, causative agent of typhoid fever.
- Typhoid bacillus was 1st observed by Eberth (1880) in mysentric node and spleen of foetal cases of typhoid fever and was isolated by Gaffky (1884).
- It came to be known as the Eberth-Gaffky bacillus or Eberthella Typhi.
- In 1885 Salmon and Smith described the bacillus and redesignated as S. Typhi.
- Salmonella comprises above 2000 serotype, all of them are potential pathogens.
- The Genus Salmonella are killed at 55oC in 1 hour or at 60oC in 15 minutes.
- Boiling, chlorination of water, pasteurization of milk destroys the organism.
- They can survive for weeks in polluted water and soils and for months in ice.
- Cultures may be viable for years if prevented from drying.
- They are killed within 5 minutes by mercury chloride (1:500) or 5% phenol.
- For practical purpose, they may be divided into two groups:
- i. Enteric fever group:
- Consisting of typhoid and paratyphoid bacilli.
- These are exclusively human parasite.
- ii. Food poisoning group:
- These are essentially parasites animal parasite but can also infect human beings producing gastroenteritis, septicemia and localized infections.
Morphology of Salmonella:
- Salmonella is Gram Negative rods, measuring 2-4 X 0.6 μm.
- Non-acid fast
- Non-capsulated and non-sporing
- Most serotypes are motile with peritrichous flagella
- S. galinerum and S. pullorum are non-motile.
- Most strains of most serotypes produce type-I fimbriae.
Antigenic structure of Salmonella:
- Salmonella possess following antigens:
- Flagella antigen (H)
- Somatic antigen (O)
- Surface antigen (Vi)- found in some species
- Several strains carry fimbriae.
- Fimbrial antigens are not important in identification but may cause confusion due to their non-specific nature and widespread sharing among enterobacteria.
- Flagellar (H) antigens:
- These antigens are determinant groups on the flagellar protein.
- They are heat labile and alcohol labile but are well preserved in 0.04-0.2% formaldehyde.
- Heating at 60oC of temperature detach the flagella from the bacteria and detachment of all flagella is achieved by heating for 30 minutes at 100oC.
- Somatic (O) antigens:
- It is a phospholipid protein polysaccharide complex which forms an integral part of the cell wall of bacteria.
- They are hydrophilic and enable the bacteria to form stable homogenous suspensions in saline.
- Over 60 different O-antigens have been recognized.
- The o-antigens are heat stable being unaffected by heating for 2.5hrs at 100oC and also alcohol stable withstanding treatment with 96% ethanol at 37oC for 4 hrs.
- The heat treatment destroys flagellar and fimbrial antigen while alcohol treatment detaches flagella from bacteria.
- The O-antigens are unaffected by suspension of bacteria in 0.2% formaldehyde.
- O-antigen is identical with endotoxin.
- It can be extracted from the bacterial cell by treatment with trichloro acetic acid, at first shown by Boivin.
- Therefore, it is also named as Boivin antigen.
- When treated with phenol, it splits off the protein moiety eliminating the antigenicity but retaining the toxicity of the complex.
- The O-antigen is less immunogenic than H-antigen and the titer of O-antibody is generally low than H-antibody.
- Surface antigen (vi):
- It is supposed to be virulent factor.
- Almost all isolated Salmonella form vi-antigen as a covering layer outside their cell wall.
- This antigen is an acidic polysaccharide.
- When fully developed, it reduces the bacteria agglutinable by vi-antibody and in-agglutinable by O-antibody.
- Many strains of Salmonella (S. Typhi) fails to agglutinate with O-antiserum.
- When freshly isolated, this is due to presence of a surface polysaccharide antigen enveloping the O-antigen.
- Felix and Pitt who first described this antigen found out that it was associated to virulence and thus named it vi-antigen.
- Freshly isolated strain of S. Typhi rich in vi-antigen (v-form) produce more opaque colonies than strains lacking vi-antigen (w-form).
- The vi-antigen tends to be lost on serial subculture (v-forms becomes w-form).
- The vi-polysaccharide acts as a virulence factor by inhibiting phagocytosis, resisting complement activation and bacterial lysis by the alternative pathway and peroxidase mediated killing.
- In human volunteer experiments, strains possessing vi-antigens were found to cause clinical disease more constantly.
- The vi-antigen is heat labile, it can be removed from bacteria by heating suspension for 1 hr at 100oC and centrifuging the bacteria from the vi-containing fluid.
- Alternatively, burn without heating, vi-antigen gradually separate from the bacteria in a saline suspension.
- The bacteria in-agglutinable with the O-antiserum become agglutinable after boiling or heating at 60oC for 1 hr.
- It is also destroyed by 0.5N NaOH.
- It is unaffected by alcohol.
Cultural characteristics of Salmonella:
- Aerobic and facultative anaerobes
- Growing radially on simple media over a range of pH 6-8 and temperature (optimum 37oC).
- Colonies are large 2-3 mm diameter, circular, low convex and smooth, more translucent than coliform colonies.
