Microbiology- Bacteriology - Salmonella, Shigella & Proteus

Microbiology, Bacteriology, Gram-Negative Bacilli (GNB),
Gram-negative Rods (GNR).

Salmonella - Infection, disease, Significance, Infective dose, History, Morphology of Salmonella, Cultural characteristics of Salmonella, Laboratory diagnosis of Salmonella.

Shigella - Distribution, Colony appearance, infection site, infective dose, Morphology of Shigella, Cultural characteristics of Shigella, Laboratory diagnosis of Shigella.

Proteus- Distribution & Habitat, Culture Colony Appearance, Infection Site, Morphology of Proteus, Cultural characteristics of Proteus, Laboratory diagnosis of Proteus.

1. Salmonella, Shigella & Proteus By SANDEEP KUMAR Master of Science in Medical Laboratory Science Delhi Skill and Entrepreneurship University, Dwarka Sec-9, New Delhi

2. Bacteria Morphology of Bacteria C ultural characteristics of Bacteria L aboratory diagnosis of Bacteria Salmonella, Shigella, Proteus

3. Salmonella Bacteria

4. Salmonella Introduction: Infective Dose: High infective dose (10 2 – 10 5 Bacilli) Morphology : Salmonella is a rod-shaped bacterium (bacillus). Unlike other strains of bacilli, salmonella does not produce spores. On MacConkey agar, salmonella colonies appear colorless and transparent, sometimes with dark centers . Diseases: Salmonella causes two diseases in humans: Enteric fever ( typhoid) & Gastroenteritis , Doctors refer to both diseases as “salmonellosis.” Significance : Salmonella is a common foodborne pathogen . It causes zoonotic infections (transmitted between animals and humans). History: Named after American veterinary surgeon Daniel Elmer Salmon.

5. Salmonella Morphology: Gram Negative Bacilli (Rod Shaped). Size - 2-4 X 0.6 μ m. (length X diameter) Non Spore forming bacterium Non Capsulated bacteria Motile bacteria – Have peritrichous flagella which help in movements.

6. Salmonella Culture Characteristics: Aerobic and facultatively anaerobic Salmonellae Growth Conditions: Optimum temperature: 37°C (range: 15–41°C), pH range: 6-8 MacConkey’s Agar and Deoxycholate Citrate Agar (DCA): Colonies - Colorless due to non-lactose fermentation (NLF) Wilson and Blair Bismuth Sulphite Medium: Colonies: Jet black with metallic sheen (due to hydrogen sulphide production), Exception: S. paratyphi A and other non-H2S-producing species form green colonies Xylose Lysine Deoxycholate (XLD) Agar: Colonies - Red with black centers (due to H2S production) , H2S-negative Salmonella serotypes: Red colonies without black centers . Enrichment Broths (Selenite F and Tetrathionate): Used for inoculating specimens, especially feces

7. Salmonella Laboratory Diagnosis: Duration of illness Specimen, Test done 1 st Weak Culture of Blood Culture of Bone marrow aspirate Culture of Duodenal aspirate 2 nd & 3 rd Weak Serum For Antibody (Ab) detection by Widal. For Antigen (Ag) detection. Stool & Urine Culture 4 th Weak Stool & Urine Culture Carriers Stool & Urine Culture Serum for detection of Abs to Vi Ag. Sewage culture – Indirect way

8. Salmonella Laboratory Diagnosis: Continued ……………… Specimen: Blood, Serum, Stool and urine, Bone marrow aspirate, duodenal aspirate, sewage sample (Indirect Test). Gram Staining: Gram Negative (-ve) Bacilli, red color. Motility Test: By hanging drop method, Salmonella are motile with peritrichous flagella . Culture Isolation Blood & Bone marrow culture: (in the first week of illness): in blood culture bottle (BHI broth, automated BACTEC)

9. Salmonella Laboratory Diagnosis: Continued ……………… 2. Stool culture (in 3-4 weeks of illness): Enrichment broth - Selenite F broth, tetrathionate broth and gram-negative(-ve) broth Low selective media - e.g. MacConkey Agar Media - Non-lactose fermenting (NLF) do not ferment lactose, produces a colorless or pale colony, translucent colony. Highly selective medium: 3. Urine culture (in 3-4 weeks of illness) - done on MacConkey agar Agar Media Appearance Deoxycholate Citrate Agar (DCA) NLF pale colonies with black center. Xylose Lysine Deoxycholate (XLD agar) Red colonies with black center. Shigella-Salmonella agar (SSA) Colorless colonies with black center. Hektoen Enteric Agar (HEA) Blue Green colonies with black center. Wilson Blair's bismuth sulphite medium jet black colonies

