By Ms. Kostiuk from Microbiology department

1. Lesson N6: MICROBIOLOGIC DIAGNOSIS OF SALMONELLA GASTROENTERITIS
1.Scientifically methodical ground of theme
Salmonellae are a major cause of food-borne infection in the worldwide. In the UK and USA, reports of salmonella
infections increased steadily since the early 1969s, and this increase has recently accelerated.
2.Educational purpose
Students must know:
1. Structure, staining, fermentative properties and cultivation of Salmonellae – causative agents of
salmonella gastroenteritis
2. Epidemiology and pathogenesis of salmonella gastroenteritis.
3. Methods of laboratory diagnosis of disease, main methods of prophylaxis and treatment.
Students should be able to:
– prepare the smears from tested material;
– stain the smears by Gram’s technique;
– make microscopical examination of the smears;
– value the growth of Salmonellae in different media;
– value the biochemical properties of Salmonellae according to Hiss’ media;
3.Chart of topic content.
Salmonellae — Causative Agents of Food Toxinfections. The genus Salmonella comprises many
species and types of bacteria, which possess properties similar to those of S. schottmuelleri. In 1885 in America D.
Salmon isolated the bacterium S. cholerae-suis, which was long considered the causative agent of plague in pigs.
Later it was shown, that it associated with human toxinfections. In 1888 during a large-scale outbreak of
toxinfections in Saxony A. Gartner isolated S. enteritidis bacteria from the flesh of a cow, which had to be killed,
and also from the spleen of a dead person. The organisms proved to be pathogenic for mice, guinea pigs, rabbits,
sheep, and goats. In 1896 in Breslau K. Kensche and in 1898 in Ertike G. Nobel discovered S. typhimurium
(Bacillus Breslau) in cases of food poisoning and isolated a pure culture of the organism. It is now known that
among the large number of organisms, which comprise the salmonella group, about 440 species and types are
pathogenic for humans and cause food poisoning (toxinfections).
Morphology. Morphologically Salmonella organisms possess the general characteristics of the family
Enterobacteriaceae. They are motile and peritrichous.
Cultivation. The organisms are facultative aerobes, the optimum temperature for growth being 37° C.
They grow readily on ordinary nutrient media.
Fermentative properties. Salmonellae do not liquefy gelatine and do not produce indole. The majority of
species produce hydrogen sulphide and ferment glucose, maltose, and mannitol, with acid and gas formation.
Toxin production. Salmonellae produce no exotoxin. Their ability to cause diseases in animals and
humans is associated with an endotoxin which is a gluco-lipo-protein complex and is characterized by its high
toxicity.
Antigenic structure. As was mentioned above, all salmonella are divided into 65 groups according to their
serological properties.Thus, according to the Kauffmann-White Scheme, S. enteritidis belongs to group D, S.
typhimurium to group B, and S. cholerae-suis to group C.
Classification. The organisms are classified according to their antigenic, cultural, and biological
properties
Virulence Factors of Salmonella Organisms. It is surprising that virulence factors for organisms that have
caused so much disease still are largely unknown. However, the ability to invade and grow inside of non-
phagocytic cells undoubtedly comprises the major virulence determinant of the Salmonella because this
intracellular location provides a compartment where they can replicate and avoid host defences. The mechanism
whereby these bacteria accomplish this invasion is complex and only beginning to unfold.
Using various mutants of Salmonella typhimurium, John Pace and colleagues at the State University of New
York determined that invasion of a host cell occurs in two separable steps: (1) adhesion to the host cell, and (2)
invasion of the host cell. Furthermore, they found that invasion required that the organisms activate a growth factor
receptor on the host cell known as epidermal growth factor receptor (EGFR). Mutants that could adhere, but not
invade, were unable to activate EGFR. However, if EGF was added to the host cell-bacterium mixture, the EGFR
was activated and the noninvasive mutant was internalized.
