Salmonella – Citrobacter

Salmonella - CitrobacterHISTORY:

– In 1820, Bretonneau showed the contagiousness of typhoid fever that was called dothiénentérite.

– In 1880, Eberth bacillus first observed in the organs of a dead sick of typhoid.

– In 1884, Graffky successful culture of this bacillus.

– In 1896, Widal showed that the serum of patients with typhoid fever agglutinated cultures of the bacillus of Eberth.This was the first sero-diagnostic technique which we know the ultimate success for the help in the diagnosis of infectious diseases.

– In 1930, Kauffmann and White developed a classification of neighboring bacteria bacillus of Eberth based on the identification of their antigens.

– In 1939, Reilly showed the role of the autonomic nervous system in the pathogenesis of typhoid.


The most recent taxonomic studies have shown that the genus Salmonella consists of only one species Salmonella enterica with 7 subspecies refer to the former subgenus I, II, III and IV of Kauffmann. The name S. enterica was preferred to that originally proposed 5 ‘. choleraesuis which also means a serovar. The different subspecies are:

subspecies enterica subspecies I Salmonella enterica

subspecies Salmonella enterica subspecies II salamae

subspecies IIIa Salmonella enterica subspecies arizonae

subspecies IIIb Salmonella enterica subspecies diarizonae

subspecies Salmonella enterica subspecies IV houtenae

subspecies Salmonella enterica subspecies V bongori

subspecies enterica subspecies indica VI Salmoenlla

These subspecies are further subdivided into serovars (or serogroups) on the basis of antigenic components (0, H and Vi). The subspecies 1 represents 99.5% of the strains isolated in pathology. The designation of the serovars that previously corresponded to species names, especially those of the subspecies I, was chosen as the syndrome (5 ‘.Typhi), host specificity (typhimurium choleraesuis) and on . the geographical origin of the first new strain of serovar(dublin) These names have been preserved, but the writing is amended subspecies is not mentioned because all serovars are subspecies and 1 name starts with a capital letter (Salmonella Typhimurium, S.

Montevideo) and is not italicized. Serovars of the other subspecies are designated only by their antigenic formula (S. salamae 1, 9, 12: 1, w: e, n, x).

1987, 2213 serovars 1299 in the subspecies I, 445 in the subspecies II, 296 in the IIIb, 91 in the IIIa in 59 IV, 14 V and 9 in the VI were identified. The strains of the subspecies one generally come from warm-blooded animals, and this subspecies is practically the only one to have a medical interest. The other subspecies are generally isolated from cold-blooded animals or the environment.


S. Typhi and S. Paratyphi A are strictly adapted to humans. It is not possible to reproduce the typhoid fever in animals by oral administration.

The transmission of typhoid fever from human to human is through food or water (shellfish) soiled with feces of sick or convalescent healthy carriers.

In France S. Typhi is usually isolated as S. Paratyphi. There are no more major epidemics but localized outbreaks;however it is estimated at over a thousand the number of the cases observed in a year. The increase in the number of cases contracted in Mediterranean countries is noteworthy. There is no in France of indigenous cases of salmonellosis caused by S. Paratyphi A. Contamination of patients is done abroad.

Salmonella others are primarily parasites of the digestive tract of humans and animals. Serotypes, unlike previous lack of host specificity, are called ubiquitous. After illness, some subjects are healthy carriers for several months and eliminate Salmonella in their feces. Salmonella is found in the environment in sewage in particular. Salmonella are also commonly found in fish meal or bone powder used for animal feed.

Contamination of the man is orally. The frequency of Salmonella infections is increasing. It is favored by the development of meals in community where food is prepared well before consumption and in which the bacteria can multiply.

S. Typhimurium is found in all countries. It is most often identified in the National Salmonella Centre at the Institut Pasteur. It is isolated in humans, animals and the environment.

S. Typhimurium ranked first in the etiology of foodborne illness. Virtually all foods can harbor Salmonella few, but the animal denrpes play the main role. Defects in food preservation (the so-called cold chain) allows the multiplication of some Salmonella may be present.

Ingestion of 106 bacteria causes food poisoning.

