Salmonella: An Overview

The History of Discovery

  • In 1880 the German scientist K. Eberth first described the bacterium – causative agent of enteric typhoid fever. Later in 1884 it was isolated and thoroughly investigated by G. Gaffky.
  • The causative agent of a similar disease, paratyphoid fever, was initially isolated by C. Archard and R. Bensaude and studied in detail by H. Schottmuller in 1900.
  • It was named later Salmonella paratyphi B or S. schottmuelleri. Another paratyphoid bacterium or S. paratyphi A was investigated by A. Brion and H. Kayser in Germany.
  • All these salmonellae were found to cause diseases in humans. Also, many salmonella species were isolated in animal diseases.
  • D. Salmon in 1885 revealed the causative agent of pig’s plague, S. choleraesuis. Then various salmonellae of animal origin were demonstrated to cause food poisoning or infections in humans.
  • In 1888, A. Gartner isolated S. enteritidis both from cow’s meat and patient, died from toxinfection.
  • In 1896-1898, К. Kensche and E. Nobel discovered another significant agent of food poisoning, S. typhimurium.
  • Finally, it was proven that the great number of salmonella species, isolated from animals, can cause human food poisoning and in some cases septicemia.

Classification

  • Salmonellae pertain to the family Enterobacteriaceae. In the past, more than two thousand species were described within the Salmonella genus.
  • Recent genetic studies revealed only two species of salmonellae – S. enterica and S. bongori with a vast number of antigenic salmonella variations.
  • S. enterica species is further divided into several distinct subspecies.
  • S.enterica subsp. enterica comprises more than 99% of salmonella that causes diseases in humans.
  • The complete name of distinct salmonella isolates includes species name and the name of serovar (former species designation).
  • Antigenic variant (serovar) is designated with starting upper-case letter and non-italicized straight font. For instance, the causative agent of enteric typhoid fever is classified as S. enterica serovar Typhi or S. Typhi for short.
  • The limited number of salmonella serovars affects humans only. The most serious disease is enteric typhoid fever, caused by S. Typhi.
  • S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C are the agents of paratyphoid enteric fevers. The latter diseases are generally regarded as anthroponoses, but these bacteria may be also isolated from animals.
  • Numerous salmonella serovars are the causative agents of salmonelloses. Usually, salmonelloses are contracted from animal sources and appear in two major clinical forms- food poisoning (food toxinfection) and septicemia.
  • Septicemia is a more rare but severe clinical condition that predominantly affects children. S. Typhimurium and S. Enteritidis are the most virulent and frequently isolated agents, causing these infections. Many other variants (e.g., S. Choleraesuis, S. Derby, or S. Heidelberg) can also cause salmonelloses.

Structure and Properties of Salmonellae

Morphology

  • All salmonellae are very similar. Their morphology is typical for Enterobacteriaceae family members (gram-negative small or medium-size rods without spores).
  • Salmonellae possess peritrichous flagella and multiple pili. Virulent strains carry needle complex, or injectisome – type III secretion system structures.
  • The strains, isolated from carriers, frequently produce capsule-like polysaccharide substances.

Structure of salmonella

Cultivation

  • Salmonellae easily grow on basic nutrient media within the temperature range from 15 to 40°C with an optimum of 37°C at pH 7.0.
  • The growth in meat peptone agar results in round semitransparent middle-size colonies. S. Paratyphi B colonies produce edge mucous swelling.
  • Salmonellae are resistant to bile salts and several antiseptics, e.g. brilliant green, sodium selenite, or sodium tetrathionate. Thus, they are cultivated on various selective and enrichment media that inhibit E. coli growth.
  • Among them is a meat-peptone broth with bile salts, selenite broth, tetrathionate broth, Wilson-Blair agar (i.e., bismuth sulfite agar), composed of MPA, glucose, bismuth sulfite, ferrous sulfate, and brilliant green.
  • Growing on bismuth sulfite agar, salmonella produce black colonies due to the formation of iron sulfide, except serovar S. Paratyphi A.
  • As salmonellae don’t ferment lactose, they form lactose-negative colonies on McConkey agar, EMB (Levine) agar, etc.

Biochemical properties

  • Salmonella is facultative anaerobes. S. Typhi ferments various carbohydrates (glucose, maltose, mannitol, dextrin, glycerol, and others) with acid production.
  • Other salmonellae, e.g. S. Paratyphi A and B, S. Typhimurium, S. Enteritidis, etc., utilize carbohydrates with acid and gas end products. All salmonella are lactose-negative bacteria.
  • Pathogenic salmonellae, except S. paratyphi A, reveal proteolytic activity with hydrogen sulfide formation. They reduce nitrates to nitrites.
  • As all the members of the Enterobacteriaceae family, salmonellae are oxidase-negative, but catalase-positive bacteria.

Antigenic structure

  • Salmonellae possess somatic O- and flagellar H-antigens. S. Typhi strains, predominantly isolated from microbial carriers, synthesize outer capsule-like Vi-antigen. Temperate phage transduction can influence the expression of salmonella antigens.
  • Lipopolysaccharide heat-stable O-antigen displays endotoxin activity. Flagellar H-antigen is heat-labile. Polysaccharide complex Vi-antigen is also a heat-labile substance. It is readily destroyed by boiling for 10 minutes.
  • Vi-antigen partially covers O-antigen, and thereby hampers microbial agglutination by anti-O antibodies. It is almost solely found in S. Typhi strains and rare cases in S. Paratyphi C and S. Dublin.
  • Polysaccharides of Vi-antigen bind to a vast number of specific bacteriophages. As the result, about 100 distinct phagotypes are determined in Vi-Ag-expressing S. Typhi.
  • F. Kauffmann and P. White elaborated the classification of salmonellae according to their O- and H-antigen variations.
  • O-antigen is shown to be group-specific. It is heterogeneous and contains specific and several non-specific antigenic determinants. About 70 serogroups were distinguished by a specific fraction of salmonellae O-antigen.
  • H-antigen is found to be in two phases, encoded by different genes.
  • The onlyfirst phase of H-antigen appears to be “species”- or variant-specific. Phase 2 antigens are agglutinated by group-specific sera. More than 2500 serovars of salmonellae are identified by specific phase H-antigen.
  • Thus, serological typing of certain Salmonella strains, despite their tremendous diversity, is reduced to a simple two-step procedure: once the serogroup was determined by specific O-antigen agglutination, serovar identification is accomplished by agglutination with specific anti-H serum.

Virulence factors

  • Salmonellae produce various virulence factors that actively participate in disease pathogenesis.
  • Since bacteria can persist intracellularly, they express multiple adhesins and invasive proteins, which promote microbial invasion, intercellular spread, and final impairment of host cellular immune response.
  • At least 10 genetic Salmonella pathogenicity islands (SPI) are detected that encode microbial virulence factors. They are found both in bacterial nucleoids and plasmids.
  • Many of them were delivered to bacterial cells with temperate bacteriophages upon transduction. Besides, S. Typhi harbors additional genetic elements known as a major pathogenicity island.
  • Genes located in chromosomal pathogenicity islands SPI-1 and SPI-2 as well as in major pathogenicity islands of S. Typhi play a crucial role in the pathogenesis of salmonella-associated infections.
  • Genes of SPI-1 and SPI-2 code for the structures of type III secretion system with bacterial needle complex or injectisome. Using injectisome, salmonellae deliver invasive effector proteins into intestinal cells and phagocytes.
  • Genes of major pathogenicity islands encode the capability of S. Typhi to produce capsular Vi-Ag that promotes microbial survival in worsened surroundings (e.g., within phagocytes or in the gallbladder of carriers).
  • One of the most potent virulence factors of salmonellae is thermostable LPS endotoxin. It activates macrophages and T cells that are followed by proinflammatory cytokine release and subsequent tissue damage.
  • Endotoxin action provokes deep disorders of the patient’s gastrointestinal tract, cardiovascular system, and CNS. Bacteria of the typho-paratyphoid group can produce large amounts of endotoxin.
  • Certain salmonella serovars, e.g- S. enteritidis, are able to produce potent enterotoxin. It activates enterocyte adenylate cyclase elevating intracellular cAMP concentration that results in diarrhea with the massive secretion of water and chlorides into the intestinal lumen.
  • Several genetic regions within nucleoid and plasmids of salmonellae contain genes of multidrug resistance to antibiotics.

Resistance

  • Salmonellae reveal marked stability in the environment. They can survive for several weeks and even months in soil, contaminated by bacteria, as well as in various foodstuffs, where they can propagate (dairy products, meat, bread, etc.)
  • The bacteria stay viable upon contaminated fruits and vegetables for up to 1-2 weeks. In water, they maintain viability for 3-4 months. Salmonellae readily withstand drying and long-time freezing.
  • S. Typhi and S. Paratyphi A are inactivated at 56°С within an hour, while other bacteria are relatively resistant to heating at 60-70°С. Boiling rapidly inactivates bacteria.
  • Microbial endotoxin is heat-stable and can cause food poisoning even in absence of live salmonella.
  • S. Typhi is sensitive to conventional disinfectants (e.g., chlorine-containing chemicals or phenol).

