Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies

Abstract

Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72 degrees C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.

FIG. 1.

Effect of high temperature on the viability of L. lactis bacteriophages suspended in whole-fat milk. (A) Treatment of representative phages belonging to the 936 (bIL170), c2, and P335 quasi-species in a water bath. (B) Comparison of the behavior of each representative phage (□) with the behavior of four milk isolates (▪) at the temperatures indicated above the graphs. (C) Pasteurization of phage-contaminated milk in a plate heat exchanger (72°C for 15 s). Dark gray bars, control suspensions; light gray bars, treated suspensions. Experiments were carried out in triplicate (A and B) or quintuplicate (C).

FIG. 2.

(A) Restriction analysis (EcoRV) of genome DNA purified from 936-like phages. Lane 2, representative strain (bIL170); lane 3, milk isolate (1240); lane 4, whey isolate (9205). Lane 1 contained size markers in base pairs (lambda DNA restricted with PstI). (B) High-stringency hybridization with a probe made from bIL170 DNA.