Isolation and Characterization of Bacteriophages that ...

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David Kelly, Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland; Horst Neve,. Max Rubner-Institute, Federal Research ...
Isolation and Characterization of Bacteriophages That Inhibit Strains of Pediococcus damnosus, Lactobacillus brevis, and Lactobacillus paraplantarum That Cause Beer Spoilage David Kelly, Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland; Horst Neve, Max Rubner-Institute, Federal Research Institute of Nutrition and Food, Department of Microbiology and Biotechnology, Kiel, Germany; Olivia McAuliffe and R. Paul Ross, Biotechnology Department, Teagasc, Moorepark Food Research Centre, Fermoy, Cork, Ireland; Elke K. Arendt, Department of Food and Nutritional Sciences, University College Cork, Ireland; and Aidan Coffey,1 Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland

Beer has been recognized for centuries as a safe and microbiologically stable beverage. This is due to a number of factors: the presence of ethanol, a low pH, the presence of hop compounds, the reduced content of oxygen and resulting raised carbon dioxide content, and the low level of residual nutrients (2,21,23). However, in spite of these unfavorable conditions, a few bacterial genera still succeed in growing in a beer environment, due in part to their resistance to hop compounds (3,24). These beer-spoilage microorganisms can cause visible turbidity and unpleasant sensory changes in beer, affecting both the final product quality and financial profits of the brewing company. Members of the genera Pediococcus and Lactobacillus are regarded as the most common beerspoilage organisms found in modern breweries and have been reported to be responsible for approx. 70% of microbial beer-spoilage incidents (12,23). L. brevis and L. lindneri have been reported as the most common lactobacilli causing spoilage (21,23), while P. damnosus is regarded as the most common of the pediococci (12,22). Lytic bacteriophages (phages) are viruses that infect bacterial cells, disrupt bacterial metabolism, and lyse the bacterial cell, causing cell death. They possess attributes that are attractive in the context of novel methods to control foodborne pathogens and spoilage bacteria (5,10). Phages have been used for many biocontrol applications in foods and have successfully contributed to the control of Campylobacter spp., Listeria monocytogenes, Salmonella spp., and various spoilage organisms in foods (11,14). Indeed, the European Food Safety Authority recently published an extensive review of the successful use of phages in dairy products, chicken, beef, pork, seafood, and food-processing environments and on metallic surfaces (1). Regarding the stability of phages in food systems, this review stated that phage persistence in or on foods tends to vary with each bacteriophage and also with the conditions of application of the phage, including the dose, and with both physical and chemical factors relating to the food matrix. Hudson et al (11) suggest that considerations for phage stability in foods are important because phages need to be stable under the physiochemical conditions of the food to which they are applied to be useful as biocontrol agents. These authors cite tolerance to pH, temperature, visible and UV light, osmotic shock, osmotic pressure, and processing environment as the main factors to be taken into account when considering phage stability. Phages are natural and relatively inexpensive to produce (9). Thus, screening for bacteriophages that kill problematic bacteria is very worthwhile because they may have an application for control of these bacteria (4,19,20). In this study, we describe the isolation and characterization of lytic phages active against bacterial genera that cause spoilage problems in beer. In addition, from a scientific point of view, very limited if any work has been performed on phages of P. damnosus.