Some serotypes, until exceptionally isolated, can have a significant epidemic spread. This was the case in 1972 forS. Wien. In 1984, S. Goldcoast presented a “puff”, following the consumption of contaminated pie. In 1985, S.Bovismorbificans had a significant distribution in France.

Since 1987, the frequency of isolation of S. Enteritidis increases sharply in 1989 to be the second most frequently isolated serovar in humans. This outbreak of S. Enteritidis is worrying and is in most cases to the consumption of chicken eggs. Preventive measures should be applied downstream at the use of eggs with the usual precautions for the cold chain for preparations without cooking, but especially upstream in the production of eggs, complicated by the vertical transmission (transovarially) of S. Enteritidis (PT 33).

The 15 most frequent serovars, representing 84% of the strains isolated in humans are reported in Table II.


Salmonellosis may take three aspects:

A – septicemic forms:

These are the typhoid and paratyphoid fevers caused by S. Typhi, S. Paratyphi A, B and C. They are rarely septicemia lymphatic start.

In the newborn or young child, other serotypes such as S. Panama and S. Wien may be responsible for septicemia that highlight life-threatening.

B – Purely digestive salmonellosis:

Foodborne Salmonella infections are manifested by diarrhea, fever, and vomiting. The first signs occur 8-10 hours after ingestion of contaminated food. The evolution of these gastroenteritis is spontaneously favorable rule within days.

Salmonella enteritis are observed mainly in young children. Outbreaks can occur in infants communities.

The frequency of Salmonella enteritis in AIDS is noteworthy.

C – The extra-digestive forms:

They are rare; urinary tract infections, cholécystistes, meningitis, osteomyelitis, spondylodiscitis, lung infections.These forms occur more often among immunocompromised patients. Enzymatic deficits and sickle cell red blood cells are predisposing circumstances.


During typhoid fever, Salmonella ingested enter cells of Peyer’s patches and colonize the intestinal lymph nodes.They scatter through blood, realizing lymphatic starting point for sepsis. During the disease it is possible to findSalmonella in urine.

Salmonella can grow in bile and survive in the tubules and gallbladder where it is difficult to eradicate. Their persistence at this level leads to a chronic carrier state.

A part of the bacterial bodies by lysing releases endotoxin or LPS which reaches the bloodstream by autonomic centers, resulting in the tuphos and cardiovascular collapse. In experimentally injecting rabbits of endotoxin in the vicinity of the splanchnic nerve, Reilly could reproduce the gastrointestinal tract lesions, demonstrating the role of the autonomic nervous system.


A – Characteristics of the subspecies 1:

Salmonella were cropping and metabolic morphological characters described above as common to allEnterobacteriaceae. The characters that identify strains belonging to the subspecies 1 or S. enterica subsp entericaare:

– Mobile bacilli

– Producing glucose gases (except S. Typhi)

– Lactose negative and ONPG,

– Having an LDC and ODC,

– Using the Simmons citrate as sole carbon source,

– With neither urease or TDA or gelatinase

– Fermenting sucrose, raffinose and salicin,

– The Voges-Proskauer (VP) is negative.

01 phage Felix and Callow lyse 98% of strains of Salmonella and other Enterobacteriaceae not. The discovery of this lysis is simple. The procedure is as for susceptibility testing. Instead of filing of antibiotic discs on the agar surface there drops a drop of a suspension of 01 phage.

It is important to note some exceptions to these fundamental characteristics:

– A bird serovar, S. Gallinarum is stationary;

S. Paratyphi A does not produce H ^ S and negative LDC;

S. Typhi does not produce glucose gas and produces little or no H ^ S;

S. Typhi and S. Paratyphi A are not using the Simmons citrate;

S. serovar Choleraesuis does not produce H ^ S.

B – Characters other subspecies:

Due to their low incidence in medical bacteriology we refer the interested reader to the article by Le Minor, Popoff and Veron cited in the bibliography of this chapter. Indicate only that these subspecies are distinguished from subspecies 1 with the following characters: ONPG, gelatinase, use of malonate and culture medium KCN. These four characters are simultaneously negative for single strains belonging to the subspecies 1.

C – Differential diagnosis:

Three Enterobacteriaceae commensal of the human digestive tract, may in a summary identification be confused with Salmonella. These are Hafnia alvei, Citrobacter freundii and Proteus mirabilis. The differential characters withSalmonella are shown in Table II.