10. Salmonella Laboratory Diagnosis: Continued ………………

11. Salmonella Laboratory Diagnosis: Continued ……………… Catalase Test : Positive for catalase, Indicates the production of catalase enzyme. 2. Oxidase Test: Negative for oxidase., No cytochrome c oxidase production. 3. Nitrate Reduction: Nitrate reduced to nitrite, Presence of nitrate reductase enzyme. 4. Indole Test Negative for indole production, No indole from tryptophan. (ICUT) 5. Citrate Utilization Test ( TSl ): Variable result for citrate utilization, May or may not utilize citrate as a carbon source. 6. Urease Test: Negative for urease production. 7. TSI (Triple Sugar Iron) Test: K/A (alkaline/acid) result, Gas production (except in Salmonella Typhi), H2S production (positive in Salmonella Typhi, absent in Salmonella Paratyphi A, abundant in Salmonella Paratyphi B). Biochemical Test

12. Salmonella Laboratory Diagnosis: Continued ……………… Serum Antibody Detection (Widal Test): - Detects antibodies against O and H antigens. Specific antibodies for different infections. Typhi Dot (Rapid Test): Based on Immunochromatography (or ICT, lateral flow assay), detect IgM & IgG. Antigen Detection: Serum and urine antigen detection by ELISA and CIEP. Molecular Methods: PCR detecting specific genes. Nonspecific Findings: Neutropenia (reduced white blood cell count). Antimicrobial Susceptibility Testing: Determines antibiotic sensitivity.

13. Salmonella Laboratory Diagnosis: Continued ………………

14. Salmonella Laboratory Diagnosis: Continued ……………… Rapid Card test

15. Shigella Bacteria

16. Shigella Introduction: 1. Infective Dose: low infective dose (10 Bacilli) 2. Distribution: Shigella is primarily found in the human intestinal tract. It can also be present in contaminated environments and organic matter. 3. Colony Appearance: Shigella forms small, smooth, moist, and creamy colonies on culture media. Notably, it exhibits a characteristic “swarming” ability. 4. Infection Site: Shigella primarily infects the gastrointestinal tract, leading to severe inflammation and dysentery .

17. Shigella Morphology: Gram Negative Bacilli (Rod Shaped). Size - 1-3 µm X 0.5 µm (length X diameter). Non-Spore forming bacterium. Non Capsulated bacteria. Non Motile bacteria – It Does not have flagella for movements

18. Shigella Culture Characteristics: 1. Growth Conditions: Aerobes and facultative anaerobes Optimal temperature: 37°C & pH: 7.4 Tolerate temperature range: 10°C to 40°C 2. Colony Characteristics: After overnight incubation: 2 mm diameter, circular, convex, smooth, translucent. MacConkey’s agar and DCA: Colourless (non-lactose fermenting), except Sh. sonnei (pink). XLD agar : Shigella colonies appear red without black centers , while H2S-producing bacteria have black centers in colonies.

19. Shigella Culture Characteristics : Continued ……… 3. Selective Media: Salmonella-Shigella (SS) agar : Contains bile salts, sodium citrate, sodium thiosulphate, ferric citrate, lactose, and neutral red. - Shigella: Colourless (non-lactose fermenting), no blackening. - Salmonella: Colourless with black centers . Hektoen -enteric (HE) agar: Contains bile salts, lactose, sucrose, sodium thiosulphate, ferric ammonium citrate, acid fuchsin, and thymol blue - Shigella: Green (fading to periphery) - Salmonella: Blue-green with black centers

20. Shigella Laboratory Diagnosis: 1. Specimens: - Fresh stool collection is ideal. - Rectal swabs are not recommended. - Direct swab of an ulcer under sigmoidoscopic examination is preferred. 2. Transport: - Transport specimens immediately. - Use Sach's buffered glycerol saline (pH 7.0-7.4) if delay is inevitable. - Avoid alkaline transport media for vibrios (inhibitory for shigella). 3. Direct Microscopy: - Saline and iodine preparation reveals pus cells, erythrocytes, and macrophages. - Excludes parasitic causes of dysentery.

