Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence

Abstract

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 10(5) PFU ml(-1)).

FIG. 1.

Agarose gel electrophoresis of the EcoRV-generated DNA fragments of S. thermophilus phages (A) and the corresponding Southern blots hybridized with ³²P-labeled φSfi11 DNA (B), φSfi21 DNA (C), and φCP DNA (D). Lane M, 1-kb DNA ladder (Invitrogen); lane 1, φ021-5; lane 2, φCP; lane 3, φCQ210; lane 4, φCQ211; lane 5, φFcSth10; lane 6, φAbc2; lane 7, φLy1; lane 8, φLy7; lane 9, φ799-M1; lane 10, φP10.3; lane 11, φP13.2; lane 12, φSfi11; lane 13, φSfi21.

FIG. 2.

Amplification products of the highly conserved DNA fragments from the replication module of S. thermophilus bacteriophages. Lane M, 100-bp PCR EZ load molecular ruler (Bio-Rad); lane 1, φ021-5; lane 2, φCP; lane 3, φCQ210; lane 4, φCQ211; lane 5, φFcSth10; lane 6, φAbc2; lane 7, φLy1; lane 8, φLy7; lane 9, φ799-M1; lane 10, φP10.3; lane 11, φP13.2; lane 12, φSfi11; lane 13, φSfi21; lane 14, negative control. Upper row, PCR products obtained with primers A and B; lower row, PCR products obtained with primers A and C.

FIG. 3.

Dendrogram (obtained by the neighbor-joining method) for the antireceptor VR2 sequences from all the S. thermophilus bacteriophages used in this work (boldface type) and the phages whose sequences are available in the GenBank database (italics). Boxes indicate highly homologous corresponding sequences (>96% similarity) in the phages.

FIG. 4.

Amplification of the antireceptor VR2 variable region using bacteriophage-infected milk. Lane M, 100-bp PCR EZ load molecular ruler (Bio-Rad); lane 1, φ021-5; lane 2, φCP; lane 3, φCQ210; lane 4, φCQ211; lane 5, φFcSth10; lane 6, φAbc2; lane 7, φLy1; lane 8, φLy7; lane 9, φ799-M1; lane 10, φP10.3; lane 11, φP13.2; lane 12, φ0BJ; lane 13, φ021-4; lane 14, φP3.1; lane 15, φMi1; lane 16, φLy4; lane 17, φSfi21; lane 18, negative control.

FIG. 5.

Amplification of the antireceptor VR2 region using milk contaminated with different titers of S. thermophilus bacteriophage φFcSth10. Lane M, 1-kb DNA ladder (Invitrogen); lane 1, 10¹⁰ PFU ml⁻¹ (M17); lanes 2 to 10, 10¹⁰ to 10² PFU ml⁻¹ of milk; lane 11, φSfi11; lane 12, φSfi21; lane 13, negative control.