Pathogenesis and Clinical Findings in Typho-Paratyphoid Diseases

  • Enteric typhoid fever is an anthroponotic disease caused by S. Typhi and transmitted by the fecal-oral route. In developed countries, it occurs as a sporadic infection.
  • Nevertheless, from 15 to 30 million disease cases appear annually worldwide. The disease spreads predominantly in developing countries. It results in 250,000-500,000 lethal outcomes being a serious public health problem.
  • Salmonella carriers and the patients with subclinical forms of illness are the main sources of infection.
  • The infectious dose of S. Typhi is 103-105 microbial cells, i.e. it is rather low. The incubation period lasts for about 10-14 days.
  • Salmonellae, entering the gastrointestinal tract, are partially killed in the stomach. The rest of the bacteria appear in the intestine and adheres to mucosal cells. Microbial intracellular invasion is promoted by the salmonella needle complex.
  • When injected into enterocytes, SPI-1 effector proteins stimulate cytoskeleton remodeling and next membrane folding.
  • It leads to the engulfment of attached bacteria and their entry into epithelial cells by macropinocytosis. Other SPI-1 proteins activate membrane channels of epithelial cells resulting in chloride excretion and diarrhea.
  • In parallel with infection of the intestinal epithelium, salmonellae spread into the lymphatic follicles and Peyer’s patches. Microbial cells have multiple mechanisms of survival within phagocytes.
  • Certain SPI-1 proteins activate caspase-1 that stimulates the production of proinflammatory cytokines and eventually triggers phagocyte apoptosis. Inflammatory cytokines damage the intestinal tissues.
  • Effector proteins, associated with SPI-2, play an even more powerful role in microbial protection against phagocytosis.
  • Once captured by a phagocyte, salmonellae long time survive within the phagolysosome. It is related to SPI-2 effector proteins that block the enzymes of respiratory burst thereby inhibiting microbial digestion.
  • Infected phagocytes spread salmonellae throughout the body resulting in the systemic character of infection. Thus, the presence of genes of SPI-2 strongly predisposes to generalized salmonellosis.
  • Bacterial transition across the intestinal wall leads to their appearance in the bloodstream with subsequent microbial dissemination.
  • Salmonellae affect lymph nodes, spleen, liver, bone marrow, etc. Microbial death results in massive LPS endotoxin release.
  • It provokes a systemic inflammatory response and vascular damage that causes cardiovascular and CNS disorders.
  • At the end of the first week of disease high fever, headache (“status typhosus”), myalgia and roseolar skin rashes arise.
  • These symptoms are followed by hepatosplenomegalia. In the third week, salmonellae accumulate within bile ducts and gallbladder and then re-enter the intestine.
  • Multiple inflammatory reactions induced by microbial cells cause intestinal lesions and necrosis of lymphoid tissue.
  • At this time bacteria are intensively released from the patient’s intestine with feces. Also, they are excreted with urine.
  • As the disease confers both cellular and humoral immunity, the immune reactions ultimately eliminate bacteria promoting the patient’s recovery. The immunity is rather stable, but sometimes reinfections occur.
  • Nevertheless, appropriate conditions for salmonella survival especially within the gallbladder maintain microbial persistence and often cause the development of carrier state. Expression of capsular Vi-Ag increases bacterial resistance to bile salts.
  • Long-term (sometimes – lifelong) salmonella carriers are proven to be the most frequent sources of S. Typhi infection.
  • Paratyphoid infections caused by S. Paratyphi A, S. Paratyphi B, or S. Paratyphi C are characterized by a similar but modest clinical course with a favorable prognosis.
  • Salmonelloses pertain to a large widespread group of diseases, caused by non-typhoidal salmonellae. They are transmitted by fecal-oral and contact roots.
  • The infected and sick animals are regarded as the main sources of infection. The incubation period is short – from 2-6 hours to 2-3 days.
  • The infectious dose is higher than that of enteric fever agents – about 106-108 microbial cells.
  • The disease usually evolves after ingestion of contaminated foodstuffs (poultry – about 50% of disease cases, also eggs, meat, dairy products, etc.)
  • Various serovars of salmonellae (e.g., S. Enteritidis, S. Choleraesuis, S. Anatum, S. Derby, and many others) can cause these diseases.
  • The symptoms of severe food poisoning (enterocolitis, fever, vomiting, diarrhea, collapses, etc.) can appear even in few hours after infection onset due to the large microbial load.
  • Endotoxin is released from destroyed bacterial cells. Toxin triggers inflammatory reactions and affects the gastrointestinal tract and cardiovascular system. Some bacterial serovars (e.g. S. Enteritidis) express enterotoxin, which causes profuse diarrhea.
  • Mild forms of diseases are assumed to be self-limited, but severe intoxications lead to generalization of infection with septicemia.
  • Another type of salmonellosis resulting in systemic disease (septicemia) can develop in newborns or immunocompromised patients.
  • The disease is transmitted from human carriers or sick persons. It usually occurs as a hospital-acquired infection. Very often it is caused by multiple antibiotic-resistant strains of S. Typhimurium or S. Enteritidis and finally results in endotoxemia and septicemia.
  • The systemic character of infection is largely related to bacterial pathogenicity island SPI-2 encoding effector proteins that inhibit phagocytosis.
  • Salmonellosis with septicemia has a serious prognosis and may be fatal, especially in infants.
  • Post-salmonellosis immunity is weak, short-term, and has low specificity.

