Salmonella – Morphology, Antigenic structure, Cultural and Biochemical Characteristics

What is Genus Salmonella?

• The genus Salmonella consists of rod-shaped, gram-negative bacteria belonging to the family Enterobacteriaceae. Named after Daniel Elmer Salmon, an American veterinary surgeon, these bacteria are widely studied due to their impact on human and animal health. The genus is divided into two primary species: Salmonella enterica and Salmonella bongori. Among these, S. enterica is further categorized into six subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica. These subspecies encompass over 2,650 serotypes, each identified based on unique combinations of somatic (O), flagellar (H), and capsular (Vi) antigens.
• Salmonella bacteria are gram-negative, motile, and non-spore-forming. Their size ranges from 0.7 to 1.5 μm in diameter and 2 to 5 μm in length. They possess peritrichous flagella, allowing movement in various environments. As chemotrophs, they derive energy through oxidation and reduction reactions using organic sources. They are also facultative anaerobes, capable of producing energy in the presence of oxygen or switching to alternative electron acceptors or fermentation processes when oxygen is unavailable.
• Pathogenicity varies among Salmonella serotypes. They are intracellular pathogens, capable of surviving and multiplying within host cells. Infections generally result from ingesting contaminated food or water. Typhoidal Salmonella serotypes, such as those causing typhoid and paratyphoid fever, are human-specific and can spread systemically, invading the bloodstream and multiple organs. These infections often lead to severe conditions, including septic shock, requiring urgent antibiotic treatment.
• Nontyphoidal Salmonella serotypes, in contrast, are zoonotic, transmitted between animals and humans. They typically infect the gastrointestinal tract, causing salmonellosis. While most cases resolve without antibiotics, invasive nontyphoidal infections can occur, particularly in regions like sub-Saharan Africa. In such cases, the bacteria may cause bacteremia, necessitating immediate medical intervention.
• The classification of Salmonella is both intricate and clinically significant. Each serotype is regarded as a unique species for diagnostic and epidemiological purposes. For instance, Salmonella enterica subsp. enterica serotype Enteritidis is commonly abbreviated as S. Enteritidis in medical and research contexts. This systematic nomenclature aids in identifying and managing Salmonella-related diseases effectively.

Scientific classification

Domain:	Bacteria
Phylum:	Pseudomonadota
Class:	Gammaproteobacteria
Order:	Enterobacterales
Family:	Enterobacteriaceae
Genus:	Salmonella

History of Salmonella

The discovery and study of Salmonella have significantly advanced our understanding of bacterial pathogens. Below is a breakdown of key historical milestones in the study of this genus.

• Early Observations (1880):
  Karl Eberth first identified Salmonella in 1880 while examining the spleens and Peyer’s patches of patients suffering from typhoid fever. This marked the initial visualization of the pathogen associated with a major human illness.
• Cultivation of the Pathogen (1884):
  Georg Theodor Gaffky successfully grew the organism in pure culture four years after Eberth’s observations. This achievement was critical for subsequent research and classification.
• Discovery of Salmonella enterica (1885):
  Theobald Smith, working under Daniel Elmer Salmon at the Veterinary Division of the United States Department of Agriculture, identified what is now known as Salmonella enterica (var. Choleraesuis). At the time, it was believed to cause hog cholera and was named “Hog-cholera bacillus.”
• Naming the Genus (1900):
  The name “Salmonella” was proposed by Joseph Leon Lignières to honor Daniel Elmer Salmon. This formalized the nomenclature for the group of bacteria initially discovered in Salmon’s research division.
• Advances in Typing (1930s-1940s):
  Nancy Atkinson, an Australian bacteriologist, established a salmonella typing laboratory in Adelaide, one of only three globally at the time. Her work led to the identification of new strains, including Salmonella Adelaide, first isolated in 1943.
• Pioneering Research Publication (1957):
  Atkinson published her comprehensive research on Salmonella, shedding light on various strains and their characteristics. Her contributions laid a foundation for modern Salmonella taxonomy and epidemiology.

Geographical Distribution and Habitat of S. Typhi Infection

S. Typhi, the causative agent of typhoid fever, is widely distributed across the globe, with a stronger presence in developing regions. Its prevalence is closely tied to sanitation conditions and water supply quality.