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2. When EGFR is activated, a signal transduction process occurs, which results in at least two major events: (1)
a rapid rise in the internal Ca2+ level occurs, and (2) enzymes are activated that lead to the synthesis of leukotriene
D4 (LTD4). It is unclear how these events trigger the entry of Salmonella into the cell, but it is known that the Ca 2+
level increase is essential because the addition of Ca2+ chelators blocked entry of the bacterium into the cell. It is
also known that the addition of LTD4 to cultured cells causes an increase in intracellular Ca2+ levels, permitting the
internalization of an invasion-deficient mutant.
One can postulate, therefore, that the mediation of Ca2+ influx by LTD4 results in the opening of a Ca2+
channel, which, in turn, causes a reorganization of the host cell cytoskeleton, permitting entry of the bacterium.
It is also of note that the inflammatory diarrhea produced by the Salmonella may result from its ability to
induce leukotriene synthesis because leukotrienes are well-known mediators of inflammation.
It is also known that a number of Salmonella, serotypes carry plasmids that greatly increase virulence in
experimentally infected mice. Although many of these plasmids are distinct, all have a highly conserved 8-kb
region that has been named the spv regulon. Interestingly, spv genes are not expressed during logarithmic growth in
vitro but seem to enhance the growth of salmonellae within host cells. In experimentally infected mice, the
expression of spv by intraccelular salmonellae in vivo has been postulated to lead to an increased rate of bacterial
growth, resulting in early bacteremia and death before the infected mice can develop immunity.
The general types of infections that may be caused by the salmonellae usually are grouped into three
categories: enterocolitis, enteric fevers, and septicemia.
Resistance. Salmonellae are relatively stable to high temperatures (60-75 °C), high salt concentrations, and
to certain acids. They with stand 8-10 per cent solution of acetic acid for 18 hours, and survive for 75-80 days at
room temperature. The endotoxins remain active within large pieces of meat for long periods (even after the meat
has been cooked) as well as in inadequately fried rissoles and other foods.
A characteristic feature of foodstuffs contaminated by Salmonellae is that they show no changes which can
be detected organoleptically.
Pathogenicity for animals. Salmonellae, the causative agents of toxinfections, are pathogenic micro-
organisms which may give rise to paratyphoid in calves, typhoid and paratyphoid in newly-born pigs, typhoid in
fowls and pullorum disease in chickens, typhoid in mice and rats, and enteritis in adult cattle.
Among laboratory animals, white mice are most susceptible to the organisms (S. typhimurium, S. enteritidis,
S. cholerae-suis, etc.). Enteral and parenteral inoculations result in septicaemia in these animals.
Pathogenesis and diseases in man. Ingestion of food contaminated by salmonellae is the main cause of
disease. Most frequently food poisoning is due to meat prepared from infected animals and waterfowls without
observance of culinary regulations. Eggs of infected waterfowls are also sources of infection. Seabirds are frequent
Salmonellae carriers. Meat may be infected while the animal is alive or after its death.
As distinct from typhoid fever and paratyphoids A and B, salmonellae toxinfections are anthropo-zoonotic
diseases. S. typhimurium, S. cholerae-suis, S. heidelberg, S. enteritidis, S. anatum, S. newport. S. derby, and others
cause clinically manifest forms
Gastroenteritis is the most common manifestation of salmonella
infection
Salmonella enteritis ingestion
Adsorbed to epithelial cells in terminal portion of small intestine
Bacteria penetrate cells and migrate to lamina propria layer of ileoceal
region
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3. Multiply in limphoid follicles causing reticuloendothelial hyperplasia and hypertrophy
Polimorphonuclear leucocytes confine infection to gastrointestinal tract
Inflammatory response also madiates release of prostaglandins
Stimulates cAMP and active fluid secretion
Diarrhoea
Fig.1 The passage of salmonellae through the body to the gut. The vast majority of salmonellae cause infection
localized to the gastrointestinal tract and don’t invade beyond the gut mucosa. They do not produce enterotoxins.