ABSTRACT J. Am. Soc. Brew. Chem. 69(1):8-12, 2011 The aim of this study was to isolate and characterize bacteriophages against Pediococcus and Lactobacillus strains that cause spoilage in brewing processes. A number of beer-spoilage bacteria were isolated from breweries and characterized by 16S rRNA typing. Five distinct P. damnosus phages and four Lactobacillus phages, which lysed both L. brevis and L. paraplantarum, were isolated from municipal sewage and farmyard slurries. A rapid DNA isolation method was used to isolate DNA of sufficient purity for restriction endonuclease digestion from de Man, Rogosa, and Sharpe (MRS) broth. Phages were analyzed using restriction digest and transmission electron microscopy and shown to be in the family Siphoviridae, with genomes ranging in size from 40 to 50 kb. All phages were characterized and shown to be distinct. This study identifies five novel phages against P. damnosus. Phages for this species are very rare in the scientific literature. Four novel phages against L. brevis and L. paraplantarum are also identified. These phages may have an application in the biocontrol of beer-spoilage bacteria. Keywords: Bacteriophage, Beer, Biocontrol, Lactobacillus, Pediococcus, Phage DNA isolation RESUMEN El objetivo de este estudio fue aislar y caracterizar los bacteriófagos contra cepas de Lactobacillus y Pediococcus que causan deterioro en los procesos de elaboración de la cerveza. Un número de bacterias de deterioro de cerveza fueron aisladas de fábricas de cerveza y se caracteriza por ARNr 16S. Cinco distintos fagos de P. damnosus y cuatro fagos de Lactobacillus, que tanto lisis L. brevis y L. paraplantarum, fueron aislados de las aguas residuales municipales y lodos de corral. Un método rápido aislamiento del ADN se utilizó para aislar el ADN de suficiente pureza para la digestión con endonucleasas de restricción del caldo de Man, Rogosa, y Sharpe (MRS). Los fagos fueron analizados mediante el resumen de la restricción y la microscopía electrónica de transmisión y se demostró que eran en la familia Siphoviridae, con genomas que varían en tamaño desde 40 hasta 50 kb. Todos los fagos se caracterizaron y se demostró que ser distintos. Este estudio identifica cinco fagos novela contra P. damnosus. Los fagos a esta especie son muy raros en la literatura científica. Cuatro nuevos fagos contra L. brevis y L. paraplantarum también se identifican. Estos fagos pueden tener una aplicación en el control biológico de bacterias de deterioro de cerveza. Palabras claves: Bacteriófago, Bacteriófago aislamiento del ADN, Cerveza, Control biológico, Lactobacillus, Pediococcus

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Corresponding author. Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland. E-mail: [email protected]; Phone: 353-214326834; Fax: 353-21-4326308.

doi:10.1094 /ASBCJ-2010-1119-01 © 2011 American Society of Brewing Chemists, Inc.

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Phage for Beer-Spoilage Lactic Acid Bacteria EXPERIMENTAL Bacterial Strains and Growth Media All bacterial strains used in this study are listed in Table I. All strains were isolated from breweries and maintained in de Man, Rogosa, and Sharpe (MRS) medium (Sigma-Aldrich) (7). They were cultivated at 30°C in MRS broth or on solid media containing 1.0% (wt/vol) bacteriological agar (Merck). Characterization of Beer-Spoilage Bacteria by 16S rRNA Typing DNA was extracted from overnight bacterial cultures (1% inoculum) using a DNA mini kit (QIA amp, Qiagen House) according to the manufacturer’s instructions. PCR amplification of the DNA was carried out using a core kit (Biotaq, Bioline). For amplification of the target 16S rRNA gene sequence from DNA extracted from pure bacterial cultures, universal primers (25) 16S-f1 (AGA GTT TGA TCC TGG CTC AG) and 16S-r5 (ACG GCT ACC TTG TTA CGA CTT) were used. The reaction mixture (50 µL) consisted of reaction buffer (5 µL), a 2.5 mM concentration of each deoxynucleoside triphosphate (5 µL), 100 pmol each primer (1 µL each), 50 mM MgCl2 (4 µL), sterile water (28.5 µL), bacterial DNA (5 µL), and 0.5 µL of DNA polymerase (Biotaq, Bioline) at 5 U/µL. The amplification program consisted of an initial “hot start” of 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 61°C for 30 sec, 72°C for 1 min, and 72°C for 7 min. This was followed by holding at 4°C. Amplified target sequences were purified with a PCR purification kit (QIA quick, Qiagen House), and after sequencing (MWG Biotech), identifications were made using the BLAST database. Bacteriophage Isolation All beer-spoilage strains listed in Table I were used as hosts for phage isolation. A 10-g sample of farmyard slurry or municipal sewage (Table II) was added to a 15-mL volume of MRS broth containing 100 µL of all target strains within the bacterial genus. In each propagation, all strains were included to increase the chance of amplifying phages. In all, 50 µL of CaCl2 at 1 mol L–1 was also added to the tube, which was incubated at 30°C overnight to allow specific phages to propagate if present. The culture was then centrifuged at 3,000 rpm for 10 min, and the supernatant was filter-sterilized (0.45-µm filter) and stored at 4°C. The supernatant was then subjected to a plaque assay using each bacterial strain individually that had been used in that propagation. Plates were then examined for plaque formation. Phage Assays Phage propagations were performed as described by O’Flaherty et al (16). Briefly, 100 µL of the appropriate overnight culture and 20 µL of CaCl2 (1 mol L–1) were added to 10-mL volumes of MRS broth. After incubation at 30°C for 2 hr, 1 mL of purified phage solution was added, and the sample was incubated at 30°C until the TABLE I Beer-Spoilage Bacterial Strains Used in This Study and rRNA Typing Matches with Relevant Type Strains Bacterial Strain Lactobacillus brevis L1105 L. brevis L1033 L. paraplantarum L913 Pediococcus damnosus P82 P. damnosus P120

