Salmonella: Salmonella are widespread pathogens of vertebrates including man.
Mycobacterium tuberculosis
General Characteristics of Salmonella:
Habital: Most salmonella are found in the intestines of animals especially of pigs, cows, goats, sheep,
rodents, hens, ducks and other poultry. S. Typhi and S. Paratyphi however are usually found only in humans.
Morphology: Gram-ve rods, non-sporing, non capsulated bacteria (except S. Typhi)
 Measures about (2-4)x(0.6µm)
 Most strains are motile due to the presence of peritrichous flagella except S. Gallinarum and S.
Pullorum.
 Salmonella are not killed by drying and therefore survive in products such as dried eggs or
bone-meal fertilizers.
 Salmonella are aerobics and facultative anaerobes. They grow between 15-45 0C with an
optimum temperature of 370C.
 Salmonella may be divided into two groups: typhoidal (enteric fever group consisting the
typhoid and paratyphoid bacilli) and non-typhoidal (food poisoning group)
Antigenic Structure: Salmonella possess the following structure based on which they are classified and
identified as; Flagellar antigen(H), Somatic antigen(O),Surface antigen(Vi).
H antigen: This antigen present on the flagella is a heat labile protein.
 It is destroyed by boiling or by treatment with alcohol but no by formaldehyde.
 When mixed with antisera, H suspensions agglutinate rap[idly, producing large, loose clumps.
 Many H antigens are diphase i.e. they can occur in 2-antigenic forms referred to as phase I and
phase II.
O antigen: The somatic O antigen is phospholipid-protein-polysaccharide complex which forms an integral
part of the cell wall.
 Salmonella are grouped by their O antigens. The groups are designated A to Z. The O antigen is
unaffected by boiling alcohol or weak acids.
 When mixed antisera, O antigen suspensions from compact, chalky, granular clumps.
Vi antigen: These surface(K) antigens can be found on S. Typhi.
 It is associated with virulence and can be detected using Vi antiserum. These are surface
polysaccharide antigen enveloping the O antigen.
 It is heat table, is destroyed by IN HCL and 0.5N NaOH.
 Vi antigen can interfere with O antigen testing.
Virulence Factors:
 Endotoxin: Lipopolysaccharides (LPS)
 Invasions: Invasions are proteins that mediate adherence to, and penetration of, intestinal
epithelial cells. These proteins (type III protein secretion systems) are produced by inv genes.
 Resistance to acid pH: Salmonella are protected from the stomach acid and the phagosome
by acid tolerance response (APR) gene of the chromosome.
 Resistance to phagocytosis: Catalase and superoxide dismutase produced from bacteria
protect them from intracellular killing by neutralizing oxygen cells.
Pathogenesis
 After ingestion of food or water, typhoid organisms go into the stomach. S. Typhi must survive
the gastric acid barrier to reach the small intestine. S. Typhi is protected from stomach acid by
ATR gene of it.
 Then through the pylorus (a small circular opening between the stomach and the duodenum),
bacteria reach the small intestine. In the small intestine, the bacteria adhere to mucosal cells
and then invade or penetrate the intestinal epithelial cells.
 The M cells, specialized epithelial cells overlaying payer’s patches (organized lymphoid
nodules usually found in the lowest portion of the small intestine, the ileum are probably the
site of the internalization of S. Typhi.
 S. Typhi is transported to the underlying lymphoid tissue and multiplication of bacteria occurs
into the lymph nodes (payer’s patches). Lymph nodes contain macrophages. Catalase and
superoxide dismutase produced from bacteria protect them from phagocytosis. Bacteria
survive and are released into the bloodstream where they disseminate widely (transient
primary bacteraemia) or systematically to colonize the liver and spleen.
 As bile is a good culture medium for the bacterium, it multiplies abundantly in the gall bladder
and is discharged continuously into the intestine and can lead to re-infection of the intestinal
tract, causing inflammation, ulceration and necrosis. Haemorrhage form ulcers during the
third week of illness can cause generalized peritonitis and septicaemia, the commonest cause
of death in typhoid fever.