Immunity acquired after salmonellosis is of low grade and short duration. Low titres of agglutinins (from
1:50 to 1 :400 and, rarely, up to 1:800) appear in the blood of convalescents during the second week.
Laboratory diagnosis. Specimens of food remains, washings from objects, stools, vomit, lavage water,
blood, urine and organs obtained at autopsy are carefully collected and examined systematically. In the beginning,
the specimens are inoculated into nutrient media employed for diagnosis of typhoid fever and paratyphoids A and
B. Then the cultural, serological, and biological properties of the isolated cultures are examined.In some cases the
biological test is performed not only with the cultures, but also with remains of the food WHICH CAUSED?the
poisoning.
For retrospective diagnosis blood of convalescents is examined for the presence of agglutinins on the
eighth-tenth day after the onset of disease. This is performed by the Widal reaction with suspensions of the main
diagnostic bacterial species which cause food toxinfections.
Differential laboratory diagnosis between S. typhimurium and S. schottmuelleri is particularly difficult
since they have group, somatic, and flagellar phase 2 antigens in common. Pathogenicity for white mice and
appearance of mucous swellings and daughter colonies on agar serve as differential criteria.
Bacteriological examination. To isolate a haemoculture of salmonellae, the blood is introduced into a bile
broth. The vomit, faeces, section material, pus, cerebrospinal fluid, foodstuffs, and washings off are inoculated into
plates with Ploskirev's medium and in enrichment media (bile broth and selenite medium) from which
subinoculation is made into Ploskirev's medium in 6-10 hrs. The inoculated cultures are incubated at 37 "C for 24
hrs. after which they are examined, colourless lactose-negative colonies are selected and transferred to OIkenitsky's
triple sugar medium or to an agar slant to enrich for pure culture. On the third day of the investigation, the isolated
pure cultures are identified: they are inoculated into Hiss' cultures and the agglutination test with adsorbed group
sera (A, B, C, D, E) is performed. If a positive result has been obtained with one of serum groups, one makes the
agglutination test with the adsorbed 0-sera typical for the given group and then with monoreceptor H-sera (non-
specific and specific phases) in order to determine the species and serovars of bacteria. For example, if the studied
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4. culture has agglutinated with a group B-serum, it is necessary to perform the agglutination test with sera against O,
and OB antigens, which are typical of this group. If agglutination has been positive, the H-monoreceptor sera are
utilized.
On the fourth day of the investigation, changes in Hiss' media are assessed. The causative agents of
salmonella gastroenteritis, similar to the salmonellae responsible for paratyphus A and B, do not ferment lactose
and sucrose, split glucose, mannitol, and maltose with the formation of acid and gas, do not form indol and, with
minor exceptions, release hydrogen sulphide.
Salmonella cultures can most frequently be isolated from patients' faeces, somewhat less commonly, from
vomit and stomach washings, and even less often from blood, urine, and bile. The results of bacteriological
examination of various biosubstrates are of varying diagnostic significance. Isolation of salmonellae from the
blood, bone marrow, cerebrospinal fluid, vomit, and waters from the stomach lavage is a definite confirmation of
the diagnosis. On the other hand, detection of salmonellae in the faeces, urine, and bile may be related to a bacteria
carrier-state. The aetiological role of salmonellae in the development of gastroenteritis is corroborated by an
increased titre of specific antibodies in an agglutination reaction with an autestrain.
Biological examination. Salmonellae of food poisoning, in contrast to salmonellae of paratyphi A, are
pathogenic for white mice. This property is used for the differentiation between the two types. On the first day of
examination, along with inoculation of the pathological material and foodstuffs, white mice are infected per os.