16S RNA Type BLAST Result L. brevis strain ATCC14869 16S rRNA gene, partial sequence (99% identity) L. brevis strain ATCC14869 16S rRNA gene, partial sequence (99% identity) L. paraplantarum strain DSM10667 16S rRNA gene, partial sequence (99% identity) P. damnosus strain DSM20331 16S rRNA gene (99% identity) P. damnosus strain DSM20331 16S rRNA gene (99% identity)

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phage cleared the broth. Samples were then centrifuged at 3,000 rpm, filter-sterilized (0.45-µm filter), and stored at 4°C. Phage plaque assays and spot tests were performed as described previously (18). Bacteriophages were generally subjected to three rounds of plaque purification prior to acquisition of experimental data. Electron Microscopy Phage stocks were prepared from MRS broth lysates to achieve titers in excess of 108 PFU/mL. Each sample was stained negatively with 2% (wt/vol) uranyl acetate on freshly prepared carbon films. Grids were analyzed in a transmission electron microscope (Tecnai 10, FEI Company) at an acceleration voltage of 80 kV. Micrographs were taken with a CCD camera (MegaView II, SIS). Phage dimensions were calculated as means of 8–22 measurements. Preparation of Phage DNA from Pediococcus and Lactobacillus Phages DNA was extracted from MRS broth phage stock solutions as follows. An aliquot of 750 µL of MRS phage suspension at 108 PFU/mL was incubated at 37°C for 10 min with RNase (50 mg/mL; SigmaAldrich). Subsequently, 150 µL of lysis buffer (500-µL solution) containing 50 µL of 10% sodium dodecyl sulfate, 400 µL of 0.5M EDTA, 25 µL of 1M Tris (pH 8.0), and 25 µL of sterile distilled water was added, followed by 10 µL of proteinase K (10 mg/mL; Roche Diagnostics). The sample was incubated at 65°C for 30 min. Next, 750 µL of phenol/chloroform/isoamyl alcohol (25:24:1; Sigma-Aldrich) was added, and the sample was shaken well and then centrifuged at 10,000 rpm for 5 min. This step was repeated once, and the upper layer was transferred into a new microfuge tube. To the upper layer, 50 µL of chloroform/isoamyl alcohol (24:1; Sigma-Aldrich) was added, and the sample was shaken well and then centrifuged at 10,000 rpm for 5 min. The upper layer (600 µL) was transferred into a fresh microfuge tube, and 6 µL of 3M sodium acetate (pH 5.1) and 350 µL of 100% isopropanol were added. The sample was stored at –20°C for 2 hr, after which it was centrifuged at 10,000 rpm for 5 min, the supernatant was removed, and the pellet was dried under vacuum. After 70% ethanol washing, the DNA pellet was resuspended in 100 µL of 10 mM Tris and 1 mM EDTA (pH 8). Typically, 20–30 µL of DNA was used for restriction digests. Phage Host-Range Tests The host range for each phage was ascertained using the spot test and plaque assay techniques, as described previously (18). The efficiency of plaquing (EOP) for each phage on each strain was also calculated as described previously (17).

TABLE II Sources of Bacteriophages Phage Designation ckP1 mmP1 clP1 clP2 mmP2 clL1 mmL1 clL2 mmL2

Location Wastewater treatment plant, Carrick-On-Suir, Tipperary, Ireland Farm slurry pit 1, Dunmarklum, Lissarda, Cork, Ireland Wastewater treatment plant (postpress machine sample), Clonmel, Tipperary, Ireland Wastewater treatment plant (postprimary digestion sample), Clonmel, Tipperary, Ireland Farm slurry pit 2, Dunmarklum, Lissarda, Cork, Ireland Wastewater treatment plant (final tank and thickener sample), Clonmel, Tipperary, Ireland Farm slurry pit 1, Dunmarklum, Lissarda, Cork, Ireland Wastewater treatment plant (postprimary digestion sample), Clonmel, Tipperary, Ireland Farm slurry pit 2, Dunmarklum, Lissarda, Cork, Ireland