• Geographical Distribution:
  • Enteric fever is endemic in many developing countries.
  • It is especially prevalent in regions with poor sanitation and inadequate water supply.
  • Typhoid fever is most commonly found in:
    • Indian subcontinent
    • Southeast and Far East Asia
    • Middle East
    • Africa
    • Central America
    • South America
  • Global Incidence: Around 12–13 million cases of typhoid fever are reported each year, with an estimated 600,000 deaths globally.
  • Typhoid fever has been virtually eliminated in developed countries due to improvements in water supply and sanitation over recent decades.
  • Paratyphoid fever, caused by other Salmonella species, remains a health issue in both developing and developed nations.
  • Specific strains of S. Paratyphi:
    • S. Paratyphi A is common in India, other parts of Asia, Eastern Europe, and South America.
    • S. Paratyphi B is primarily found in North America, Britain, and Western Europe.
    • S. Paratyphi C is more prevalent in Eastern Europe and Guyana.
• Habitat:
  • S. Typhi and its paratyphoid counterparts (S. Paratyphi A, B, and C) are strict human pathogens, meaning they are not found in animals.
  • These bacteria primarily colonize the small intestine, particularly the ileal mucosa, in infected human hosts.
  • Long-term, asymptomatic colonization can occur, especially in the gallbladder, which may lead to chronic infection and fecal carriage.
  • Unlike S. Typhi, other Salmonella species affect various domestic animals, rodents, reptiles, and birds.
  • For instance:
    • S. Typhimurium has a broad host range, infecting humans, animals, and birds.
    • Some species like Salmonella Abortus-equi are specific to horses, while Salmonella Abortus-oris infects sheep, and S. Gallinarum primarily affects poultry.

Morphology of Salmonella

Salmonella species exhibit distinct structural features that play a crucial role in their survival, movement, and pathogenicity. Here’s a breakdown of their key morphological characteristics:

• Gram-negative Structure:
  Salmonella are Gram-negative bacteria, meaning their cell walls have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides.
• Shape and Size:
  These bacteria are rod-shaped (bacilli), with dimensions typically ranging from 1 to 3 micrometers in length.
• Motility:
  Most Salmonella species are motile due to the presence of peritrichous flagella, which are distributed across the surface of the cell. Exceptions include Salmonella Gallinarum and Salmonella Pullorum, which lack motility.
• Spore and Capsule Formation:
  Salmonella do not produce spores or capsules, setting them apart from some other bacterial genera.
• Fimbriae Presence:
  While some strains of Salmonella have fimbriae, which are hair-like projections aiding in adhesion to host surfaces, not all strains possess this feature. For instance, most Salmonella Paratyphi A strains, along with a few strains of Salmonella Paratyphi B, Salmonella Typhi, and Salmonella Typhimurium, are nonfimbriated.

Human Infections Caused by Salmonella

Salmonella infections in humans can lead to a range of illnesses, from gastroenteritis to more severe systemic diseases like typhoid fever. Different Salmonella serotypes are responsible for various types of infections, with varying severity.

• Typhoid Fever:
  Salmonella Typhi is the primary causative agent of typhoid fever, a systemic illness that can spread throughout the body. In some cases, this can progress to Salmonella bacteremia, where the bacteria enter the bloodstream.
• Paratyphoid Fever:
  Salmonella Paratyphi A, B, and C cause paratyphoid fever, a disease similar to typhoid fever but generally less severe. These strains also have the potential to cause Salmonella bacteremia, leading to serious complications if left untreated.
• Salmonella Bacteremia:
  Some serotypes, like Salmonella Choleraesuis, can directly result in Salmonella bacteremia, where the bacteria spread through the bloodstream, potentially affecting organs and causing life-threatening complications.
• Gastroenteritis:
  The most common Salmonella infections in humans are gastrointestinal. Several serotypes, including Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Hadar, Salmonella Heidelberg, Salmonella Agona, Salmonella Virchow, Salmonella Seftenberg, Salmonella Indiana, Salmonella Newport, and Salmonella Anatum, are known to cause Salmonella gastroenteritis. This condition is typically characterized by symptoms like diarrhea, vomiting, abdominal cramps, and fever, and often resolves without the need for antibiotics.

Classification and Typing of Salmonella

Salmonella strains are classified and typed using several methods, each providing a unique approach to identifying and categorizing the bacteria. These methods allow for precise identification, which is crucial for understanding the epidemiology of infections and tracing sources of outbreaks.