Laboratory Diagnosis of Enteric Typhoid Fever and Salmonelloses

  • Specimen collection for diagnosis of enteric typhoid fever depends on the pathogenesis stage.
  • The hemoculture examination is repeated from the first week of the disease. Stool specimens are examined on the second week from enteric fever onset.
  • Slightly later the urine can be taken for the investigation. Bone marrow culture is examined much more rarely.
  • For isolation of salmonellae from the patient’s blood hemoculture investigation is performed. About 10-15 ml of blood are inoculated into 100-150 ml of liquid selective medium, e.g. into bile broth.
  • After overnight incubation, the material is planted onto a differential medium (McConkey agar, EMB agar). Salmonellae grow as lactose-negative colorless colonies.
  • To isolate the pure culture of salmonellae the material from lactose-negative colonies is re-inoculated into slant agar with appropriate differential media.
  • For instance, the growth in double sugar agar or Russel’s medium (contains meat-peptone agar, 1% lactose, 0.1% glucose, and indicator) reveals the color change only within the butt of the medium that ensues from glucose fermentation.
  • Isolated culture is identified by a two-step slide agglutination test according to the Kauffmann-White scheme.
  • The serogroup is defined by specific O-antigen agglutination and microbial serovar is determined further by agglutination with specific anti-H serum.
  • The examination is accomplished by culture biochemical tests and phage typing. The latter test is elaborated with a large number of specific phages. Vi-I bacteriophage is regarded as universal and reacts with all cultures of S. Typhi bearing Vi-Ag.
  • In the case of stool specimen examination, the material is inoculated into bile broth, selenite broth, tetrathionate broth, or another selective media to inhibit concomitant flora.
  • Also, it may be planted on bismuth sulfite medium (Wilson-Blair agar) resulting in black salmonella colonies. Further investigation is similar to hemoculture isolation.
  • For serological diagnosis-specific antibodies against microbial antigens are tested. Antibody titer arises at the end of the second week of the disease.
  • Growth of specific antibodies is usually detected by indirect hemagglutination test or by tube dilution agglutination (Widal’s reaction) with typhoid and paratyphoid A and В antigenic diagnostics. The patient’s serum is regarded as positive in titer of 1:200 and higher.
  • Typhoid patients with manifested disease demonstrate high titers of antibodies both to O- and H- microbial antigens.
  • Convalescent or previously vaccinated individuals maintain the elevated level of H-antibodies for a long time.
  • For carrier state determination the indirect hemagglutination test with Vi-antigen erythrocyte diagnostic is used. Serum of salmonella carriers contains anti-Vi antibodies in titers 1:40 and more.
  • For laboratory diagnosis of salmonelloses stool specimens, vomit, food remnants, animal organs, patient’s blood, urine, etc. should be tested repeatedly.
  • The material is inoculated into bile broth, selenite, or tetrathionate selective medium or onto bismuth sulfite agar. Laboratory investigation is similar to typho-paratyphoid culture isolation and identification.
  • Genetic typing of salmonellae is performed by PCR.

Treatment and Prophylaxis of Enteric Fever and Salmonelloses

  • Various antibiotics, affecting gram-negative bacteria (primarily, third-generation cephalosporins or fluoroquinolones) are administered to patients with typho-paratyphoid diseases and salmonella-caused septicemia.
  • Most cases of food poisoning and enterocolitis in adults don’t require antibiotic treatment but need adequate infusion therapy.
  • Salmonellae reveal marked multidrug antibiotic resistance, which is conferred by the number of R plasmids.
  • The resistance is easily transmitted throughout the microbial population, thus susceptibility testing and resistance monitoring are valuable measures in disease control.
  • Specific prophylaxis of enteric typhoid fever requires further advances. Previously used killed vaccines are regarded today as inappropriate due to their short-term activity and side effects.
  • Two vaccines are implicated now for practical use. The chemical polysaccharide vaccine is derived from the capsular Vi antigen of S. Typhi.
  • Another live attenuated vaccine of S. Typhi (Ty21a) strain is the result of chemical mutagenesis. Nevertheless, they create only relatively short-term protection that lasts several years.
  • Genetically engineered and DNA vaccines, based on various recombinant S. Typhi strains are intensively designed now.
  • Non-specific prophylaxis includes the prevention of water and foodstuffs from microbial contamination with proper control of their sanitary state, maintenance of hygienic regimens, and sanitary regulations, especially in food handling.
  • All foodstuffs prone to possible microbial contamination must be thoroughly cooked or sterilized. The patients and salmonella carriers should be timely identified and treated. The infection sites require intensive disinfection.