Milk Contamination and Resistance to Processing Conditions Determine the Fate of Lactococcus lactis Bacteriophages in Dairies

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°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.     

Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment

We present here the results of an exploration of the bacteriophage content of dairy wheys collected from milk plants localized in various regions of Poland. Thirty-three whey samples from 17 regions were analyzed and found to contain phages active against L. lactis strains. High phage titer in all whey samples suggested phage-induced lysis to be the main cause of fermentation failures. In total, over 220 isolated phages were examined for their restriction patterns, genome sizes, genetic groups of DNA homology, and host ranges. Based on DNA digestions the identified phages were classified into 34 distinct DNA restriction groups. Phage genome sizes were estimated at 14-35 kb. Multiplex PCR analysis established that the studied phages belong to two out of the three main lactococcal phage types--c2 and 936, while P335-type phages were not detected. Yet, analyses of bacterial starter strains revealed that the majority of them are lysogenic and carry prophages of P335-type in their chromosome. Phage geographical distribution and host range are additionally discussed.     

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.     

Multiplex PCR for detection and identification of lactococcal bacteriophages

Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82. 2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10(4) to 10(7) PFU/ml and was 10(3) to 10(5) PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.     

Identification of a New P335 Subgroup through Molecular Analysis of Lactococcal Phages Q33 and BM13

Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.     

Evolution of Lactococcus lactis Phages within a Cheese Factory

We have sequenced the double-stranded DNA genomes of six lactococcal phages (SL4, CB13, CB14, CB19, CB20, and GR7) from the 936 group that were isolated over a 9-year period from whey samples obtained from a Canadian cheese factory. These six phages infected the same two industrial Lactococcus lactis strains out of 30 tested. The CB14 and GR7 genomes were found to be 100% identical even though they were isolated 14 months apart, indicating that a phage can survive in a cheese plant for more than a year. The other four genomes were related but notably different. The length of the genomes varied from 28,144 to 32,182 bp, and they coded for 51 to 55 open reading frames. All five genomes possessed a 3′ overhang cos site that was 11 nucleotides long. Several structural proteins were also identified by nano-high-performance liquid chromatography-tandem mass spectrometry, confirming bioinformatic analyses. Comparative analyses suggested that the most recently isolated phages (CB19 and CB20) were derived, in part, from older phage isolates (CB13 and CB14/GR7). The organization of the five distinct genomes was similar to the previously sequenced lactococcal phage genomes of the 936 group, and from these sequences, a core genome was determined for lactococcal phages of the 936 group.The manufacture of cheeses requires the inoculation of carefully selected bacterial cultures, known as starter cultures, at concentrations of at least 10⁷ live bacteria per ml of heat-treated milk. The purpose of this process is to control the fermentation and to obtain high-quality fermented products (29). Starter cultures are a combination of lactic acid bacteria (LAB), of which one of the most important species is Lactococcus lactis. L. lactis is a low-GC gram-positive bacterium used to metabolize lactose into lactic acid during the production of several cheese varieties. Because large amounts of lactococcal cells are cultivated each day in large-scale fermentation vats and because these cells are susceptible to bacteriophage infection, it is not surprising that most cheese factories have experienced problems with phage contamination (13). Even a single phage infecting a starter strain is enough to begin a chain reaction that can eventually inhibit bacterial growth and cause production delays, taste and texture variations, and even complete fermentation failures (1, 29).Phage infections are unpredictable in food fermentations. Their presence and persistence in a dairy factory can be explained in many ways. First, raw milk can introduce new phages into an industrial plant (25). Madera et al. (22) also reported that newly isolated lactococcal phages were more resistant to pasteurization. Whey, a liquid by-product of cheese manufacturing, is another reservoir that can spread phages in a factory environment (25). Airborne phage dissemination may also be important since concentrations of up to 10⁶ PFU/m³ have been observed close to a functional whey separation tank (32).For decades, the dairy industry has been working to curtail the propagation of virulent phages using a variety of practical strategies, including, among others, sanitation, optimized factory design, air filtration units, rotation of bacterial strains, and the use of phage resistance systems (13). Yet new virulent phages emerge on a regular basis. Indeed, large-scale industrial milk fermentation processes can be slowed down by virulent phages of the Caudovirales order. Members of three lactococcal phage groups, namely, 936, c2, and P335, are mostly found in dairy plants. The 936-like phages are by far the most predominant worldwide (3, 18, 22, 27).Phages of the 936 group have a double-stranded DNA genome and possess a long noncontractile tail connected to a capsid with icosahedral symmetry characteristic of the Siphoviridae family. Currently, six complete phage genomes of the lactococcal 936 group are available in public databases, including sk1 (6), bIL170 (10), jj50, 712, P008 (23), and bIBB29 (16). Their comparative analysis revealed a conserved gene organization despite being isolated from different countries. Most of the differences have been observed in the early gene module, where insertions, deletions, and point mutations likely occurred (16, 23). Moreover, it is assumed that these phages can also exchange DNA through recombination with other bacterial viruses present in the same ecosystem.Because new members of this lactococcal phage group are regularly isolated, a better understanding of their evolution is warranted to better control them. A cheese factory is a particular man-made niche where rapidly growing bacterial strains encounter ubiquitous phages. Such active environments provide ample opportunities for phage evolution, especially to dodge phage resistance mechanisms that may be present in host cells. Nonetheless, the evolutionary dynamics that shape the diversity of lactococcal phage populations are still not well understood.In this study, we analyzed the genome and structural proteome of six 936-group phages (SL4, CB13, CB14, CB19, CB20, and GR7) that infected the same L. lactis strains and were isolated over a 9-year period from a cheese factory.     

Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes

AIMS: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. METHODS AND RESULTS: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 microl whey samples and the detection limit of the assay was 10(2)-10(3) PFU ml(-1) as opposed to the detection limit of 10(4) PFU ml(-1) for conventional PCR performed on 1-microl whey samples. CONCLUSIONS: In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.     

Identification of four phage resistance plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage phi712 (936 phage species) and the prolate-headed phage phic2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Detection of airborne lactococcal bacteriophages in cheese manufacturing plants

The dairy industry adds starter bacterial cultures to heat-treated milk to control the fermentation process during the manufacture of many cheeses. These highly concentrated bacterial populations are susceptible to virulent phages that are ubiquitous in cheese factories. In this study, the dissemination of these phages by the airborne route and their presence on working surfaces were investigated in a cheese factory. Several surfaces were swabbed, and five air samplers (polytetrafluoroethylene filter, polycarbonate filter, BioSampler, Coriolis cyclone sampler, and NIOSH two-stage cyclone bioaerosol personal sampler) were tested. Samples were then analyzed for the presence of two Lactococcus lactis phage groups (936 and c2), and quantification was done by quantitative PCR (qPCR). Both lactococcal phage groups were found on most swabbed surfaces, while airborne phages were detected at concentrations of at least 10(3) genomes/m(3) of air. The NIOSH sampler had the highest rate of air samples with detectable levels of lactococcal phages. This study demonstrates that virulent phages can circulate through the air and that they are ubiquitous in cheese manufacturing facilities.     

Identification of Four Phage Resistance Plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage 712 (936 phage species) and the prolate-headed phage c2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Molecular taxonomy of Lactobacillus phages

Forty-eight strains of lactobacilli used as starter strains in the dairy industry were examined for lysogeny after treatment with mitomycin C. Two strains of L. delbrueckii subsp. bulgaricus were able to produce active phages. These temperate phages as well as 4 virulent phages isolated during abnormal fermentations were compared to a previously characterized phage mv4 which is temperate. All these phages were shown to be partially homologous by DNA-DNA hybridization. Genes that code for viral proteins seem to be well conserved since 2 major virion polypeptides of 18 (or 19) kD and 34 kD could be detected in the protein composition of each phage. Immunoblotting studies of the 7 phages using serum raised against phage mv4 confirmed that the proteins of the different phages were related. All these phages can be classified in the previously constituted group a, which now comprises 4 temperate and 15 virulent phages. These results show that some virulent phages appearing during abnormal fermentations and some temperate phages isolated by appearing during abnormal fermentations and some temperate phages isolated by induction of starter strains can be closely related genetically. Five virulent phages of L. helveticus were also compared according to their restriction pattern and their DNA homology. They were shown to be related to one another, but unrelated to phages of other lactic acid bacteria species.     

Assessing and analysing contamination of a dairy products processing plant by Staphylococcus aureus using antibiotic resistance and PFGE

A dairy product processing plant was studied for 2.5 years to examine contamination with Staphylococcus aureus and try to correlate the source of contamination. Cultures were submitted to an antibiotic susceptibility test (AST) and characterised by Pulsed-field Gel Electrophoresis (PFGE) analysis. Results showed that 35.2% (19/51) of food handlers were asymptomatic carriers of S. aureus, and that 90.4% (19/21) of raw milk sampled was contaminated. Staphylococcus aureus was isolated from only 10 samples among more than 3200 investigated dairy products. No S. aureus contamination was found on machinery. The AST analysis demonstrated sensitivity of tested S. aureus to oxacillin, cephalothin, vancomycin, gentamicin, and sulfamethoxazole/trimethoprim. AST analysis generated eight different phenotypic profiles, but did not allow us to identify the source of contamination in seven of ten final products. PFGE analysis proved to be a sensitive method as it generated 42 different DNA banding profiles among the 48 S. aureus investigated, demonstrating a lack of predominance of endemic strains in the plant, contrary to suggestions raised by antibiotic resistance typing. Based on PFGE genotyping, S. aureus strains isolated from four contaminated final products were similar to four S. aureus isolated from raw milk. Five final products contained S. aureus different from all other strains collected, and one showed similarity to a strain isolated from a food handler. These results suggest contamination by raw milk as the main source of contamination of the final dairy products.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group ( B. cereus, B. mycoides and B. thuringiensis ) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10°C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group (B. cereus, B. mycoides and B. thuringiensis) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10 degrees C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Factors influencing the survival and multiplication of bacteriophages in calves and in their environment

