Antibiotics Ampicillin, penicillin G, kanamycin, rifampicin and tetracycline hydrochloride were purchased from Sigma-Aldrich Inc. (St. Louis MO – USA) while cefotaxime was obtained from Labesfal-Laboratórios de Almiro SA (Amadora – Portugal). They were dissolved in distilled water and filter-sterilized using a 0.22 μm PES syringe filter from Tpp-Techno Plastic Products AG (Trasadingen – Switzerland) prior to addition to the media. Phages All phages used in this work are virulent and are listed in Table 1 along with their sizes and hosts. The phages were isolated from sewage (purified by several isolation of single plaques)

and represent the three families in the order Caudovirales, which include 96% of all observed phages [16]. The Pseudomonas fluorescens phage phi IBB-PF7A was already described by Sillankorva et al [26]. Phage dimensions were determined by Dr. selleck Hans-W. Ackermann (Université Ion Channel Ligand Library Laval, Quebec, Canada – personal communication). Table 1 Phages used. PHAGE FAMILY DIMENSIONS (nm) HOST phi PVP-SE1 Myoviridae Tail:120 × 18; head: 84 Salmonella enterica Enteritidis phi PVP-SE2 Siphoviridae Tail:125 × 8; head: 57 Salmonella enterica Enteritidis phi IBB-PF7A Podoviridae Tail:13 × 8; head: 63 Pseudomonas fluorescens phi IBB-SL58B Podoviridae Tail:13 × 9; head: 64 Staphylococcus

lentus Determination of phage titer The titer of each phage, expressed as plaque forming units (pfu), was determined using the DLA technique as described by Sambrook and Russel [27]. Briefly, 100 μl of a dilution of the phage sample was added to 100 μl of a bacterial suspension

grown overnight at 37°C, 120 rpm. This solution was added to 4 ml top agar, gently homogenized, and poured Fossariinae into a 90 mm petri dish (Plastiques-Gosselin, Borre – France) previously prepared with 10 ml bottom agar. The plates were gently swirled, dried for 10 min at room temperature and then inverted and incubated at 37°C overnight. To test the effects of antibiotics on plaque size, the corresponding antibiotic was added at the concentration desired to the bottom, top or both agar layers after sterilization of the medium. Glycerol was added to the top, bottom or both layers before sterilization. Phage plaque size Pictures of the plates were taken with a Hewlett-Packard Scanjet 3300C scanner, using a black background to avoid distortion and to allow equal light exposure and contrast conditions in all photographs. The photographs were not adjusted for brightness, contrast or colour. In order to obtain accurate dimensions, the diameter and area of the plaques were automatically determined from photographs at 4-fold magnification using the computer image analysis program Sigma Scan Pro, version 5.0.0 of SPSS Inc (Chicago – USA). Each value is the average of up to 20 plaque measurements. Microscopic observation of bacterial cells Bacterial cells were grown for 7 h in LB with or without glycerol and supplemented with an antibiotic (0.5 mg/l ampicillin, 0.06 mg/l cefotaxime or 1.5 mg/l tetracycline).