• Kauffmann–White Scheme:
  • This classification method is based on the O and H antigens found on Salmonella strains.
  • The O antigens are the core component used to group Salmonella into serogroups, with each serogroup containing serotypes sharing a common O antigen.
  • The O antigen factors were originally labeled with letters (A–Z) and later with numbers (51–67). However, there’s a trend toward using numerical designations for these groups (e.g., Group A as 2, Group B as 4).
  • Flagellar antigens play a role in identifying different serotypes within each serogroup. These are classified based on two phases of flagellar antigens, called phase 1 and phase 2.
  • The complete antigenic structure of a Salmonella serotype is described by an antigenic formula, which includes the O antigens and the phase 1 and phase 2 H antigens.
  • Over 2,400 serotypes have been identified using the Kauffmann–White system.
• Bacteriophage Typing:
  • Bacteriophage typing is a method for classifying S. Typhi, developed in 1937 by Craigie and Yen.
  • This method utilizes Vi phages, which target the Vi antigen found on S. Typhi. Phages are adapted to be specific to particular strains, and this specificity is achieved by serial propagation of the phage in a strain.
  • Phage typing is particularly useful for identifying Vi-positive strains. However, Vi-negative strains (those lacking the Vi antigen) cannot be typed using this method.
  • There are 97 Vi phage types recognized for S. Typhi, with types E1, O, and A being common in India.
  • To further refine phage typing, additional markers like Nicolle’s complementary phage typing, Kristensen’s biotyping, and antibiograms are used.
  • Phage typing helps track epidemics, providing valuable information on typhoid outbreaks at local, national, and international levels.
• Biotyping:
  • Biotyping categorizes strains of Salmonella based on their biochemical characteristics.
  • For instance, S. Typhimurium has been differentiated into 144 biotypes using 15 different biochemical traits.
  • S. Typhi can be biotyped based on its ability to ferment certain sugars like arabinose, dulcitol, and xylose.
  • Biotyping is particularly useful when phage typing cannot be applied, as it can differentiate strains that are untypeable or belong to the same phage type.
  • A combination of phage typing and biotyping provides a more thorough classification and enables a finer level of discrimination between Salmonella strains.
• Molecular Methods:
  • In advanced research, genotyping methods have become key in differentiating Salmonella strains.
  • Techniques like plasmid fingerprinting, multilocus enzyme electrophoresis, and random amplified polymorphic DNA (RAPD) analysis allow for precise genetic characterization.
  • These molecular approaches are especially valuable for epidemiological studies, as they offer a higher level of discrimination than traditional methods.
  • The National Salmonella Reference Centre in India, located at the Central Research Institute in Kasauli, supports the use of these molecular techniques.
  • Additional reference centers exist for Salmonella of animal origin, located at the Indian Veterinary Research Institute in Izatnagar, which aids in the identification of unusual serotypes.

Cultural Characteristics and Biochemical Reactions of Salmonella

Salmonella is a versatile bacterium that can grow under both aerobic and facultatively anaerobic conditions. Its cultural and biochemical properties play a key role in its identification and differentiation.

Cultural Characteristics

• Growth Conditions:
  • Salmonella thrives at an optimal temperature of 37ºC and a pH of 6–8.
  • It can grow on a variety of both nonselective and selective media.
• Nonselective Media:
  • On nutrient agar and blood agar, Salmonella forms gray-white moist colonies with a smooth, convex surface after 18–24 hours of incubation.
  • Rough strains can form colonies that are opaque and granular, with an irregular surface.
  • Certain strains of Salmonella Paratyphi B may produce large mucoid colonies due to the secretion of polysaccharide slime.
  • On MacConkey agar, Salmonella forms pale, colorless colonies because it does not ferment lactose.
  • Salmonella Typhi does not grow on MacConkey agar, and its colonies on deoxycholate citrate agar are often similar to those on MacConkey agar, sometimes showing black centers after 48 hours of incubation.
• Selective Media:
  • Wilson and Blair’s bismuth sulfite agar is particularly effective for isolating Salmonella spp., especially S. Typhi. This medium inhibits the growth of Shigella, Proteus, and coliforms.
    • On this agar, Salmonella produces jet-black colonies surrounded by a metallic sheen, indicating hydrogen sulfide (H2S) production.
    • Strains like Salmonella Paratyphi A, which do not produce H2S, form green colonies instead.
  • XLD agar (xylose, lysine deoxycholate agar) also selects for Salmonella, where H2S-producing strains form pink colonies with black centers. Non-H2S producing strains produce red colonies.
• Enrichment Media:
  • Selenite F broth and tetrathionate broth are often used to enrich samples for Salmonella.
  • Selenite F is commonly used for clinical specimens but can inhibit the growth of certain strains, such as S. Paratyphi B and Salmonella Choleraesuis.
  • Tetrathionate broth, while useful, can sometimes allow the growth of other bacteria like Shigella and Proteus. Adding brilliant green to tetrathionate broth helps inhibit Proteus growth but might hinder some Salmonella strains.

Biochemical Reactions

• Fermentation Patterns:
  • Salmonella ferments glucose, mannitol, and maltose, producing acid and gas as a result.
  • Salmonella Typhi is an exception, as it does not ferment these sugars.
  • It does not ferment lactose, sucrose, or salicin.
• Other Biochemical Features:
  • Salmonella does not produce indole, a reaction commonly used for bacterial differentiation.
  • Most strains produce hydrogen sulfide (H2S), except S. Paratyphi A and S. Choleraesuis, among a few others.
  • Salmonella does not hydrolyze urea, which can be a key factor in distinguishing it from other bacteria.
  • It is MR positive (methyl red test), VP negative (Voges-Proskauer test), and citrate positive. However, strains like S. Typhi and S. Paratyphi require tryptophan as a growth factor and do not grow on Simmon’s citrate media.
• Amino Acid Decarboxylation:
  • Salmonella strains typically decarboxylate lysine, ornithine, and arginine but not glutamic acid.
  • Exceptions include S. Typhi, which does not decarboxylate ornithine, and S. Paratyphi A, which does not decarboxylate lysine.
• Catalase and Oxidase Reactions:
  • Salmonella is catalase positive and oxidase negative, two tests that help differentiate it from other Gram-negative bacteria.