Seven phages were fairly susceptible in vitro to the lethal effect of acidified whey, more so than the enteropathogenic Escherichia coli strains on which they were active. The low acidity that prevailed in the abomasum contents of calves shortly after a milk feed had little harmful effect on orally administered organisms of these phages; they flooded into the small intestine. The high acidity that prevailed later was lethal to orally administered phage organisms; few entered the small intestine. The lethal effect could be counteracted by giving CaCO3 in the feed. Low concentrations of phage-neutralizing antibodies were found in some serum samples from human beings, cattle and pigs. Antibodies to one of the seven phages were common in the human samples and antibodies to another, phage B44/1, were common in the cattle and pig samples and in bovine colostrum. Phage B44/1 antibodies in a sample of colostral whey were destroyed at pH 3.25 or less. Giving colostrum containing phage B44/1 antibodies with CaCO3 to a calf greatly reduced the numbers of orally administered phage B44/1 organisms in its alimentary tract. Antibodies to another phage were induced in the serum of a calf suffering from E. coli diarrhoea by treating it with that phage. The phages were as susceptible as the E. coli strains to the lethal action of formaldehyde and sodium hypochlorite. In contrast to the E. coli strains, they were almost completely resistant to phenol and chloroxylenol. The in vitro virulence of 21 phages varied according to the temperature at which tests were performed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Leuconostoc bacteriophage infecting flavor producers of cheese starter cultures

Dairy siphovirus φLmd1, which infects starter culture isolate Leuconostoc mesenteroides subsp. dextranicum A1, showed resistance to pasteurization and was able to grow on 3 of the 4 commercial starter cultures tested. Its 26,201-bp genome was similar to that of Leuconostoc phage of vegetable origin but not to those of dairy phages infecting Lactococcus.     

A Strategy for Rotation of Different Bacteriophage Defenses in a Lactococcal Single-Strain Starter Culture System

A new strategy for starter culture rotations was developed for a series of phage-resistant clones genetically derived from a single strain of Lactococcus lactis subsp. lactis. Phage-resistant derivatives carrying different defense systems were constructed via conjugation with various plasmids encoding abortive infection (Abi/Hsp) and/or restriction and modification (R/M) systems of different specificity. The plasmids included pTR2030 (Hsp⁺ R⁺/M⁺), pTN20 (Abi⁺ R⁺/M⁺), pTRK11 (R⁺/M⁺), and pTRK68 (R⁺/M⁺). Selected phage-resistant transconjugants or transformants were evaluated in different rotation sequences through cycles of the Heap-Lawrence starter culture activity test in milk contaminated with phage and whey from the previous cycle. When used in consecutive sequence, derivative strains carrying the R/M systems encoded by pTN20, pTRK11, and pTRK68 retarded phage development when the initial levels of phage contamination were below 10² PFU/ml but not when levels were increased to 10³ PFU/ml. Use of a derivative bearing pTR2030 (Hsp⁺ R⁺/M⁺) at the beginning of the rotation prevented phage development, even when the initial levels of phage contamination were high (10⁶ PFU/ml). Alternating the type and specificity of R/M and Abi defenses through the rotation prevented phage proliferation and in some cases eliminated contaminating phages. A model rotation sequence for the phage defense rotation strategy was developed and performed successfully over nine cycles of the Heap-Lawrence starter culture activity test in the presence of high-titer commercial phage composites. This phage defense rotation strategy is designed to protect a highly specialized Lactococcus strain from phage attack during continuous and extended use in the dairy industry.     

Use of the molecular typing methods to evaluate the control ofListeria monocytogenes contamination in a raw milk and dairy products

Nineteen serogroup 1/2a Listeria monocytogenes strains isolated from raw milk, dairy products and salt water in one dairy were analyzed. Pulsed field gel electrophoresis (PFGE) and ribotyping were used to determine whether these strains isolated over a 8-month period are epidemiologically related. The samples of raw milk were contaminated by different L. monocytogenes clones. The clones isolated from dairy products (with the exception of one sample) and salt water were identical. Comparative genetic analysis of the clones isolated from raw milk, salt water and dairy products revealed the source of contamination and identified the L. monocytogenes strain involved in this process.     

Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.