- Colonies morphology of Salmonella on different medias:
- i. Nutrient Agar (NA) and Blood Agar (BA):
- In Nutrient Agar and Blood agar, after 24hr at 37oC, colonies of most strain of Salmonella are moderately large 2-3mm in diameter, grey white, moist, circular disc with smooth convex surface and entire edge.
- Their size and degree opacity vary with the serotypes.
- ii. Peptone water and Nutrient Broth (NB):
- In liquid medium (Nutrient broth), most strains give abundant growth with uniform turbidity of their surface pellicle usually forms on prolong incubation. R variants granular deposit and sometime a thick pellicle.
- iii. MacConkey bile-salt lactose Agar:
- After 24 hours at 37oC, colonies are pale yellow or nearly colorless, 1-3 mm in diameter.
- iv. Brilliant green MacConkey Agar:
- The addition to MA of Brilliant green 0.004gm/liter which is inhibitory to E. coli, Proteus and other commensal Enterobacteria likely to outnumber the Salmonella in faeces, makes these an excellent selective medium as well as differential medium for S. Typhi.
- Salmonella colony appear as low convex, pale green translucent colonies 1-3 mm is diameter.
- Lactose fermenting bacteria produce the purple colonies.
- Deoxycholate Citrate agar (DCA):
- Colonies of Salmonella in DCA are similar to or slightly smaller in size than those on MA.
- They are pale or nearly colorless, smooth, shiny and translucent.
- Sometimes they have black center and sometimes they are surrounded by a zone of clear medium.
- But these characteristics need 48hrs of incubation for their development.
- DCA is also selective for Shigella which give colonies similar to those of Salmonella and for this reason is probably the most widely used plating medium for isolation of intestinal pathogen from faeces.
- Unfortunately, Proteus spp grow well on DCA and produce a colony that may be mistaken with Salmonella or Shigella.
- Wilson and Blaia’s Brilliant green Bismuth sulfide Agar (BBBA):
- Especially for S. Typhi.
- Here, the crowded colonies about 1 mm is diameter may take up the dye from the medium and appear green or pale brown.
- Large discrete colonies have a black center and clear edge.
- All Salmonella may produce H2S which causes the colonies to the surrounded by a metallic sheen.
- Xylose Lysine Deoxucholate (XLD):
- XLD was developed by Taylor (1965) as a selective medium for Shigella because he found that Shigella dysenteriae and Shigella flexneri were liable to be inhibitory bu the concentration of Deoxycholate brilliant green, selenite and tetrathionate in the selective media most used for Salmonella.
- Colonies of Salmonella and Shigella are red (alkaline to phenol red) because Shigella do not form acid from the Xylose, lactose and sucrose in the medium within 24hrs and because Salmonella neutralize acid, they form from the limited amount of xylose by decarboxylating the lysine.
- Most Salmonella (Edwardsiella) are distinguished from Shigella because they produce H2S which react with ferric ammonium citrate to produce black center.
- Yellow (acid) colonies are formed by most other enterobacteria namely:
- Those e.g. E. coli that form so much acid from lactose and sucrose as not to be neutralizable by decarboxylation of lysine.
- Those e.g. Proteus that do not ferment lactose or sucrose but from failing to decarboxylate do not neutralize acid, they form from xylose. The acid reaction delay blackening of colonies of H2S producing strain of Proteus until after 24hrs.
- Enrichment media for Salmonella:
- Tetrathionate broth: enriches Salmonella including S. Typhi and sometimes Shigella but permits the growth of Proteus which may reduce tetrathionate and thus impairs the selectivity for Salmonella.
- Salenite F-broth: It is probably most used enrichment broth for specimen that may contain Salmonella or Shigella.
- Strontium chloride broth:
- Malachite green magnesium chloride broth
- Tetrathionate broth with brilliant blue
Biochemical tests for Salmonella:
i. Fermentation tests:
- Carbohydrates are generally fermented with the production of acid and gas.
- Salmonella typhi, S. galinerum and anaerogenic variants in other serotypes. E.g. s. typhimurium form acid only.
- Typically, glucose, mannitol, arabinose, maltose, dulcitol and sorbitol are fermented but not lactose, sucrose, salicin or adonitol.
- ONPG test for β
-galactosidase is negative.
ii. Decarboxylase tests:
- Salmonella decarboxylase the amino-acids: lysine, ornithine and arginine but not glutamic acid.
- S. Typhi is exceptional in lacking ornithine decarboxylase and S. paratyphi A is lacking lysine decarboxylase.
iii. Other biochemical tests:
- Most Salmonella have following characteristics:
- Indole -ve, MR +ve, VP -ve, Citrate utilize (Except by S. Typhi and S. Paratyphi A).
- Malonate not utilized, gluconate not utilized, urease -ve, H2S produced in Ferrous chloride gelatin medium except by S. Paratyphi A.
- No growth in KCN medium, gelatin not liquify.