 M. tuberculosis is the causative agent of most cases of tuberculosis (TB).
 Acid-fast detection techniques are used instead, so it is Acid-fast bacillus (AFB). However,
mycobacterial cell was has characteristics of Gram Positive.
 It is highly aerobic and require high levels of oxygen.
 These are also called tubercle bacilli.
Virulence Factors:
 Cording Factor (glycolipid derivative of mycolic acid) present on outer surface of M. tuberculosis is
responsible for inhibition of migration of macrophages and encourages granuloma information.
 Cell surface glycoloipids inhibits phagolysosome formation permitting intracellular survival of
mycobacterial after ingestion by macrophages.
Pathogenesis
 Tuberculosis is the infection of the lower respiratory tract.
 Infection occurs when a person inhales droplet nuclei containing tubercle bacilli that reach the alveoli
of the lungs.
 These tubercle bacilli are ingested by alveolar macrophages; the majority of these bacilli are
destroyed or inhibited.
 A small number may multiply intracellularly in alveolar macrophages and are released when the
macrophages die. Survived organisms are carried to the nearest lymph node. In the lymph node, the
bacteria slowly multiple within macrophages.
 Bacteria multiply to a critical mass within the protected environment of the macrophages. Having
reached a critical mass, the organism spill out of the destroyed macrophages, through the lymphatics,
and into the bloodstream, producing mycobacteria and carrying tubercle bacilli to many part of the
body.
 Sometimes, a small reservoir of live bacteria may be left in areas of normally high oxygen
concentration, such as the apical (top) portion of the lung.
 Within 2-8 weeks, macrophages surround the tubercle bacilli to form a barrier shell, called a
granuloma, that keeps the bacilli contained and under control and this condition is known as latent
tuberculosis infection (LTBI).
 If the immune system cannot keep the tubercle bacilli or LTBI under control the bacilli begin to
multiply rapidly to cause tuberculosis (TB disease). This process can occur in different areas in the
body, such as the lungs, kidneys, brain or bone.
Laboratory Diagnosis
Specimens:
 Screw-cap, leak-proof specimen containers are used.
 Sputum, not saliva is required to detect AFB. Examination of up to three specimens (at least one as an
early morning specimen) may be required to detect the organisms.
 In AIDS patients, it is sometimes possible to detect AFB in buffy coat smears prepared from EDTA anti-
coagulated blood.
Microscopy/Morphology
 M. tuberculosis is a non-sporing, non-capsulated straight or slightly curved, slender rod, measuring 1-
4µm x 0.2-0.6 µm.
 Although it does not Gram stain well due to its waxy surface, the organism has a Gram positive cell
wall.
 M. tuberculosis is best demonstrated using the Ziehl-Neelsen staining technique or a fluorescence
technique.
 Ziehl-Neelsen staining of M. tuberculosis: When stained by the Ziehl-Neelsen technique, M.
tuberculosis is acid fast and stains red. This is due to the mycolic acids (fatty acids) in the cell
wall which form a complex with carbol fuchsin and cannot be removed by the acid in the
decolorizing reagent.
 Detecting AFB by fluorescence microscopy: Sputum smears can be examined rapidly using a
40X objective (or 25X objective) in fluorescence microscope. This increases the possibility of
finding AFB especially when they are a few.
Culture
 Cultural techniques for detecting M. tuberculosis, although more sensitive, are slow and expensive.
 It is possible to detect tuberculosis at an earlier stage of infection when AFB are too few to be
detected in direct sputum smears. Culture is considerably more sensitive than microscopy,
detecting 10-100 viable organisms of sputum.
Identification of M. tuberculosis cultures.
 The minimum investigations required to identify M. tuberculosis isolates are:
 Testing the pigment production: This is done by leaving the culture in light for 24 hours (avoiding
direct sunlight), reincubating it at 35-37 0C overnight, and then examining the colonies for the
development of a yellow pigment. M. tuberculosis is a non-chromogen, i.e, it does not produce
pigment in light or darkness.
 Incubation of a subculture of the organism at 250C: M. tuberculosis will not grow at 250C.