Biochemical Reactions of Common Salmonella Species

Different species of Salmonella display distinct biochemical reactions that are critical for their identification in laboratory settings. Here’s a breakdown of the key biochemical traits for common Salmonella species:

General Biochemical Reactions

• Glucose Fermentation:
  • All Salmonella species, including S. Typhi, S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C, ferment glucose.
  • They produce acid and gas during this process.
• Mannitol Fermentation:
  • Like glucose, mannitol is fermented by all the Salmonella species listed.
  • Acid and gas are produced during fermentation.
• Lactose Fermentation:
  • Salmonella species do not ferment lactose.
  • This helps distinguish them from other bacteria that can ferment lactose.
• Sucrose Fermentation:
  • These species also do not ferment sucrose.
• Indole Production:
  • None of the common Salmonella species listed produce indole.
  • This is important in differentiating Salmonella from other Enterobacteriaceae.
• Citrate Utilization:
  • Citrate is not utilized by Salmonella Typhi and Salmonella Paratyphi A.
  • However, other species like Salmonella Paratyphi B and Salmonella Paratyphi C show the ability to use citrate.
• Methyl Red (MR) Test:
  • The MR test is positive for all common Salmonella species, indicating that they ferment glucose via the mixed acid pathway.
• Voges-Proskauer (VP) Test:
  • The VP test is negative for Salmonella Typhi, Salmonella Paratyphi A, Salmonella Paratyphi B, and Salmonella Paratyphi C, reflecting the absence of acetoin production.
• Hydrogen Sulfide (H2S) Production:
  • Most Salmonella species produce hydrogen sulfide (H2S).
  • This is an essential feature for identifying Salmonella, as they often show black centers on selective media like XLD agar due to H2S production.
• Xylose Fermentation:
  • Salmonella Typhi does not ferment xylose, while other species such as S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C can ferment it, producing acid and gas.
• D-Tartrate:
  • Salmonella Typhi produces acid when incubated with D-Tartrate, while other species show no reaction.
• Mucate:
  • Similar to D-Tartrate, Salmonella Typhi can ferment mucate, while other species typically show no activity on this substrate.

Cell Wall Components and Antigenic Structure of Salmonella spp.

The cell wall of Salmonella species is a complex structure that plays a critical role in the bacteria’s survival and virulence. One of the key components of the Salmonella cell wall is lipopolysaccharide (LPS), which is responsible for several important functions related to the bacteria’s pathogenicity. Alongside LPS, Salmonella species also have various antigens that are used for identification and understanding their virulence factors.

Lipopolysaccharide (LPS)

• LPS structure: Like all Gram-negative bacteria, Salmonella has a lipopolysaccharide (LPS) structure embedded in its cell wall.
• Components of LPS:
  • The outer part is the O polysaccharide (also called O antigen), which is made up of repeating sugar units and gives the bacteria its antigenic specificity. The presence of this sugar repeat sequence is important in determining the virulence of Salmonella.
  • The R core is the middle part of the LPS structure, acting as a bridge between the O antigen and lipid A.
  • The lipid A component anchors the LPS to the outer membrane and is responsible for the endotoxin activity of Salmonella.
• Virulence and Endotoxin Activity:
  • The O antigen helps distinguish different serotypes and is crucial for virulence. Salmonella strains lacking the full complement of O sugar units are referred to as “rough” strains and are less virulent.
  • The lipid A acts as an endotoxin, contributing to fever, activation of the complement system, and promoting septic shock in systemic infections.
  • Antibodies against the R core (common enterobacterial antigen) can provide protection against various Gram-negative bacteria due to shared components in the core structure.

Major Antigens of Salmonella

There are three primary types of antigens present on the surface of Salmonella bacteria: H antigen, O antigen, and surface antigens.

1. H Antigen (Flagellar Antigen):
   • Found on the flagella, the H antigen is heat and alcohol-labile but is preserved under formaldehyde treatment.
   • It is genus-specific, meaning it is unique to Salmonella and is not shared with other enterobacteria.
   • The H antigen is highly immunogenic, triggering antibody production after infection or vaccination.
   • Salmonella can exhibit phase variation in H antigens, switching between two forms: phase I and phase II, making it important for identifying specific serotypes.
2. O Antigen (Somatic Antigen):
   • The O antigen is part of the outer membrane and is an integral part of the LPS complex.
   • It is heat-stable and resistant to boiling and alcohol, which makes it a stable antigen for identification.
   • Unlike the H antigen, the O antigen is less immunogenic but is crucial in serotyping Salmonella species.
   • Salmonella strains are classified into serogroups based on the O antigen, with 67 different O antigens described so far.
3. Surface Antigens:
   • Vi Antigen: Found over the O antigen, this surface antigen is associated with virulence and is typically seen in serotypes like Salmonella Typhi. It is heat labile and can be destroyed by boiling, but remains intact under formaldehyde treatment. It masks the O antigen, preventing agglutination with O antisera.
   • M and N Antigens: These are also surface polysaccharide antigens. They can prevent agglutination by O antiserum and are destroyed by extended boiling.
   • F Antigens: Located on the fimbriae, these antigens appear in two phases: one where they are present and one where they are absent. Their presence is related to the bacteria’s ability to form colonies with specific characteristics.

Antigenic Variation

Salmonella species exhibit several types of antigenic variation, allowing them to evade host immune responses and adapt to different environments. These variations include:

• OH–O Variation: Salmonella can switch between flagellated (OH) and nonflagellated (O) strains, typically in response to changes in growth conditions. This is a phenotypic variation and can be reversed when conditions return to normal.
• V–W Variation: Salmonella Typhi and some other serotypes can switch between the Vi antigen (V form) and the loss of the Vi antigen (W form). This is related to subculturing and can be reversible.
• S–R Variation (Smooth-to-Rough): Mutations can lead to a loss of the O antigen, changing the colony morphology from smooth to rough. Rough strains are less virulent than smooth strains.
• Phase Variation: Salmonella flagellar antigens exist in two phases (phase 1 and phase 2). The bacteria may alternate between these phases, and identifying both phases is essential for accurate serotyping. This variation is important for immune evasion, as different antigens can be expressed at different times.
• Variations in O Antigens: Alterations in O antigens can occur due to lysogenization by converting phages, which can lead to changes in bacterial serotype.

Virulence Factors of Salmonella spp.

Salmonella spp. have a range of virulence factors that contribute to their ability to cause infection. These factors are encoded on both the bacterial chromosome and large plasmids. The major virulence factors include:

• Type III Secretion Systems (TTSS):
  • These systems play a critical role in how Salmonella secretes its virulence factors into host cells.
  • SPI-1 facilitates nonphagocytic cell invasion, allowing the bacteria to enter epithelial cells.
  • SPI-2 is involved in survival and replication within macrophages.
  • Salmonella strains lacking these pathogenicity islands (SPI-1, SPI-2) become avirulent and lose the ability to infect.
• Endotoxin:
  • The endotoxin, a component of the bacterial cell wall, is responsible for many systemic manifestations of Salmonella infections, such as fever and septic shock.
• Fimbriae:
  • Fimbriae are species-specific structures that allow Salmonella to bind to M cells in the Peyer patches of the small intestine.
  • M cells are specialized to transport foreign antigens to macrophages, helping the bacteria to interact with the immune system and initiate infection.
• Acid Tolerance Response (ATR) Gene:
  • The ATR gene enables Salmonella to survive in highly acidic environments, such as the stomach.
  • It also helps the bacteria resist the acidic pH inside phagosomes, providing a means for survival within host immune cells.
• Enzymes (Catalase & Superoxide Dismutase):
  • Catalase and superoxide dismutase are enzymes that help Salmonella survive the oxidative stress within macrophages.
  • These enzymes protect the bacteria from intracellular killing, a key defense mechanism employed by immune cells.

Together, these virulence factors equip Salmonella spp. with the tools necessary to invade host cells, survive within the host, and evade immune responses. Without these factors, Salmonella would be far less effective at causing disease.

Pathogenesis of Enteric Fever

Enteric fever, commonly caused by Salmonella Typhi and Salmonella Paratyphi, results from the complex interaction between the infecting bacteria and the human host. The severity of the disease depends on the virulence of the strain and factors related to the host’s immune response.