Citrobacter freundii has no LDC and ONPG (+);

Hafnia alvei does not produce H2S, is VP (+) at 22 ° C and is generally ONPG (+);

Proteus mirabilis urease and has a tryptophan-deaminase.

Not being Salmonella, none of these three species is lysed by phage 01.

TABLE I: Biochemical diagnosis of Salmonella
TABLE I: Biochemical diagnosis of Salmonella


It is based on the determination by agglutination, antigens 0, H and Vi. There are over 2,000 serotypes, but with a limited number of typing sera, any laboratory can typify most strains of Salmonella isolates it. Typing rare serovar requires the intervention of a reference laboratory.

A – Antigens 0:

The specificity of each of the 67 antigens listed 0 is determined by its composition, that is to say, by the structure of the bacterial cell wall polysaccharides.

Forms A. These are mutants, nonpathogenic, who lost by deletion of a large part of the charge of the polysaccharide chain specificity 0. These strains are not and are serotypeable autoagglutinable in physiological saline.

Forms T (transition). These strains are rare. They give colonies with the S appearance, but they lost their specificity 0, as R. forms

Bacteriophages called converters. They may lysogeny produce changes in the antigenic structure of Salmonella 0. 0 The antigenic factors which are related to phage conversion may be present or absent. They are highlighted in the table Kauffmann-White.

B – H antigens:

Flagella consist of a protein molecule, the flagellin, the composition of amino acids determines the antigenic type H. This composition is encoded by a structural gene H) for phase 1 and a H2 gene for phase 2.

Some serotypes are monophasic. They can not synthesize flagellin only one specificity.

Most serotypes are biphasic. They can synthesize H antigens either Phase 1 or Phase 2.

Phase 1 of the antigens are designated by letters: a, b, c … z. As the alphabet was not enough, the most recently recognized are designated by a z followed by a number. Antigens of phase 2 are designated by numbers.

Phase inversion. When in a culture, the majority of bacteria, for example, in phase 1, the amount of phase 2 antigens is too low to be detected. Phase inversion is to inoculate the strain whose phase 1 is known in soft agar in the presence of serum for this Phase 1.

Only bacteria that are not immobilized by the serum, which are therefore of the other phase, can migrate in soft agar and be collected at a distance of the seed item. This fully incorporated population of bacteria in phase 2 is used for the determination of the second phase. This technique is known as the method name Sven-Gard.

C – Vi Antigen:

The capsular polysaccharide is found so fickle in three serotypes S. Typhi, S. Paratyphi C, S. Dublin.

Vi + strains that produce a significant amount of Vi antigen are 0-inagglutinable. They usually become 0-agglutinating after heating to 100 ° C, which passed the Vi antigen in the supernatant.

TABLE II antigenic formulas Enteritica Salmonella serovars MOST Frequently Encountered in France (Extract Kauffmann-White of the table)
TABLE II antigenic formulas Enteritica Salmonella serovars most frequently encountered in France (Extract Kauffmann-White of the table)

D – Table Kauffmann-White:

This table shows for each serovar antigens 0, Vi and H whose determination is useful for serological typing.Each serovar is antigenic formula. For example, S.Virchow 6.7 r 1.2.

In this table, serovars that have common characteristics 0 antigens are combined to form a group 0 designated by letter A, B, C, D etc.

Example: serovars group B antigen were all 04 and group D antigen 09.

Within each group 0 serovars appear according to the alphabetical order of phase 1 of their antigen H.

Factors parentheses may be absent.

The figures in the N column correspond to the order of frequency of isolation of the most common serotypes that account for 84% of human strains isolated in France in 1989 (BEH 1990,16,69).


They are used to compare the strains belonging to the same serovar to find the origin of a contamination.Several types of marker are used.

– Antibiotypie. By simple susceptibility, a strain with many resistance traits can be distinguished from a susceptible strain.

– Biotypie. It is to compare some variables metabolic characters within the same serotype. Example: the use of the d-tartrate S. Paratyphi B, Java variety.

– Phage. strain to be tested is subjected to the action of a series of virulent bacteriophages active on the group to which the strain. The phage sensitivity requires the presence of specific surface receptors which are stable traits for each strain. The PT is the list of lytic phages for the strain studied.

