Characterization of Streptococcus thermophilus lytic bacteriophages from mozzarella cheese plants P. Zinno a , T. Janzen b , M. Bennedsen b , D. Ercolini a , G. Mauriello a, ⁎ a Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II – 80055 Portici, Italy b Strains Department, Innovation, Chr. Hansen A/S, Hørsholm, Denmark abstract article info Article history: Received 22 September 2009 Received in revised form 2 December 2009 Accepted 5 December 2009 Keywords: Bacteriophage Streptococcus thermophilus Mozzarella Phage infection still represents the main cause of fermentation failure during the mozzarella cheese manufacturing, where Streptococcus thermophilus is widely employed as starter culture. Thereby, the success of commercial lactic starter cultures is closely related to the use of strains with low susceptibility to phage attack. The characterization of lytic S. thermophilus bacteriophages is an important step for the selection and use of starter cultures. The aim of this study was to characterize 26 bacteriophages isolated from mozzarella cheese plants in terms of their host range, DNA restriction profile, DNA packaging mechanism, and the variable region VR2 of the antireceptor gene. The DNA restriction analysis was carried out by using the restriction enzymes EcoRV, PstI, and HindIII. The bacteriophages were distinguished into two main groups of S. thermophilus phages (cos- and pac-type) using a multiplex PCR method based on the amplification of conserved regions in the genes coding for the major structural proteins. All the phages belonged to the cos- type group except one, phage 1042, which gave a PCR fragment distinctive of pac-type group. Furthermore, DNA sequencing of the variable region VR2 of the antireceptor gene allowed to classify the phages and examine the correlation between typing profile and host range. Finally, bacterial strains used in this study were investigated for the presence of temperate phages by induction with mitomycin C and only S. ther- mophilus CHCC2070 was shown to be lysogenic. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Phage attack is a main cause of fermentation failure during the manufacture of mozzarella cheese. Dairy fermentations are vulnerable to phage infection for several reasons, i) contaminating phages may be naturally present in fluid milk; ii) repeated use of defined culture under non-aseptic processing conditions provides a constant host for phage proliferation (Klaenhammer and Fitzgerald, 1994; Neve et al., 1995); and iii) lysogenic bacteria may also be a source of new phages (Shimuzu-Kadota et al., 1983; Moineau et al., 1986; Relano et al., 1987; Tremblay and Moineau, 1999). The dairy industry has imple- mented many methods to reduce the consequences of phage infection such as ordinary disinfection of equipment, direct vat inoculation, propagation of starter cultures in phage inhibitory media, strain rotations and application of phage-resistant multiple strain starters (Everson, 1991). Streptococcus thermophilus strains are predominant in starter cultures used in the mozzarella cheese production and it is well known that they are often susceptible to phage attack resulting in slow lactic acid fermentation and loss of product quality (Mills et al., 2007). Since, beside Lactococcus lactis, S. thermophilus is considered the most technologically important lactic acid bacterium by the dairy industry, the characterization of its phages is an important advancement for the selection of starter cultures. Removing the multi-sensitive strains and replacing them with strains with different phage sensitivities is an efficient approach for correct management of a defined culture rotation system. S. thermophilus phages belong to B1 Bradley's group, having a hexagonal capsid and a long non-contractile tail. They are divided into two groups (cos and pac-types) based on the number of major structural proteins and the encapsidation mechanism of double- stranded DNA (Le Marrec et al., 1997). Some bacteriophages can infect different bacterial strains and the definition of the host range is an important feature of bacteriophages to assess. The phage-host interactions were widely studied, and Duplessis and Moineau (2001) identified the phage genetic determinant (antireceptor) likely to be involved in the recognition of S. thermophilus hosts. They, in fact, characterized the antireceptor gene (orf18) of the tail morphogenesis module of the phages MD4 and DT1, finding the variable region VR2 responsible for the host specificity. The VR2 sequence was also used to classify S. thermophilus phages and verify the correlation between typing profile and host range (Binetti et al., 2005). The aim of this study was to define some specific features of 26 S. thermophilus bacteriophages for their following use in the selection of starter cultures. International Journal of Food Microbiology 138 (2010) 137–144 ⁎ Corresponding author. Tel.: + 39 0812539452; fax: + 39 0812539407. E-mail address: giamauri@unina.it (G. Mauriello). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.12.008 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro