4. Salmonella spp, Shigella spp.

Laboratory Diagnosis of Infectious Diseases

Overview

  • Focus on bacteria from family Enterobacteriaceae: genus Salmonella and genus Shigella.

  • Presented by Assist. Prof. Denitsa Tsaneva-Damyanova, MD.

Genus Salmonella

Characteristics

  • Gram-negative rods from the family Enterobacteriaceae.

  • Non acid-fast bacteria.

  • Morphology: Size ranges from 0.3-1 μm (width) and 1.0-6.0 μm (length).

  • Motility: Flagella are peritrichous (8-20), uniformly distributed.

  • Capsules: Do not generally form capsules; S. typhi has a microcapsule.

Taxonomic Classification

Challenges in Classification

  • Taxonomy of Salmonella is controversial due to historical naming based on clinical cases (e.g., Salmonella typhi, Salmonella choleraesuis).

  • DNA studies show most clinical isolates belong to Salmonella enterica.

  • Over 2,500 serotypes and 51 groups identified from this species.

  • Correct nomenclature: Salmonella enterica, serovar Typhi (capitalized serotype).

Subspecies

  • Seven subspecies proposed: enterica I, salamae II, arizonae IIIa, diarizonae IIIb, houtenae IV, bongori V, indica VI.

  • S. typhi, S. paratyphi A & B belong to subspecies enterica I.

Physiology

Growth Conditions

  • Facultative anaerobes.

  • Non-fastidious.

  • Optimal temperature: 37°C, pH: 7.2-7.4.

  • Growth on various media types: essential, selective, enriched, differential.

  • Colonies on solid media: sizes 2-4 mm, classified as S (smooth) and R (rough).

Media Selection

Selective Media

  • Transparent colonies with black centers (due to H₂S), except S. paratyphi.

  • Common selective media:

    • MacConkey agar: selective for Gram-negative bacteria; differentiates lactose fermenters (pink) from non-fermenters (translucent).

    • Kauffman media, Rappoport media, Möller media, Apocholate-citrate agar, Hektoen enteric agar, Brilliant Green agar.

Differential Media

  • Forms lactose-negative colonies on differential media:

    • McConkey agar: differentiates lactose fermenters from non-lactose fermenters.

    • Additional media: Endo agar, Gassner agar, Levine agar, EMB agar for Gram(-) bacteria.

Additional Common Media

  • Frequently used for Salmonella: SS agar, bismuth sulfite agar, Hektoen enteric (HE) medium, Brilliant Green agar, XLD agar.

  • Growth indication: black areas indicate H₂S production under alkaline conditions.

Hektoen Enteric Agar (SS-Agar)

  • Inhibits Gram(+) bacteria.

  • Differentiates for lactose fermentation and hydrogen sulfide production.

  • Appearance: bluish-green colonies with black centers for Salmonella; bluish-green colonies for Shigella.

Colonial Growth Patterns

  • S. choleraesuis subsp. arizonae and S. Typhimurium on blood agar and HE agar respectively.

  • S. Typhimurium: blue-green on HE agar, indicating no lactose fermentation and H₂S production.

Enrichment Media

  • Selenite Cystine Broth Base: selective enrichment for Salmonella.

  • Rappoport-Vassiliadis Broth: selective for Salmonella isolation.

Biochemical Activity

  • Rich enzyme set, ferments glucose, maltose, mannitol producing acid.

  • Lactose fermentation: negative (ONPG test negative), saccharose negative.

  • Methyl red test: positive.

  • Voges-Proskauer test: negative.

  • Citrate utilization: positive (growth on Simmons' citrate agar).

  • Lysine decarboxylase: positive.

  • Urease: negative.

  • Indole: negative.

  • H₂S from thiosulfate; gelatine hydrolysis: negative.

Antigenic Structure

Types of Antigens

  • Somatic (O), Capsular (K or Vi), Flagellar (H) antigens.

  • O-antigens are heat-stable, alcohol-resistant; multiple serological identification factors present.

  • Vi antigen present in some serovars (e.g., Typhi, Paratyphi C).

Flagellar Staining

  • Motility through peritrichous flagella.

  • Differentiates Salmonella from non-motile Shigella by motility tests and flagellar stains.

Virulence Factors

  1. Endotoxin: activity based on lipid A component of LPS, released during cell lysis.

  2. Capsule: protects from phagocytosis; hinders antibody binding.

  3. Antigenic Phase Variation: somatic O, capsular K, and flagellar H antigens can be expressed or not.

  4. Type III Secretion System: common in various bacteria for delivering virulence factors.

  5. Resistance to Serum Killing: bacterial capsule prevents complement binding.

  6. Antimicrobial Resistance: often encoded on transferable plasmids.

Epidemiology

  • Reservoirs: Colonization in animals; transmission through contaminated food (poultry, eggs).

  • Serotypes: S. Typhi and S. paratyphi are highly adapted to humans, causing person-to-person infections.

