🔴 Cronobacter spp. (milk powder)
Detection of Cronobacter spp. (formerly Enterobacter sakazakii) in milk powder and infant formula by pre-enrichment in BPW, selective enrichment in mLST/vancomycin and isolation on chromogenic CCI agar.
Overview
Cronobacter spp. (formerly Enterobacter sakazakii) is an opportunistic pathogen posing serious threat to newborns and infants, especially premature babies and children with reduced immunity. Cronobacter infections can lead to meningitis, septicemia and necrotizing enterocolitis — mortality reaches 40–80% in newborns. The main source of infection is contaminated milk powder (powdered infant formula — PIF).
The PN-EN ISO 22964 standard describes a horizontal method for detecting Cronobacter spp. in food, with particular focus on milk powder, infant formula and environmental samples from facilities producing these products. The method includes three stages: pre-enrichment in buffered peptone water (BPW), selective enrichment in modified lauryl sulfate tryptose broth with vancomycin (mLST/vancomycin), and isolation on chromogenic agar (CCI — Chromogenic Cronobacter Isolation Agar).
Cronobacter shows characteristic α-glucosidase activity, which hydrolyzes chromogenic substrate in CCI agar, forming blue-green (or turquoise) colonies. Vancomycin in mLST broth inhibits Gram-positive bacteria growth, enabling selective Cronobacter enrichment.
Cronobacter is particularly dangerous because it survives in low water activity environments (milk powder, flours, herbal teas). Testing is required by Regulation (EC) No 2073/2005 for powdered infant formula (criterion: absence in 10 g). "Detected" result leads to product batch withdrawal from market.
Method principle
Three-stage method: (1) Pre-enrichment — 25 g sample in 225 mL BPW, incubation 37°C/18 h — resuscitation of damaged cells. (2) Selective enrichment — 0.1 mL transfer to 10 mL mLST with vancomycin, incubation 44°C/24 h — Cronobacter selection (thermophilic growth), vancomycin inhibits Gram-positives. (3) Isolation — plating on chromogenic CCI agar (Chromogenic Cronobacter Isolation Agar), incubation 44°C/24 h. Cronobacter produces α-glucosidase, which hydrolyzes chromogenic substrate — blue-green colonies. Confirmation: sugar fermentation, yellow pigmentation on TSA, biochemical or molecular identification.
Applications
- Infant milk powder (PIF) quality control — Regulation (EC) 2073/2005
- Powdered infant formula testing
- Environmental monitoring of milk powder production facilities
- Raw materials for infant formula production control
- Nutritional supplements and food for special medical purposes testing
- Herbal teas, starches and flours monitoring (Cronobacter reservoirs)
- Production processes and cleaning systems (CIP) validation
Key parameters
| Parameter | Value |
|---|---|
| Pre-enrichment | 37 ± 1°C, 18 ± 2 h in BPW |
| Selective enrichment | 44 ± 1°C, 24 ± 2 h in mLST/vancomycin |
| Isolation | 44 ± 1°C, 24 ± 2 h on CCI agar |
| Isolation medium | CCI Agar (Chromogenic Cronobacter Isolation Agar) |
| Colony type | Blue-green (turquoise) — α-glucosidase activity |
| Sample size | 10 × 25 g (total 250 g for PIF) |
Standard
- Standard number
- PN-EN ISO 22964:2017
- Title (PL)
- Mikrobiologia łańcucha żywnościowego — Horyzontalna metoda wykrywania Cronobacter spp.
- Title (EN)
- Microbiology of the food chain — Horizontal method for the detection of Cronobacter spp.
Step-by-step procedure
1. Sample preparation
Weigh 25 g milk powder (PIF) into stomacher bag. Add 225 mL buffered peptone water (BPW) preheated to 45°C. Mix until dissolved. For PIF, test 10 × 25 g (total 250 g).
2. Pre-enrichment in BPW
Incubate suspension at 37 ± 1°C for 18 ± 2 h. Resuscitation phase — damaged Cronobacter cells regain growth capability.
3. Selective enrichment in mLST/vancomycin
Transfer 0.1 mL enriched culture to 10 mL mLST broth with vancomycin (10 mg/L). Incubate 44 ± 1°C for 24 ± 2 h.
4. Isolation on CCI agar
From yellowed mLST broth, streak onto CCI agar plate. Incubate 44 ± 1°C for 24 ± 2 h.
5. Colony reading
Look for typical Cronobacter colonies — blue-green (turquoise), diameter 1–2 mm. Other Enterobacteriaceae may grow as colorless or pink colonies.
6. Preliminary confirmation
Select min. 5 typical colonies. Subculture on TSA and incubate 25°C for 48–72 h. Cronobacter forms yellow/yellow-orange colonies (pigment production).
7. Biochemical identification
Confirm isolates using API 20E, VITEK 2 GN or molecular methods (PCR). Cronobacter: oxidase (−), catalase (+), VP (+), arginine dihydrolase (−).
8. Reporting
Result: "Cronobacter spp. detected/not detected in 25 g (or 10 × 25 g) sample". Detection in PIF = immediate notification of supervisory authorities and batch withdrawal.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Laboratory incubator 37°C | Binder BD 56, Memmert INB 200 | 5,000–18,000 PLN |
| Laboratory incubator 44°C | Binder BD 56, Memmert INB 200, POL-EKO CLN 53 | 5,000–18,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage, ESCO Airstream | 25,000–60,000 PLN |
| Stomacher / homogenizer | Seward Stomacher 400 Circulator | 12,000–25,000 PLN |
| Water bath 44–47°C | Memmert WNB 14, Julabo TW 12 | 3,000–10,000 PLN |
| Laboratory autoclave | Tuttnauer 2540M, Systec VX-65 | 15,000–45,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Buffered peptone water (BPW) | — | For pre-enrichment; peptone, NaCl, Na₂HPO₄, KH₂PO₄; 500 g package |
| mLST Broth (modified Lauryl Sulphate Tryptose Broth) | — | Selective broth; sodium lauryl sulfate, tryptose, NaCl; 500 g package |
| Vancomycin (supplement for mLST) | 1404-93-9 | Antibiotic inhibiting Gram-positive bacteria; concentration 10 mg/L; 10 vials package |
| CCI Agar (Chromogenic Cronobacter Isolation Agar) | — | Chromogenic agar with substrate for α-glucosidase; Cronobacter colonies blue-green; 500 g package or 20 plates |
| TSA (Tryptone Soya Agar) | — | Non-selective medium for confirmation — yellow pigmentation of Cronobacter after 48–72 h at 25°C |
| Biochemical tests API 20E or VITEK | — | For biochemical identification of confirmed isolates |
Health and safety (OHS)
- Cronobacter — BSL-2 pathogen, particularly dangerous for newborns and immunosuppressed persons
- Work exclusively in class II laminar flow cabinet — strict aseptic principles observance
- Vancomycin — potentially allergenic antibiotic, use gloves
- Autoclave used materials before disposal (121°C/20 min)
- Surface disinfection with 70% ethanol or 0.5% sodium hypochlorite