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Kelly, D., Neve, H., McAuliffe, O., Ross, R. P., Arendt, E. K., and Coffey, A. RESULTS

16S RNA Typing of Bacteria All bacteria used in the study were subjected to 16S RNA typing and compared with type strains to confirm identity. The type strain for P. damnosus was P. damnosus strain DSM20331, the type strain for L. brevis was L. brevis ATCC14869, and the type strain for L. paraplantarum was L. paraplantarum DSM10667. The BLAST results shown in Table I indicate that both pediococci showed 99% identity with their type strain, DSM20331, while the lactobacilli

Fig. 1. Phage DNA digested with HindIII restriction endonuclease. A, Pediococcus phage DNA. From left to right: lane 1, 10-kb ladder (Bioline); lane 2, ckP1; lane 3, mmP1; lane 4, clP1; lane 5, clP2; lane 6, mmP2. B, Lactobacillus phage DNA. From left to right: lane 1, 10-kb ladder (Bioline); lane 2, clL1; lane 3, mmL1; lane 4, clL2; lane 5, mmL2.

also showed 99% identity with either L. brevis or L. paraplantarum. This confirmed species identity (Table I). Isolation of Phages from Farmyard Slurry and Municipal Wastewater After enrichment in the presence of a mixture of two strains of P. damnosus, 9 of 19 farmyard slurry and municipal sewage samples produced nonturbid plaques in lawns of P. damnosus P120. These were also capable of forming plaques on P. damnosus P82. After enrichment in the presence of two strains of L. brevis and one strain of L. paraplantarum, 10 samples produced nonturbid plaques in lawns of L. paraplantarum L913. These were also capable of forming plaques on L. brevis. Phages were isolated from these plaques and prepared for extraction of phage DNA for restriction analysis. A variety of restriction endonucleases were used, but HindIII gave the best quality digests with discrimination between phages (Fig. 1A and B). Comparison of digests indicated that there were five distinct Pediococcus phages and four distinct Lactobacillus phages. All phages were named according to the geographic location where the samples were sourced and the host bacterium on which they were propagated. The genome sizes of the phages were estimated based on the different restriction digests, and all were in the region of 40 to 50 kb, which is typical of the family Siphoviridae (15). Characterization of Phages The plaque-forming ability of all phages was compared. All phages formed clear plaques that were 0.5–1 mm in diameter on host strains, indicating that all phages were lytic on the host bacteria used. Morphological analysis of phages by electron microscopy allowed each to be classified into its respective viral family and order. All phages had obvious tails and, hence, belonged to the order Caudovirales. The Pediococcus phages had noncontractile tails and isometric heads (Fig. 2). Head dimensions ranged from

Fig. 2. Electron micrographs of Pediococcus and Lactobacillus phages negatively stained with 2% uranyl acetate: a, ckP1; b, mmP1; c, clP1; d, clP2; e, mmP2; f, clL1; g, mmL1; h, clL2; i, mmL2. Arrows indicate appendices at the tail tips (obscured on phage mmL1 [g]). Scale bar represents 100 nm.

Phage for Beer-Spoilage Lactic Acid Bacteria 58 × 53 to 59 × 54 nm (Table III). Tail lengths ranged from 173 to 176 nm, while tail diameters ranged from 9 to 10 nm (Table III). Differences in plaque size were also observed: phages mmP1, clP2, and mmp2 had plaque diameters of 0.8 mm, whereas phages ckP1 and clP1 had plaque diameters of 0.9 and 0.7 mm, respectively (Table IV). As with the Pediococcus phages, the Lactobacillus phages had noncontractile tails and isometric heads (Fig. 2). Dimensions for these phages were also similar, with head dimensions ranging from 57 × 52 to 60 × 55 nm (Table III). Tail lengths ranged from 166 to 178 nm, and tail diameters were approx. 11 nm. Tail tip appendices were visible for phages clL1, clL2, and mmL2 and ranged in length from 16 to 19 (Fig. 2; Table III). Phage mmL1 had similar fine structures, albeit to a lower degree of resolution on the transmission electron micrographs. As with the Pediococcus phages, some differences in plaque diameter were noted. Phages mmL1 and mmL2 had plaque diameters of 0.5 mm, whereas phages clL1 and clL2 had diameters of 0.9 and 1 mm, respectively (Table IV). All phages were classified into the family Siphoviridae based on the guidelines of the International Committee on Taxonomy of Viruses (15).