• Infective Dose:
  • Infection begins with ingestion of food or water contaminated with Salmonella. The required infective dose (ID50) for infection in humans ranges from 10³ to 10⁶ bacilli.
  • A larger inoculum is needed to overcome stomach acidity and compete with the gut flora. Higher doses often lead to more severe illness and shorter incubation periods.
  • Smaller doses can still cause infection under certain conditions, such as:
    • Co-ingestion with food that speeds up gastric transit (e.g., liquids).
    • Co-ingestion with foods that increase stomach pH (e.g., milk, cheese).
    • Use of antacids.
    • Ingestion by individuals with compromised immune systems.
• Attachment and Invasion:
  • Once in the intestine, Salmonella attaches to the mucosal lining using fimbriae or pili.
  • The bacteria selectively target M cells in the Peyer patches of the ileum. These cells play a crucial role in the immune system, as they transport antigens to macrophages.
  • Using a Type III secretion system (TTSS), Salmonella introduces invasion proteins (Sips or Ssps) into the M cells, causing membrane ruffling. This leads to the engulfment of the bacteria by the host cells, allowing them to replicate intracellularly in the phagosome.
  • Other factors, such as the acid tolerance response (ATR) gene, protect Salmonella from the acidic conditions of the stomach and phagosome, enhancing its survival.
  • Catalase and superoxide dismutase also help protect the bacteria from the immune system’s attempts at intracellular killing.
• Spread Within the Host:
  • After replication within the M cells, the bacteria are released into the lamina propria, a layer of tissue just beneath the mucosal lining.
  • Here, the immune response is triggered: Typhoidal strains like S. Typhi attract macrophages, while nontyphoidal strains provoke neutrophil recruitment.
  • S. Typhi and other virulent strains enter the bloodstream via the mesenteric lymph nodes and the thoracic duct, initiating a bacteremic phase. From the blood, the bacteria can spread to various organs, especially the liver, spleen, bone marrow, gallbladder, and Peyer patches in the terminal ileum.
• Organ Invasion and Effects:
  • The liver becomes a key site of infection, leading to cholecystitis, which often remains subclinical but results in positive stool cultures.
  • Chronic biliary infections can occur, especially in individuals with pre-existing gallbladder disease, leading to long-term fecal carriage of the bacteria.
  • Peyer patches may undergo hyperplasia, and the chronic inflammation can lead to tissue necrosis, ulcer formation, and complications like hemorrhage or peritonitis due to transmural perforation.
• Fever and Toxemia:
  • The prolonged fever seen in enteric fever is likely caused by pyrogens and other inflammatory mediators produced at the sites of infection. However, the exact mechanisms behind the fever and toxemia remain poorly understood.

Clinical Syndromes of S. Typhi Infection

S. Typhi causes typhoid fever, a severe, systemic infection with a hallmark set of symptoms. It shares similarities with paratyphoid fever, caused by other Salmonella species, but typhoid fever tends to be more severe. The infection progresses in phases, with both gastrointestinal and systemic manifestations.

• Incubation Period:
  • The incubation period typically ranges from 7 to 14 days, though it can extend anywhere from 3 to 56 days.
• Initial Symptoms:
  • Symptoms begin gradually, often starting with:
    • Headache
    • Malaise
    • Anorexia
    • Coated tongue
    • Abdominal discomfort (may include constipation or diarrhea)
• Fever and Toxemia:
  • A hallmark feature of enteric fever is a step-ladder pyrexia (rising fever with peaks), often accompanied by relative bradycardia (slow heart rate).
  • Toxemia is common, contributing to systemic symptoms.
• Abdominal and Organ Involvement:
  • During the progression, the patient may develop:
    • Abdominal symptoms: gastrointestinal distress may emerge after the initial fever phase.
    • Palpable spleen (splenomegaly) and enlarged liver are common physical findings during the acute phase of infection.
• Bacteremic Phase:
  • This phase occurs early and involves the spread of S. Typhi through the bloodstream. The bacteria then colonize the gallbladder and reinfect the intestines, continuing the cycle of infection.
• Complications:
  • Intestinal perforation and severe hemorrhage are major complications of enteric fever, leading to high morbidity and potential mortality.
  • Other complications include:
    • Circulatory collapse
    • Toxic encephalopathy
    • Cerebral thrombosis
    • Hepatitis
    • Pancreatitis
    • Arthritis
    • Myocarditis
• Relapse:
  • Relapse is common, occurring in 10–20% of patients who receive antibiotic treatment. It usually happens about 1 week after stopping treatment but can even occur 2 months later.
  • The relapse tends to be milder and shorter than the initial infection.
  • Blood cultures and serum H, O, and Vi antibodies can test positive during relapse, confirming the reinfection.
• Paratyphoid Fever:
  • Paratyphoid fever, caused by S. Paratyphi A, B, and C, has similar symptoms to typhoid fever but is generally milder. However:
    • S. Paratyphi C may cause more severe septicemia with suppurative complications.
    • S. Paratyphi A and B cause a form of fever that resembles typhoid fever but with less severity.

Reservoir, Source, and Transmission of S. Typhi Infection

The transmission of S. Typhi is heavily reliant on human carriers and contaminated food or water sources. There is no animal reservoir for typhoidal salmonellae, making human-to-human transmission the primary route.