– Bactériocinotypie or colicinotypie The initially described in E. substances. coli, are produced by some strains and are able to lyse other strains of the same species or related species. Detection of bacteriocins and determination of phage are performed only by specialized laboratories.


We must always try to isolate the germ during a salmonellosis. This allows the precise characterization for an epidemiological survey and study of its sensitivity to antibiotics. Serodiagnosis of Widal and Felix is ​​only useful for typhoid and paratyphoid fevers. Serodiagnosis is only an indirect method and cross-reactions are possible even with bacteria other than Salmonella.

Epidemiological markers used by the reference center for phage typing and enteric biotypie
Epidemiological markers used by the reference center for phage typing and enteric biotypie

A – Direct diagnosis; Isolation of Salmonella:

1. Blood cultures:

. They are made ​​preferably during thermal ascents. It is desirable that each sample collects about 10 ml of blood because the number of bacteria per ml of blood is often low during the typhoid.

The realization poses no technical problems: Salmonella grow on usual media. Blood cultures are especially useful during typhoid and paratyphoid fevers. During the first sevenfold, it is positive in 90% of cases. The positivity rate drops to 40% for the third septenary.

Blood cultures may be positive during enteritis in young children. Its positivity is exceptional in adult enteritis and food poisoning.

2. Stool Cultures:

a / notes:

Stool cultures should be performed as soon as is suspected Salmonella infection.

During fever typhoid and paratyphoid, it remains longer than positive blood culture.

– During enteritis and food poisoning, stool culture is positive while the blood cultures remain negative.

– After treatment, the stool to verify that the patient remains healthy carrier. The elimination of Salmonella can be intermittent, it is therefore appropriate to make two stool cultures to a week away.

– An annual routine stool culture is recommended for persons employed in kitchens or in the food industry.

b / Technique:

In Salmonella, the germ excretion in the stool may be low.

More Salmonella are fewer in commensal species Escherichia coli and Proteus Therefore, to isolate, use both enrichment media and selective media..

– At the levy arrival an enrichment medium and selective medium were sown. A large number of media have been proposed. Including food bacteriology. Here we list the most commonly adopted in medical bacteriology environments.

Enrichment media

They allow using selective antiseptic inhibiting other bacteria to increase the proportion of Salmonella. A broth Muller-Kauffmann tetrathionate or selenite broth are inoculated and subcultured after incubation for 18 hours at 37 ° C.

Selective media

These agar media containing antiseptics, bile salts that prevent the growth of certain bacterial species and inhibit invasion by Proteus. They are used to identify suspicious colonies by fermentation of certain sugars (lactose) and the production of H2S. The Hektoen agar is generally preferred in the middle SS (DCA agar).

The second day, after incubation of the media at 37 ° C:

– The enrichment medium is transplanted on a selective medium which will be discussed the next day.

Five suspect colonies (lactose (-) and H2S (+)) marked on the selective medium seeded the day before, are the subject of a succinct biochemical characterization, then, if the characters are from a precise identification ofSalmonella and finally for serotyping.

Diagram of Salmonella isolation from stool
Diagram of Salmonella isolation from stool

3. Other charges:

Bile mark Salmonella is analogous to the stool.

In other pathological products, urine, pus variety, the presence of Salmonella is usually an incidental finding of the laboratory.

Analysis of the contaminated food using selective media and enrichment media is the best way to isolate aSalmonella responsible for food poisoning.

B – Indirect diagnosis: the serodiagnosis of Widal and Felix

It is only useful for the diagnosis of typhoid and paratyphoid fevers A, B and C and has no interest in other salmonellosis.

When the patient has been late or has received antibiotics applied to the blind, isolation of the bacterium is often not possible. It was then that the serodiagnosis of Widal and Felix interest. It lets you search for anti-0 and anti-H serum.But many causes of errors exist and interpretation is sometimes tricky. This is why the prescription of a serology Widal and Felix must always be accompanied by that of a stool or blood culture to isolate Salmonella.

1. Principle and realization:

II is to search in the serum of patients agglutinins 0 corresponding to somatic antigens and flagellar antigens ofSalmonella Typhi H and

Salmonella paratyphi A, B and C.

