Decreasing Enterobacter sakazakii (Cronobacter spp.) food contamination level with bacteriophages: prospects and problems

Abstract

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen, which can cause rare, but life-threatening infections in neonates and infants through feeding of a contaminated milk formula. We isolated 67 phages from environmental samples and tested their lytic host range on a representative collection of 40 E. sakazakii strains. A cocktail of five phages prevented the outgrowth of 35 out of 40 test strains in artificially contaminated infant formula. Two E. sakazakii phages represented prolate head Myoviridae. Molecular tests identified them as close relatives of Escherichia coli phage T4. The remaining three phages represented isometric head Myoviridae with large genome size of 140 and 200 kb, respectively, which belonged to two different DNA hybridization groups. A high dose of 10(8) pfu ml(-1) of phage could effectively sterilize a broth contaminated with both high and low pathogen counts (10(6) and 10(2) cfu ml(-1)). In contrast, broth inoculated with 10(4) phage and 10(2) bacteria per ml first showed normal bacterial growth until reaching a cell titre of 10(5) cfu ml(-1). Only when crossing this threshold, phage replication started, but it could not reduce the contamination level below 100 cfu ml(-1). Phages could be produced with titres of 10(10) pfu ml(-1) in broth culture, but they were not stable upon freeze-drying. Addition of trehalose or milk formula stabilized the phage preparation, which then showed excellent storage stability even at elevated temperature.

Figure 1

E. sakazakii host strain differentiation. From left to right: Dendrogram of 39 strains of E. sakazakii (A) (the scale bar shows the percentage of similarity), obtained after restriction with EcoRI. Groups 1–4 indicate assignment of the strains to 16S rRNA groups (Iversen et al., 2007a). The FSM strain designation (with reference strains in italics) is shown (B). The star symbol (*) indicates the presence of a prophage and the strain designation in parenthesis corresponds to the indicator strain used to reveal it. Origins (N, the Netherlands; F, France; S, Switzerland; M, Malaysia; G, Germany; U, USA) of the selected strains (C). Susceptibility of the E. sakazakii strain from the corresponding row to infection with E. sakazakii phages F, 23, 81, 82 and 83 as determined by the plaque assay using 10⁵ pfu ml⁻¹ on BHI‐agar; ‘X’ indicates observation of phage plaques (D). Enterobacter sakazakii challenge test in infant formula using a cocktail of the five phages showing the broadest host range on E. sakazakii; ‘X’ indicates prevention of outgrowth of 10² cfu ml⁻¹E. sakazakii when phage was added at 10⁸ pfu ml⁻¹ (E).

Figure 2

Negative‐staining electron microscopy of CsCl‐purified E. sakazakii phages constituting the 5‐phage cocktail. (A) phage F; (B) phage 23; (C) phages 81; (D) phage 82 and (E) phage 83. The negative stain was ammonium molybdate/bacitracine. Scale bars, 100 nm.

Figure 3

Molecular characterization of the phage genomes from the 5‐phage cocktail.
A. Pulsed‐field gel electrophoresis of E. sakazakii phages. (1), phage F; (2), phage 23; (3), phage 81; (4), phage 82; (5), phage 83; and (M), size marker (phage concatemers, 50 kb DNA ladder, Promega).
B. PCR analysis on phage DNA with primers Mzia1 and CAP8 amplifying the major head gene g23 (Tétart et al., 2001). (M), Smartladder (Eurogentec); (+), T4 positive control; (1), phage F; (2), phage 23; (3), phage 81; (4), phage 82; (5), phage 83; and (−), negative control without DNA.

Figure 4

Restriction analysis and Southern hybridization of E. sakazakii phages. (A) DraI restriction digests of E. sakazakii phage DNA. (M), Smartladder (Eurogentec); (T4), T4 phage; (1), phage F; (2), phage 23; (3), phage 81; (4), phage 82; and (5), phage 83. (B, C) Corresponding Southern blot hybridization using DIG‐labelled phage T4 DNA (B) or phage F DNA (C) as a probe respectively.

Figure 5

Challenge tests.
A. Prevention of the outgrowth of 10⁶ cfu ml⁻¹E. sakazakii strain 316 in the presence of 10⁸ pfu ml⁻¹ phage F (filled squares). The growth of the strain 316 in the absence of phage is documented by empty squares. The squares represent OD₆₀₀ readings. The replication of phage F on the infected strain 316 is documented as pfu ml⁻¹ readings (filled circles).
B. A parallel experiment demonstrates the effect of phage 82 on E. sakazakii strain 368.
C. Prevention of the outgrowth of 10² cfu ml⁻¹E. sakazakii strain 316 in the presence of 10⁸ pfu ml⁻¹ phage F (filled squares). The growth of the strain 316 in the absence of phage is documented by empty squares. Bacterial and phage growth are documented as cfu ml⁻¹ and pfu ml⁻¹ (filled circles) respectively.
D. Prevention of the outgrowth of 10² cfu ml⁻¹E. sakazakii strain 316 in the presence of 10⁴ pfu ml⁻¹ phage F (filled squares). The growth of the strain 316 in the absence of phage is documented by empty squares. Bacterial and phage growth are documented as cfu ml⁻¹ and pfu ml⁻¹ (filled circles) respectively.

Figure 6

Stability of E. sakazakii phages towards freeze‐drying. The bars represent data from phages F, 23, 81, 82 and 83 before (first bars) and after (second bars) freeze‐drying in phage buffer and before (third bars) and after (fourth bars) freeze‐drying in phage buffer plus 5% trehalose. Values are the average of three replicates and standard deviations are indicated.