One-two days later the mice die of septicaemia. Post-mortem examination demonstrates a sharply enlarged spleen
and, occasionally, liver, while inoculation of the blood from the heart and samples from the internal organs permits
isolation of salmonella culture.
The agglutination reaction and indirect haemagglutination test are employed for serological diagnosis.
These may be carried out from the first days of the disease and should be repeated in 7-10 days to determine
whether the titre of specific antibodies tends to increase. In conducting these tests, salmonellal polyvalent and
group (group A, B, C, D, E) diagnosticums (corpuscular and erythrocyte) are utilized.
A two-four-order elevation of the antibody titre is of diagnostic importance.
Salmonella Septicemia
Septicemia caused by Salmonella is a fulminating blood infection that does not involve the gastrointestinal
tract. Most cases are caused by S. choleraesuis and are characterized by suppurative lesions throughout the body.
Pneumonia, osteomyelitis, or meningitis may result from such an infection. Salmonella osteomyelitis is especially
prevalent in persons who have sickle cell anemia, and focal infections, particularly on on vascular prosthesis, also
are common.
Treatment. Diarrhoea is usually self-limitng and resolves without treatment. Fluid and electrolit
replacement may be required particularly in the very young and elderly. Ulness there is avidence of invasion and
septicemia; antibiotics should be positively discouraged because they do not reduce the symptoms or shoter the
illness, and may prolong excretion of salmonellae in the faeces.
Prophylaxis of salmonellae toxinfections is ensured by veterinary and sanitary control of cattle, slaughter-
houses, meat factories and fish industries, laboratory control of meat intended for sale, and sterilization of meat
which otherwise may not be sold. The medical hygiene service identifies carriers among people working in food
factories, catering houses, and other food-processing establishments and controls the sanitary regulations at food
enterprises, shops, store-houses, and in catering houses.Following an episode of salmonella diarrhoea, people may
continue to carry and excrete organisms in their faeces for several weeks. Although in the absence of symptoms the
organism will not be dispersed so liberally into invironment, proper hand washing prior to food handling is
essential. Persons employed as food handlers are excluded from work until three specimens of faeces have failed to
grow salmonella.
Serological Classification of Bacteria of the Genus Salmonella
Group and species Antigenic structure
(type) somatic flagella antigen
antigen
phase I phase II
Group A
S. paratyphi A 1, 2, 12 a
Group B
S. schottmuelleri 1, 4, 5, 12 b 1, 2
S. abony 1, 4, 5, 12 b e, n, x
S. typhimurium 1, 4, 5, 12 i 1, 2
S. stanley 4, 5, 12 d 1, 2
S. heidelberg 4, 5, 12 r 1, 2
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5. S. abortivoequina 4, 12 – e, n, x
S. abortus ovis 4, 12 c 1, 6
S. abortus bovis 1. 4, 12, 27 b e, n, x
Group C (1, 2)
S. hirschfeldii 6, 7, Vi c 1, 5
S. cholerae-suis 6, 7 c 1, 5
S. typhi-suis 6, 7 c 1, 5
S. thomson 6, 7 k 1, 5
S. duesseldorf 6, 8 Z4, Z24, –
S. newport 6, 8 e, h 1,2
S. albany (8), 20 Z4, Z24, –
Group D1
S. typhi 12. Vi d –
S. enteritidis 9, 12 g, m –
S. dublin 9, 12 g, p –
S. rostock 9, 12 g, p, u –
S. moscow 12 g, q –
S. gallinarum and oth. 9, 12 i –
Group E (1, 3)
S. london 10 i, v 1. 6
S. anatum 10 e, h 1. 6
S. harrisonburg and oth. (3) (15), 34 z10 1, 6
Salmonella Septicemia
Septicemia caused by Salmonella is a fulminating blood infection that does not involve the gastrointestinal
tract. Most cases are caused by S. choleraesuis and are characterized by suppurative lesions throughout the body.