Search agglutinin Vi has no practical interest

For this we use antigenic suspensions TO, TH, AO, AH, BO, BH, CO and CH. These antigenic suspensions are consituées killed bacteria, treated with either alcohol destroying antigens H or formalin destroying antigens 0.

In a series of tubes, each antigenic suspension is the presence of increasing dilutions of the serum of the patient to determine the titer of agglutinins.

The reading is made after centrifugation for 10 minutes (rapid method) or after 2 hours in an oven at 37 ° C.

Changing the title of agglutinins typhoid and paratyphoid in the fevers
Changing the title of agglutinins in the typhoid and paratyphoid fevers

2. Normal results:

0 agglutinins appear to the 8th day of the disease, and H agglutinins to 10-12e day.At the state period, there has simultaneously agglutinins 0 and H.

The title of the latter is higher (for example TO = 1/200; TH = 1/800).

0 agglutinins normally disappear in 2 to 3 months. H agglutinins persist several years after infection or vaccination antityphoparatyphoïdique A and B. The presence of agglutinins only 0 indicates recent infection.

Typhoid fever can sometimes be seen in a vaccinated: in this case, there is the simultaneous presence of agglutinins TH, AH, BH and agglutinin 0.

There is no relationship between the title agglutinins and severity of the disease.

3. False positive results:

The presence of only agglutinins TO may be due to infection with a Salmonella having a 0 common antigen with S.typhi but different H antigens. It is usually from 5 ‘. Enteritidis.

– Similarly the presence of agglutinins only BO may be due to infection with S. Typhimurium, for example.

– Some strains of Yersinia pseudotuberculosis, because of antigenic communities, can give an agglutination with BO (serogroup II) or TO (serogroup IV)

– False positive reactions can be observed in certain diseases: malaria, typhus, dysglobulinémies (myeloma, collagen, cirrhosis), and various infections with Enterobacteriaceae.

4. False negative results

Widal’s agglutination reaction is negative in the first septenary.

– Early treatment with antibiotics or corticosteroids can prevent the rise of antibody levels.

– N are rare cases of typhoid authentic without elevation of antibody levels.


Susceptibility testing is performed on any strain of Salmonella isolated as much as to characterize this strain to guide antibiotic treatment is not systematic for all digestive salmonellosis.

A – Typhoid and paratyphoid:

The strains isolated in France remain generally well sensitive to antibiotics, contrary to what was observed during epidemics in Mexico, Vietnam and Peru.

Antibiotics of choice are those which have a good concentration in lymph nodes: chloramphenicol or trimethoprim-sulfamethoxazole.

Ampicillin has a lower lymphatic concentration, against it has a good biliary elimination.

Antibiotics administered orally provides better lymphatic concentration. This is the case of fluoriquinolones.

Contrary to the general rule antibiotics, antibiotics are typically given dosage gradually to avoid a massive microbial lysis and the disadvantages due to the sudden release of endotoxin.

B – enteritis and food poisoning:

Only severe forms in infants and in the elderly are treated with antibiotics. In other cases where the prognosis is favorable, symptomatic treatment is sufficient.

C – healthy carrier:

Persons who, in course of the disease, continue to eliminate Salmonella in their feces should not be treated with antibiotics. These select resistant strains and have no effect on the duration of the carrier. Only hygiene is advocating.


A – Vaccination:

TAB vaccine injection, consisting of inactivated whole bacteria, poorly tolerated and giving a poor quality of protection is no longer a nightmare for young incorporated into the French army and the few categories of people for which this protection is mandatory.

Two vaccines have been developed. One is a live attenuated vaccine (the mutant strain Ty 2), used orally. The other, available in France (TYPHIM Vi®) is an injectable vaccine consists of the capsular polysaccharide of S. Typhi or Ag obtained purified Vi in its native 0-acetylated form. Safety and efficacy are the qualities of this vaccine which helped give 72% and 64% protection areas with a high incidence (Nepal, South Africa).

B – Hygiene:

Collective hygiene. This is preventing fecal peril by bacteriological monitoring the effectiveness of the drinking water distribution network and the installation of sanitation.

It is also monitoring the bacteriological quality of the food.

Personal hygiene. This is the detection of healthy carriers including among staff kitchens or food industries.