Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine)

Abstract

The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

FIG 1

Phages and phage tails. Transmission electron micrographs of negatively stained bacteriophages from L. innocua F25 (phage; arrow) (A) and from L. monocytogenes EGD-e (phage tails; arrow) (B). The samples were stained using uranyl acetate as described in Materials and Methods. Bacterial lysis was observed at the deposited phage or phage tail spot (20 μl) on agar plates. (C to F) Induction by phage, >50 PFU (C) and 19 PFU (D), and by phage tails, diffuse lysis zone (F). (E) Control (no bacterial lysis).

FIG 2

Effects of pH and Na₂HPO₄ on EGD-e phage tail induction in BHIM broth. Cultures were performed at 30°C for 24 h in BHIM broth supplemented with different Na₂HPO₄ concentrations and initial pH (pHi) values (5.5, 6.5, and 7.5). Twofold serial dilutions of the Listeria culture filtrates were applied on L. ivanovii RR3 lawns. The level of phage tail induction is expressed as the highest dilution factor of filtrate that induced a detectable RR3 lysis zone. The results are representative of three experiments. Different letters above the bars indicate values with significant differences (2 orders of dilution magnitude).

FIG 3

Highest dilution factor of the EGD-e culture performed in the presence of LiCl with detectable induction of phage tails. The bars show induction of EGD-e phage tails at different LiCl concentrations in BHIM broth for 24 h at 30°C. Bacterial concentrations were determined both by measuring the OD₆₀₀ and by counting CFU/ml. The results are expressed as the means ± standard deviations for triplicate experimental cultures for the bacterial concentrations, and the curve representing the threshold dilution with detectable lysis activity is representative of three experiments.