 Growth on Lowenstein Jensen medium containing 500g/ml 4 (p)-nitrobenzoic acid; M. tuberculosis
will not grow on 4 (p)-nitrobenzoic acid (PNB) medium.
Antimacribial susceptibility (treatment)
 The first line drugs (antibiotic): isoniazid, rifampicin, pyrazinamide, ethambutol.
 The second line drugs: streptomycin, capreomycin, cycloserine, thiacetazone and ethionamide.
 Vibrio Cholera: V. cholerae was first isolated as the cause of cholera by Italian anatomist Filippo Pacini in 1854,
but his discovery was not widely known until Robert Koch, working independently 30 years later (1883),
publicized the knowledge and the means of fighting the disease.
 It is a short, curved, cylindrical rod (comma shaped), about 3-4 x 0.5µm in size, with rounded or slightly
pointed ends.
 It is an aerobic/facultative anaerobic organism.
 It is actively motile and has a flagellum at one cell pole (polar flagella)
 They tolerate alkaline media that kill most intestinal commensals, but they are sensitive to acid.
 Some strains of V. cholerae cause the disease cholera.
Virulence Factors(of V. cholerae O1 and O139)
 Enterotoxin: V. cholerae secretes the enterotoxin choleragen (cholera toxin: CT) which is very similar to the
E. coli LT, the CT consists of A and B subunits. The A region, responsible for biologic activity of the
enterotoxin, is linked by non-covalent interactions with the B region. B region is composed of five identical
non-covalently associated peptide chains.
 Pili: These help in adherence to mucosal cells.
 Haemagglutination protease (mucinase): It helps to release bacteria from mucosal cells.
Pathogenesis
 Vibrios are sensitive to acid, and most die in the stomach.
 Surviving virulent organisms may adhere to and colonize the small intestine, where they secrete the CT.
 The toxins bind to the GM1 ganglioside receptor on intestinal epithelial cells by means of subunit B.
 Then disulphide bond that joins A1 and A2 is broken and the A1 subunit is activated. The A1 fragment
activates adenylyl cyclase in the enterocyte to form increased level of cyclic adenosine 5’
monophosphate(cAMP) within the cell.
 The marked increase of cAMP within the intestinal cells results in intense and prolonged hypersecretion of
water, chlorides and bicarbonate and inhibits the reabsorption (lysis and assimilation) of sodium. The
intestinal lumen or gut becomes full of fluid and consequent diarrhoea occurs.
 In hospitalized patients, this can result in losses of 20L or more of fluid per day.
 The stool of an actively purging. Severely ill cholera patient can resemble rice water-the supernatant of
boiled rice.
 Because the stool can contain 10 8 viable vibrios per ml, such a patient could shed 2x10 12cholera vibrios per
day into the environment.
 These symptoms usually start suddenly, half a day to five days after ingestion of the bacteria.
Laboratory Diagnosis
Specimen
 A faecal specimen is required to test directly for V. cholerae antigen, and to isolate V. cholerae in culture.
Microscopy
 V. cholerae is a Gram negative motile usually curved rod (vibro), measuring 3-4 x 0.5 µm with a single
flagellum at one end.
 V. cholerae 0139, unlike V. cholerae 01, is capsulated.
 Motility: V. cholerae is highly motile with a distinctive rapid to and fro movement which has been
likened gnats. Vibro mobility is best seen using dark-field microscopy, but the vibrios can also be seen
using transmitted light microscopy.
Culture
 V. cholerae is an aerobic and facultative anaerobe.
 It can grow over a wide temperature range of 16-40 0C with an optimum temperature of 37 0C.
 It grows best at an alkaline pH (pH 8.2).
 V. cholerae is non-halophilic i.e. it does not grow, like most other Vibrio species in media containing 6-
10% sodium chloride.
 Alkaline peptone water: V. cholerae grows rapidly, producing growth (turbidity) on and just below the
surface of the medium, usually within 4-6 hours. It is enrichment medium and its alkalinity suppresses
the growth of intestinal commensals.