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novel ways to control foodborne pathogens and spoilage bacteria (11). The problematic spoilage bacterial strains used in this study were hop-resistant beer bacteria found in German breweries in 2007. Our 16S rRNA typing results confirmed the identity of the spoilage strains as P. damnosus (strains P120 and P82), L. brevis (strains L1033 and L1105), and L. paraplantarum (strain L913). Five distinct Pediococcus phages and four distinct Lactobacillus phages were isolated from farmyard slurry and municipal wastewater in different locations in southern Ireland in 2008. All phages were assigned to the family Siphoviridae based on morphology. All phages formed clear plaques, indicating that phages were undergoing a lytic cycle on these hosts. In the case of the five Pediococcus phages, HindIII digests indicated there were apparent genotypic similarities between them. Although all five showed distinct banding patterns, phages mmP1, clP1, clP2, and mmP2 had a number of electrophoretic restriction bands of identical molecular mass, suggesting a common ancestor. Phage ckP1 showed less similarity with the other four. Examination of electron micrographs of these five phages also indicated a similar morphology, with minor differences in dimensions noted. In the case of the Lactobacillus phages, analysis of HindIII digests showed that phages clL2, clL1, mmL1, and mmL2 had distinct banding patterns, but some similarities were apparent, indicating these phages were related at the genotypic level. Electron micrographs also indicated a morphological similarity, with all phages having similar head and tail dimensions. These phages were interesting in that they lysed two distinct species of Lactobacillus (L. brevis and L. paraplantarum), suggesting these species are closely related with regard to phage susceptibility. It is noteworthy that this is apparently the first report of isolation and characterization of P. damnosus phages. Yoon et al (26) isolated and characterized a lytic phage against a Pediococcus starter culture used in cucumber fermentations. This phage, while also belonging to the family Siphoviridae, had a smaller head diameter (51.2 nm) and a tail that was significantly shorter (129.6 nm) yet slightly larger in diameter (11.6 nm) than the Pediococcus phages

Bacteriophage Host Range All Pediococcus phages were assessed for their ability to lyse the two beer-spoilage P. damnosus strains, and all Lactobacillus phages were assessed for their ability to lyse the three beer-spoilage lactobacilli used in the study, as previously described (18). All Pediococcus phages lysed the two spoilage pediococci tested regardless of the host strain on which they were grown; all Lactobacillus phages similarly lysed the three spoilage lactobacilli. EOP values and spot test results are shown in Table IV. DISCUSSION Bacteriophages have significant potential as antibacterial agents (13) and also possess attributes that are useful to those looking for

TABLE III Dimensions of Pediococcus and Lactobacillus Bacteriophages Bacterial Strain Bacteriophage

Head (nm)

Tail Length (nm)

Tail Diameter (nm)

Tail Tip Appendices Length (nm)a

P. damnosus ckP1 mmP1 cIP1 cIP2 mmP2

58.8  1.1 × 53.6  2.7 (n = 11) 58.0  2.4 × 52.8  2.7 (n = 11) 59.0  2.9 × 53.4  2.6 (n = 8) 57.7  4.2 × 52.5  3.3 (n = 9) 57.9  2.9 × 53.7  2.1 (n = 11)

174.3  8.0 (n = 11) 175.7  5.3 (n = 13) 174.5  5.3 (n = 8) 173.2  5.1 (n = 9) 176.0  4.7 (n = 11)

9.8  1.2 (n = 11) 9.5  0.9 (n = 13) 9.6  0.8 (n = 8) 8.7  0.7 (n = 9) 9.7  0.9 (n = 11)

– – – – –

Lactobacillus cIL1 mmL1 cIL2 mmL2

58.9  4.1 × 52.4  4.5 (n = 12) 59.0  3.8 × 53.0  2.9 (n = 8) 56.8  4.9 × 52.0  3.9 (n = 20) 59.7  3.7 × 54.8  3.4 (n = 22)

