🦠 Vibrio spp. in food

Microbiology PN-EN ISO 21872-1

Overview

Vibrio is a genus of Gram-negative, halophilic (salt-loving) bacteria, naturally occurring in marine and estuarine waters. Three main enteropathogenic species are: V. parahaemolyticus — most common cause of bacterial food poisoning associated with seafood (diarrhea, cramps, fever, 12–24 h after consumption); V. cholerae — cholera causative agent (O1 and O139 strains) and milder gastroenteritis (non-O1/non-O139 strains); V. vulnificus — most dangerous species, causing septicemia with >50% mortality in persons with liver diseases.

The PN-EN ISO 21872-1 standard describes a horizontal method for detecting these three Vibrio species in food (mainly seafood: clams, oysters, shrimp, fish) and environmental samples. The method includes selective enrichment in alkaline saline peptone water (ASPW — Alkaline Saline Peptone Water), isolation on TCBS (Thiosulfate Citrate Bile Salts Sucrose Agar) agar and second selective medium, and biochemical and/or molecular (PCR) confirmation.

ASPW provides alkaline (pH 8.6) and saline (NaCl 2–3%) conditions favoring Vibrio growth, while inhibiting many other bacteria. TCBS agar is highly selective medium — bile salts and sucrose differentiate Vibrio species: V. parahaemolyticus (green-blue colonies, sucrose-negative), V. cholerae (yellow colonies, sucrose-positive), V. vulnificus (green colonies, sucrose-negative).

Vibrio testing is gaining importance due to climate warming — Vibrio are spreading to new water bodies (Baltic Sea, North Sea). In EU, testing is required as part of official control of seafood and shellfish.

Method principle

Sample is enriched in two sequential portions of ASPW (Alkaline Saline Peptone Water, pH 8.6, NaCl 2–3%). First enrichment stage: 41.5°C/6 h and/or 37°C/6 h. Second stage: transfer to fresh ASPW, 41.5°C/18 h (V. parahaemolyticus, V. cholerae) or 37°C/18 h (V. vulnificus). High pH and salinity of ASPW selectively promote Vibrio growth. After enrichment, plate on TCBS agar (37°C/24 h) and second medium. On TCBS V. parahaemolyticus forms green-blue colonies (sucrose −), V. cholerae — yellow (sucrose +), V. vulnificus — green (sucrose −). Confirmation: biochemical tests (oxidase +, growth with NaCl, Voges-Proskauer reaction) and/or PCR (toxin genes: tdh, trh for V. parahaemolyticus; ctx for V. cholerae).

Applications

• Seafood quality control — clams, oysters, mussels, shrimp
• Fish and fish products testing, especially raw (sushi, sashimi)
• Monitoring marine and estuarine water in shellfish harvesting areas
• Official control of imported seafood
• Epidemiological investigation in outbreaks after seafood consumption
• Climate monitoring — tracking Vibrio spreading to new water bodies (Baltic)
• Salads and dishes with seafood testing (ready-to-eat)

Key parameters

Standard

Standard number

PN-EN ISO 21872-1:2017

Title (PL)

Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda oznaczania Vibrio spp. — Część 1: Wykrywanie potencjalnie enteropatogennych Vibrio parahaemolyticus, Vibrio cholerae i Vibrio vulnificus

Title (EN)

Microbiology of the food chain — Horizontal method for the determination of Vibrio spp. — Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

Step-by-step procedure

1. Sample preparation

   Weigh 25 g sample (seafood) into stomacher bag. Add 225 mL ASPW (pH 8.6). Homogenize for 60–120 s.

   ⏱ Time: 10 min

2. Enrichment — stage I

   Incubate at 41.5 ± 1°C for 6 h (V. parahaemolyticus, V. cholerae). In parallel, you can incubate second portion at 37 ± 1°C for 6 h (V. vulnificus).

   ⏱ Time: 6 h • 🌡 Temperature: 41.5°C / 37°C

3. Enrichment — stage II

   Transfer 1 mL from enriched culture to 10 mL fresh ASPW. Incubate 41.5°C/18 h (V. parahaemolyticus, V. cholerae) or 37°C/18 h (V. vulnificus).

   ⏱ Time: 18 h • 🌡 Temperature: 41.5°C / 37°C

4. Isolation on TCBS

   Streak from enriched broth onto TCBS agar plate and second selective medium (e.g., chromogenic). Incubate 37 ± 1°C for 24 ± 2 h. NOTE: Do NOT autoclave TCBS agar — dissolve by boiling and pour into plates.

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

5. Colony reading on TCBS

   Look for typical colonies: V. parahaemolyticus — green-blue, 2–3 mm; V. cholerae — yellow, smooth, 2–3 mm; V. vulnificus — green, 1–2 mm.

   ⏱ Time: 15 min

6. Subculture on non-selective medium

   Select min. 3–5 typical colonies from each plate. Subculture on agar with NaCl (TSA + 1% NaCl). Incubate 37°C/24 h.

   ⏱ Time: 24 h • 🌡 Temperature: 37°C

7. Confirmation tests

   Perform: oxidase test (+), growth test with different NaCl concentrations (0%, 3%, 6%, 8%, 10%), sucrose fermentation test, Voges-Proskauer reaction, string test. Or confirm by PCR (genes: tlh/tdh/trh for V. parahaemolyticus; ctx/ompW for V. cholerae; vvhA for V. vulnificus).

   ⏱ Time: 24–48 h

8. Reporting

   Result: "V. parahaemolyticus / V. cholerae / V. vulnificus detected/not detected in 25 g". State species and virulence factors profile (if PCR performed).

   ⏱ Time: 10 min

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• V. cholerae — BSL-2 pathogen (non-O1/non-O139 strains) or BSL-3 (O1/O139), follow biological safety guidelines
• V. vulnificus — >50% mortality in persons with liver diseases, special caution with cuts
• Work exclusively in class II laminar flow cabinet
• Bile salts in TCBS — irritating, use gloves and goggles
• Autoclave used materials before disposal (121°C/20 min)
• Surface disinfection with 70% ethanol — Vibrio sensitive to desiccation

Frequently asked questions