 Thiosulphate-citrate bile salt sucrose(TCBS) agar: This is an excellent selective medium for the primary
isolation of V. cholerae. On TCBS agar, V cholerae 01 and 0139 produce 2-3mm in diameter sucrose-
fermenting yellow colonies after overnight incubation at 35-370C.
 TSIA: V. cholerae produces a red-pink slope and yellow butt. Gas is not formed and H 2S is not produced.
 Media containing bile salts: Most strains of V. cholerae are able to grow in MacConkey agar, producing
small non-lactose fermenting colonies after overnight incubation.
 Blood agar: V. cholerae 01 and 0139 grow on blood agar, (subcultured from alkaline peptone water),
often producing (beta) haemolytic colonies.
Biochemical Tests: V. cholerae 01 and 0139 reactions
 Oxidase positive: All vibrio species are strongly oxidase positive.
 Do not ferment L-arabinose. This test is of value in differentiating V. cholerae from V. fluvialis (both
produce yellow colonies in TCBS sugar). V. fluvialis ferments L-arabinose.
Laboratory Diagnosis
Specimen: (for typhoid fever)
 Blood, faeces and urine for culture.
Microscopy/Morphology:
Salmonella are Gram negative rods. They are non-sporing and with the exception of S. Typhi, non-capsulate.
Culture:
 Salmonella are aerobes and facultative anaerobes. They grow between 14-45 0C with an
optimum temperature of 370C. A selective medium is required to isolate salmonellae from
faecal specimens.
 Isolation of S. Typhi from blood: To isolate enteric fever salmonellae, 10% of ox-gall in distilled
water is recommended (add 5 ml of whole blood to 50 ml of sterile ox-bile medium).
Subculture after overnight incubation onto blood agar.
 The use of Columbia agar-broth diphase medium is recommended because this can be used
for all the salmonellae and other pathogens that cause bacteraemia.
 Blood cultures are sub-cultured on to blood agar.
 Blood agar (Subculture): Salmonellae produce grey-white 2-3 mm diameter non-
haemolytic colonies, similar in appearance to those of many other enterobacteria. Some
strains appear mucoid.
 XLD agar: Hydrogen sulphide (H2S)-producing Salmonella form pink-red colonies 3-5 mm in
diameter with black centres. [Salmonella that do not produce H 2S, e.g. most strains of S.
Paratyphi A, form pink-red colonies without black centres, similar in appearance to Shigella.]
 MacConkey agar: Salmonella produce non-lactose fermenting pale coloured colonies on MA.
Biochemical Tests
 Triple Sugar iron agar: This medium is used to help identify salmonellae following isolation on
a primary selective medium. Salmonella produce:
 Pink-red (alkaline) slope and yellow (acid) butt, indicating fermentation of glucose but not
lactase.
 Cracks in the medium if serotype produces gas from glucose fermentation (S. Typhi no gas)
 Blackening in the medium due to H 2S unless serotype does not produce H 2S. e.g, S.
Paratyphi A. Only a small amount of blackening is seen with S. Typhi.
 Urease and indole negative.
 Lactose negative
 Gas produced from glucose fermentation (S. Typhi does not produce gas)
 Citrate positive (S. Typhi and S. Paratyphi A are citrate negative)
 Lysine decarboxylase (LDC) positive (S. Paratyphi A is LDC negative)
 Beta-galactosidase (ONPG) negative
 S. Typhi: This can be biochemically differentiated from other salmonellae by being citrate
negative not producing gas and forming only small amount of H2S.
 Isolates of S. Typhi can be identified serologically.
Serological Diagnosis: Serological tests currently in use to assist the diagnosis of enteric fever include: Widal
test, IgM antibody immunoassays
 Widal test
 IgM antibody assays to diagnose typhoid fever: These assays detect IgM antibodies to S.
Typhi which develop early in acute typhoid. They suggest current infection, are more sensitive
and specific than the Widal test, and can be performed more rapidly. In the absence of culture
facilities, IgM antibody tests are more useful in helping to diagnose typhoid in endemic areas
particularly after 7 days following the onset of fever. Commercially available IgM antibody test
include Enterocheck-WB, TYPHI rapid and IDL Tubex.