Pneumonia, osteomyelitis, or meningitis may result from such an infection. Salmonella osteomyelitis is especially
prevalent in persons who have sickle cell anemia, and focal infections, particularly on vascular prosthesis, also are
common.
4.Student’s independent study program
1. Morphology, structure, staining properties of causative agents of salmonella gastroenteritis. Cultivation.
2. Biochemical properties of causing agents of salmonella gastroenteritis.
3. Antigenic structure of salmonellae. Principles of Kauffman– White classification of Salmonellae
according their antigenic structure. Practical value of Kauffman– White classification
4. What are the differences in pathogenicity of S. typhі and salmonellae – the causing agents of
salmonellosis?
5. The sources of infectious agents and main mechanism of transmission of salmonellosis,factors of
transmission.
7. The clinical features of salmonella gastroenteritis. Treatment and prophylaxis.
8. Laboratory diagnosis
a – bacteriological method (tested materials, stages of examination)
b – value of hemoculture investigation;
c – serological method (IHAT, CFT).
5. Students’ practical activities:
1. To study under microscope morphology of causative agents of salmonellosis.
2. To perform presumptive agglutination test with groups and monoreceptor sera and unknown culture of
Salmonellae for determination of their species.
3. To familiarize with peculiarities of carbohydrates fermentation by Salmonellae on Hiss’ media.
4.To familiarize with biological preparation, which are used for salmonellosis diagnosis.
5. To inoculate feces (from patient with diarrhea) onto Ploskirev’s medium.
6. Control questions and tests:
Select the correct answers.
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6. 1. Causing agents of food poisonig are: a – S. typhі; b – S. enterіtіdіs; c – S. choleraesuіs; d – S.
schottmuellerі; e – S. typhіmurіum.
2. The salmonellae have such properties: a – peritrichates; b – amphitrichate; c – gram-positive; d – gram-
negative; e – have O- and Н-antigens.
3. Biochemical properties of salmonellae: a – produce indole; b – produce a hydrogen sulfide; c – do not
ferment lactose; d – ferment saccharose; e – ferment carbohydrates with acid and gas.
4 Causative agents of salmonella gastroenteritis have such properties: a – pathogenic only for man; b –
pathogenic for animals and man; c – pathogenic for white mice; d – do not cause bacteriemia; e – cause a
bacteriemia.
5.For diagnosis of salmonella gastroenteritis such methods are used: a –isolation of bacteria from roseolas; b
– isolation of hemoculture; c – isolation of bacteria from bile; d – isolation of bacteria from feces; e – serologic
method.
6. For identification of salmonellas it is necessary: a – to determine serogoups according O- antigen; b – to
determine of group according Н–antigen; c – to determine species according Н–antigen in the limit of group; d –
to determine species according Vi–antigen in the limit of group; e – to have O– group and Н–monoreceptor sera.
Real-life situation to be solved:
8. A woman of 32 years old was admitted to the infectious diseases hospital after 2 days of disease with
diagnosis of an acute gastroenterocolitis. It was acute onset of disease in an hour after usage of pelmeni (meat
dumplings). A sudden pain in the upper part of abdomen, sense of nausea, recurrent vomiting were appeared. Body
temperature was 39.5 °C.
A. What tested material is it necessary to take for examination?
B. What methods of diagnosis do you propose?
C. Onto what media is it necessary to inoculate tested material?
C. How can we determine a species of causative agent?
9. Several patients from the same plant were hospitalized to the infectious diseases hospital during twenty -
four hours with diagnosis of acute gastroenteritis. The disease began from high temperature, sense of nausea,
vomiting, abdominal pain. Diarrhea was appeared. The day before they ate meat salad in their plant’s snack bar.
A. What is it necessary to send to the bacteriological laboratory for examination?
B. What bacteria can be disease agents?
C. Make the schema of diagnosis.
D. How could these workers be infected?
7. List of literature:
1. I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,
chapter26, p.189-196, 206-207.
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