171.8  4.2 (n = 13) 178.3  7.0 (n = 9) 173.3  3.7 (n = 20) 166.3  6.9 (n = 22)

10.6  0.9 (n = 13) 11.0  0.8 (n = 9) 11.1  1.2 (n = 20) 11.2  0.8 (n = 22)

19.1  2.9 (n = 14) n.m. 16.2  3.0 (n = 16) 17.5  2.2 (n = 22)

a

– = not present; n.m. = not measurable (low resolution). TABLE IV Host Range of Pediococcus and Lactobacillus Bacteriophages, Efficiency of Plaquing (EOP), and Plaque Diameters Bacteriophage EOP (Plaque Diameter) Pediococcus

Bacterial Strain P. damnosus P120a P. damnosus P82 L. paraplantarum L913a L. brevis L1105 L. brevis L1033 a

Propagating host strain.

Lactobacillus

ckP1 (0.9 mm)

mmP1 (0.8 mm)

clP1 (0.7 mm)

clP2 (0.8 mm)

mmP2 (0.8 mm)

clL1 (0.9 mm)

mmL1 (0.5 mm)

clL2 (1 mm)

mmL2 (0.5 mm)

1 0.95 … … …

1 0.45 … … …

1 0.7 … … …

1 1.1 … … …

1 0.62 … … …

… … 1 0.64 0.56

… … 1 0.90 0.66

… … 1 0.45 0.51

… … 1 0.76 0.81

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isolated in this study. The phage of Yoon et al (26) also had a smaller genome size (24.1 kb) than the phages isolated in this study (40– 50 kb). Overall, this study highlighted the relative abundance in sewage of phages for pediococci and lactobacilli. Their activity against prominent beer-spoilage bacteria provides an opportunity for application in this area. In this context, bacteriophages are known to be stable at typical beer pH values ranging from 3.8 to 4.7 (1). Indeed, it is well known that phages of lactic acid bacteria frequently propagate and lyse starter cultures in lactic acid dairy fermentations (6), and thus, their activity in beer would not be compromised by the pH of this environment. With regard to the genetic stability of Lactobacillus phages during this application, Desiere et al (8) have reported that stringent barriers prevent the transfer of phage genes across Lactobacillus spp. Although information regarding the genetic stability of Pediococcus phages is lacking (because they are rare in the scientific literature), the situation is likely to be similar to that of the Lactobacillus phages. Nevertheless, we are embarking on genome sequencing of the Pediococcus phages in order to provide a more thorough knowledge of their genetic makeup. ACKNOWLEDGMENTS This research was funded in part by the Irish Research Council for Science, Engineering and Technology (IRCSET) and also by the Food Institutional Research Measure (FIRM), Irish Department of Agriculture. We thank John Maher for his cooperation. LITERATURE CITED 1. Anonymous. The use and mode of action of bacteriophages in food production. EFSA J. 1076:1-26, 2009. 2. Asano, S., Suzuki, K., Iijima, K., Motoyama, Y., Kuriyama, H., and Kitagawa, Y. Effects of morphological changes in beer-spoilage lactic acid bacteria on membrane filtration in breweries. J. Biosci. Bioeng. 104:334-338, 2007. 3. Behr, J., Gaenzle, M. G., and Vogel, F. R. Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport. Appl. Environ. Microbiol. 72:6483-6492, 2006. 4. Bigwood, T., Hudson, J. A., and Billington, C. Influence of host and bacteriophage concentrations on the inactivation of food-borne pathogenic bacteria by two phages. FEMS Microbiol. Lett. 291:59-64, 2009. 5. Coffey, B., Mills, S., McAuliffe, O., Coffey, A., and Ross, R. P. Phage and their lysins as biocontrol agents for food safety applications. Annu. Rev. Food Sci. Technol. 1:449-468, 2010. 6. Coffey, A., and Ross, R. P. Bacteriophage resistance systems in dairy starter strains: Molecular analysis to application. Lactic acid bacteria, genetics metabolism and applications. Antonie Leeuwenhoek 82:303321, 2002. 7. De Man, J. C., Rogosa, M., and Sharpe, M. E. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135, 1960. 8. Desiere, F., Lucchini, S., Canchaya, C., Ventura, M., and Brüssow, H. Comparative genomics of phages and prophages in lactic acid bacteria. Antonie Leeuwenhoek 82:73-91, 2002.

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