21. Shigella Laboratory Diagnosis: Continued ……… 4. Culture: - Inoculate specimen on selective media (e.g., MacConkey's agar, DCA, XLD agar). - Use Selenite F broth (0.4%) for enrichment. - Subculture on solid media from Selenite F broth. - Incubate at 37°C for 24 hours. 5. Colony Morphology and Staining: - Colorless (NLF) colonies on MacConkey's agar. [Non-Lactose fermenting (do not ferment lactose)]. 6. Gram staining - Shigellae are Gram-negative 7. hanging drop preparation - Shigellae are non-motile.

22. Shigella Laboratory Diagnosis: Continued ……… 8. Biochemical Reactions: - Confirm non-motile bacillus (urease, citrate, H₂S, KCN negative) with biochemical tests. 9. Slide Agglutination Test: - Confirm shigella with polyvalent and monovalent antisera. - Use type-specific antisera for agglutination (subgroups A, B, C). 10. Colicin Typing: - Done for subgroup D (Sh. sonnei ) strains.

23. Shigella Laboratory Diagnosis: Continued ……… XLD agar : Shigella colonies appear red without black centers , SS Agar Shigella: Colourless (non-lactose fermenting), no blackening MacConkey’s agar: Colourless (non-lactose fermenting), SS

24. Proteus Bacteria

25. Proteus Introduction: Distribution and Habitat: Proteus bacteria are widely distributed in nature. They occur as normal intestinal flora in humans. Found in decomposing animal matter, sewage, manure soil, the mammalian intestine, and human and animal feces . Culture Colony Appearance: Proteus bacteria exhibit a characteristic “swarming” behaviour. This allows them to migrate across surfaces. They tend to have an ammonia smell. Infection Sites: Proteus bacteria are opportunistic pathogens. Commonly responsible for urinary and septic infections, often nosocomial (hospital-acquired). Associated with infection-induced renal stones in the urinary tract.

26. Proteus Morphology: Gram Negative Bacilli (rod-shaped). Size - 1.0–3.0 μm in length and 0.4 to 0.8 μm in diameter. Non-Spore forming bacterium. Non Capsulated bacteria. Motile bacteria – Have peritrichous flagella which help in movements. Pleomorphism – It can change in shape, sometimes present as coccobacilli and sometimes in long filamentous form based on environmental conditions.

27. Proteus Culture Characteristics: Aerobic & facultative anaerobe Optimum temperature for growth – 37° C, Optimal pH for growth is around 7 (neutral). Odour – Putrefactive odor (Fruity and seminal smell). On nutrient agar media; (NAM) or blood agar media (BAM) , Pr. vulgaris and Pr. Mirabilis exhibit swarming behavior . Swarming does not occur with Pr. Morganella and Pr. Providencia species. The exact cause of swarming is not fully established but appears related to vigorous bacterial motility.

28. Proteus Culture Characteristics: Continued……….. Inhibition Methods: Methods are used to inhibit swarming. Increase agar concentration to 6% (instead of 1-2%). Addition - chloral hydrate (1:500), sodium azide (1:500), or boric acid (1:1000) in the medium. On MacConkey’s agar, Proteus forms smooth, pale, or colorless (NLF) colonies without swarming. In liquid medium (peptone water), Proteus produces uniform turbidity with a slight powdery deposit and an ammonical odor.

29. Proteus Culture Characteristics: Continued………..

30. Proteus Laboratory Diagnosis: Specimens: Midstream urine sample in UTI. Pus in pyogenic lesions. 2. Collection: - Collect specimen in a sterile container under aseptic conditions. - Transport immediately. 3. Culture: - Culture on MacConkey agar or blood agar with 6% agar to inhibit swarming. - Incubate at 37°C for 18-24 hours. - Non-lactose fermenting (NLF) colonies seen on MacConkey agar. - Inoculate peptone water.

31. Proteus Laboratory Diagnosis Continued……….. 4. Gram's Staining: Gram-negative bacilli, non-capsulated and non-sporing. 5. Hanging Drop Preparation:- Observe actively motile bacilli. 6. Biochemical Reactions: Key tests - PPA (positive in all Proteus species) and urease (positive in all Proteus species except Providencia). Other biochemical reactions can differentiate various species. 7. Agglutination Test:- Confirm strain using polyvalent group-specific sera. 8. Antibiotic Susceptibility Test:- Important due to Proteus bacilli's resistance to common antibiotics.

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