• Reservoir:
  • Infected patients and carriers serve as the main reservoirs for S. Typhi.
  • Approximately 2–4% of patients with typhoid fever become chronic carriers.
  • Chronic carriers harbor the bacteria in their gallbladder, where the bacteria can persist and be excreted in feces, known as fecal carriers.
  • A smaller percentage are urinary carriers, where the bacteria persist in the kidneys and are excreted in the urine, but this is less common.
  • Urinary carriers usually show some underlying urinary issues like calculi or schistosomiasis.
  • Carrier state is more prevalent in women than in men, and it tends to affect older adults (over 40 years) more than younger individuals.
  • Food handlers or cooks who become carriers pose a significant public health risk, as they can transmit the infection through food. One famous example is Mary Mallon (Typhoid Mary), a New York cook who caused at least seven outbreaks over 15 years, infecting more than 200 people.
• Source of Infection:
  • The primary source of infection for S. Typhi comes from human feces, specifically when food or water is contaminated by feces from infected individuals or carriers.
  • Contaminated food, such as vegetables or water, can also serve as a source when infected by human fecal matter.
  • S. Paratyphi A infection, similar to S. Typhi, occurs only in humans, whereas S. Paratyphi B can also be found in animals, such as dogs and cows.
  • The bacteria can be transmitted through poor hygiene or infected food handlers contaminating the food supply.
• Transmission:
  • The most common mode of transmission is ingestion of contaminated food or water.
  • This contamination typically occurs due to poor personal hygiene or through infected food handlers, making foodborne transmission a significant risk.
  • S. Typhi has a low infectious dose, which makes person-to-person spread more likely. Even small amounts of the bacteria are sufficient to cause infection.
  • The infectious dose is even lower for those at higher risk, including:
    • Young children or older adults.
    • Individuals with weakened immune systems, such as those with leukemia, lymphoma, or sickle cell disease.
    • People with reduced gastric acidity, which lowers the stomach’s ability to kill pathogens.
• Animal Reservoirs:
  • There is no animal reservoir for S. Typhi.
  • Transmission of nontyphoidal salmonella (such as S. Typhimurium) can occur through zoonotic transmission, where the bacteria are passed from animals to humans.
  • Animals, including poultry, livestock, reptiles, and pets, are the primary reservoirs for these nontyphoidal Salmonella species.
  • Improperly cooked foods—such as poultry, red meats, unpasteurized milk, and eggs—are common sources of infection when they are contaminated by either infected animals or infected humans.
  • Contact with infected reptiles (e.g., turtles, iguanas, tortoises) and drinking contaminated water are also possible routes of transmission.

Laboratory Diagnosis of Salmonella

The laboratory diagnosis of salmonella infections, particularly enteric fever, involves a combination of methods. These methods include culturing the bacteria, serological tests to detect antibodies or antigens, and molecular diagnostics like PCR. Each method has its strengths depending on the stage of infection and the specimen available. Here’s how it works:

Specimens Collected for Diagnosis:

• Blood and blood clot are the primary specimens used for culture.
• Bone marrow, stool, cerebrospinal fluid (CSF), and peritoneal fluid are also commonly tested.
• Other potential specimens include mesenteric lymph nodes, resected intestines, pharynx, tonsils, abscesses, bone, and urine.

Culture Methods

• Blood Culture:
  • Blood culture is highly useful in diagnosing enteric fever, especially in the first two weeks of illness.
  • The process involves collecting 5-10 mL of blood, which is then inoculated into bile broth to neutralize the bactericidal effects of blood.
  • The culture bottle is incubated at 37ºC for up to 7 days, and then subcultured onto MacConkey agar.
  • In the first week of fever, blood cultures are positive in 90% of cases, dropping to 25% towards the end of the infection. Antibiotic treatment will rapidly reduce the positivity rate.
• Castañeda’s Biphasic Blood Culture:
  • This method reduces contamination risks and is economical. It uses a culture bottle with an agar slant flooded by bile broth.
  • After inoculation, the bottle is placed upright, and subculturing is done by tilting the bottle so that the broth flows over the agar slant.
  • If Salmonella is present, colonies appear on the agar slant.
• Clot Culture:
  • A more sensitive method than blood culture. This technique avoids the inhibitory substances in serum since they are absent in the clot.
  • Blood is collected and allowed to clot, then the serum is separated for antigen or antibody detection. The clot is broken up and added to bile broth with streptokinase, which helps release bacteria trapped inside.
• Bone Marrow Culture:
  • Bone marrow culture is the most sensitive and may be positive even if blood cultures are negative, or if the patient has been on antibiotics.
  • This is particularly recommended for patients whose initial blood cultures fail.
• Fecal Culture:
  • Salmonella can be excreted in the feces during and after the illness.
  • A stool sample is inoculated on MacConkey, DCA, and Wilson-Blair media.
  • S. Typhi produces large black colonies with a metallic sheen on Wilson-Blair medium.
  • Enrichment using selenite and tetrathionate broth is used to improve culture sensitivity.

Identification of Salmonella Bacteria

• Motility and Biochemical Tests: After culturing, motility and biochemical tests help identify Salmonella.
• Slide Agglutination Test:
  • A simple test where bacterial growth is mixed with specific antisera for Salmonella serogroups.
  • Agglutination suggests the presence of Salmonella in the sample.
  • For S. Typhi, Vi antigen testing or anti-Vi serum may be necessary, as some strains do not agglutinate with O antisera.