  • Risks: Improperly cooked food, immunocompromised individuals.

  • Global Occurrence: Particularly common in warm months.

Pathogenesis and Immunity

  • Invasion: bacteria penetrate intestinal mucosa, replicate in endocytic vacuoles, may lead to septicemia through mesenteric lymph nodes.

  • Gene Regulation: Pathogenicity islands I and II control attachment, engulfment, and replication.

Clinical Diseases

  • Four infection types:

    1. Gastroenteritis

    2. Septicemia

    3. Enteric fever (Typhoid fever, Paratyphoid fever)

    4. Asymptomatic colonization.

Enteric Fever

  • Salmonella Typhi: causes typhoid fever; milder paratyphoid fever caused by S. paratyphi.

  • Symptoms: Gradual onset of fever, headache, malaise; gastrointestinal symptoms follow 1 week later.

  • Complications: myocarditis, encephalitis, sepsis.

Gastroenteritis

  • Most common salmonellosis form; symptoms appear 6 to 48 hours after consumption of contaminated food.

  • Symptoms: nausea, vomiting, diarrhea, abdominal cramps; generally resolve spontaneously in 2-7 days.

Septicemia

  • All Salmonella species can cause bacteremia, higher risk in young and elderly.

  • Clinical presentation resembles other Gram-negative bacteremias; localized infections occur in 10% of cases.

Asymptomatic Colonization

  • Human colonization by S. Typhi and S. Paratyphi; chronic in 1%-5% of patients, often in the gallbladder.

Microbiological Diagnosis of Enteric Fever

  • Materials: Blood cultures, feces, urine, bone marrow, etc.

  • Chronic colonization: gallbladder material for confirming.

S. Typhi Serology

  • Blood taken after the first week for Widal reaction; positive at thresholds of 1:100 (O agglutination), 1:200 (H agglutination), 1:10 (Vi agglutination).

  • Rapid Diagnosis: Immunofluorescence, co-agglutination.

Microbiological Diagnosis of Gastroenteritis

  • Materials: Regurgitated materials, stool specimens, urine, and blood for immunocompromised.

  • Methods: Growth on various media, biochemical activity assessment, identification, antibiogram.

Antibiotic Susceptibility

  • Treatment: Salmonella enteric fever treated with chloramphenicol, amoxicillin, ampicillin.

  • Resistance issues: Increased multiresistance in Salmonella spp.; plasmid-borne resistance is common.

  • Non-complicated gastroenteritis does not require antibiotics.

Genus Shigella

Characteristics

  • Gram-negative, facultative anaerobes, nonmotile, rod-shaped bacteria.

  • Lipopolysaccharide structure includes O polysaccharide, core polysaccharide, lipid A (endotoxin).

Shigella Classification

  • Divided into four serogroups:

    1. A: S dysenteriae (12 serotypes)

    2. B: S flexneri (6 serotypes)

    3. C: S boydii (18 serotypes)

    4. D: S sonnei (1 serotype).

Pathogenesis and Immunity

  • Genes for adherence, invasion, replication on a virulence plasmid.

  • M cells in Payer patches are the initial invasion site.

  • Type-III secretion used for delivering effector proteins; Shigella lyses phagocytic vacuoles unlike Salmonella.

  • Shiga toxin produced by S. dysenteriae strains.

Hallmarks of Shigellosis

  • Diarrhea, colonic epithelial invasion, inflammatory processes leading to dysentery symptoms.

  • Symptoms may include blood and mucus in stool.

Host Defenses

  • Inflammatory response, mucus secretion, colonic epithelium regeneration promote recovery.

  • Specific immunity induced by first infection via antibody response to LPS.

Shigella Epidemiology

  • Humans are the only reservoir; transmission occurs via fecal-oral route.

  • High risk in children; disease prevalent in developing countries with poor sanitation.

Clinical Disease of Shigellosis

  • Symptoms include diarrhea, fever, nausea, vomiting, painful bowel movements with possible blood.

  • Risk of seizures in young children; symptoms may develop after 2-4 days and last for several days.

Microbiological Diagnosis of Shigella

  • Materials: Stool, blood, intestinal contents.

  • Cultures made bedside to prevent destruction of Shigella.

  • Identification through biochemical and serological methods (Gruber, Widal).

Comparative Features of Shigella and Salmonella

Feature

Shigella

Salmonella

Disease

Bacillary dysentery

Typhoid fever

Motility

Non-motile

Motile

H₂S Production

Negative

Positive

Indole Formation

Negative

Positive

Infection Nature

GIT

Systemic

Immunity

Short period

Lasting

Endo/Exotoxin

Endotoxin

Potent endotoxin

Treatment of Shigellosis

  • Antibiotic therapy effective in shorting symptomatic disease duration.

  • Guidance from susceptibility tests recommended; empiric therapy may involve fluoroquinolone or trimethoprim-sulfamethoxazole.

  • Infection control measures include hand washing and proper handling of contaminated materials.