Serological Diagnosis

• Widal Test:
  • The Widal test detects antibodies to the O (somatic) and H (flagellar) antigens of Salmonella.
  • A fourfold rise in antibody titers between two samples, taken 7–10 days apart, is a more reliable diagnostic indicator than a single sample.
  • A titer of 1/100 or more for O antibodies and 1/200 for H antibodies suggests enteric fever.
  • However, the Widal test can be misleading due to cross-reactivity with other pathogens or prior vaccination.
• Alternative Serological Tests:
  • Tests like Indirect Hemagglutination, Co-agglutination, and ELISA are used to detect antibodies and antigens with varying levels of sensitivity.
  • These tests offer different sensitivities and can be more accurate in certain settings.

Antigen Detection

• S. Typhi Antigen in Serum:
  • The presence of S. Typhi antigen in serum and urine confirms active or recent infection, as it is absent in cured cases.
  • Counter-current immunoelectrophoresis, co-agglutination, and ELISA can detect circulating antigens for confirmation of typhoid fever.

Treatment of Salmonella Infections

Salmonella infections can be treated using several different antibiotics. The choice of treatment depends on the type of Salmonella, resistance patterns, and patient factors.

• Chloramphenicol:
  • Used since 1948 for enteric fever, specifically S. Typhi.
  • How it works: Binds to the 50S bacterial ribosomal subunit, inhibiting protein synthesis.
  • Effectiveness: Still used for sensitive S. Typhi strains due to its low cost.
  • Standard course: A 14-day treatment course with chloramphenicol, ampicillin, or trimethoprim-sulfamethoxazole.
• Fluoroquinolones:
  • Commonly used for multidrug-resistant S. Typhi infections.
  • Examples: Ciprofloxacin, pefloxacin, norfloxacin.
  • These antibiotics are effective and show low relapse rates and low carrier rates.
• Third-generation Cephalosporins:
  • Examples: Ceftazidime, ceftriaxone, cefotaxime.
  • How they work: They inhibit bacterial cell wall synthesis, preventing bacterial growth.
  • In vitro efficacy: They show good activity against S. Typhi and other salmonellae.
  • Disadvantages:
    • The need for intravenous administration.
    • Higher cost, which can be problematic in developing countries.
  • Emerging resistance: There have been reports of ceftriaxone-resistant Salmonella infections.
• Course of Treatment:
  • For S. Typhi: A 14-day course of antibiotics like chloramphenicol, ampicillin, or trimethoprim-sulfamethoxazole is commonly recommended.

Prevention and Control of Salmonella

Reducing the spread of Salmonella, particularly S. Typhi, relies on a mix of hygiene practices, safe water access, and vaccination. The most effective strategies focus on improving living conditions and preventing contamination.

• Safe Drinking Water:
  • Access to clean, safe water is essential in preventing typhoid fever.
  • Proper sanitation and the safe disposal of excreta drastically reduce the risk of Salmonella transmission.
• Food Hygiene:
  • Maintaining proper food hygiene is crucial in stopping Salmonella spread.
  • Handling and cooking food correctly can prevent contamination from infected food sources.
• Sanitary Disposal of Excreta:
  • Correct disposal of human waste ensures the environment remains free of contamination, reducing overall infection rates.
• Immunization:
  • Typhoid vaccines are an important tool in reducing the incidence of typhoidal Salmonella.
  • Routine vaccination is recommended for:
    • People with close contact to S. Typhi cases or carriers.
    • Travelers going to areas with a higher risk of Salmonella infection.
    • Microbiology lab workers handling S. Typhi samples.
  • Types of Typhoid Vaccines:
    • Killed Vaccines:
      • TAB Vaccine:
        • A whole-cell killed vaccine containing S. Typhi and S. Paratyphi A and B.
        • Given in two doses, spaced 4-6 weeks apart, followed by a booster every 3 years.
        • Efficacy: 70–90% over 3–7 years. Common side effects: fever, pain at the injection site.
        • A divalent version of this vaccine is used in India, omitting S. Paratyphi B due to its lower prevalence there.
    • Vi Capsular Polysaccharide Antigen Vaccine (ViCPS):
      • Composed of the Vi antigen, a capsular polysaccharide from S. Typhi.
      • Single-dose injection with a booster every 2 years if exposure persists.
      • Efficacy varies by region: 50–64% in South Africa and 72% in Nepal.
      • Side effects include fever, headache, and skin reactions.
      • Not recommended for children under 2 years.
    • Acetone-inactivated parenteral vaccine:
      • Available only in the United States for military use.
      • Shows efficacy between 75–94%, with boosters every 3 years if exposure continues.

Proper hygiene, clean water, and regular vaccination are the most effective measures in preventing Salmonella infections, particularly in areas with higher risk.