DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant

Aims:  To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei .
Methods and Results:  Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk.
Conclusions:  Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making.
Significance and Impact of the Study:  This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.     

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.     

Some properties ofStreptococcus thermophilus bacteriophages

The morphology, host range, structural proteins and serological properties ofStreptococcus thermophilus phages isolated from Finnish cheese plants were investigated. The results show that all the nine phages belong morphologically to Ackermann’s group B1. The host—phage reactions and plating efficiency justify the division of these phages into four specificity groups. Most of the phages showed an absolute host specificity as to their plating efficiency but were not strictly specific in the adsorption to different hosts. The electrophoretic profiles of the structural proteins appeared nearly identical. Ten to eleven well separated proteins could be detected. The antiserum raised against one of the phages contained antibodies with different neutralization capacity depending on the phage. Using an immunoblotting technique, four structural proteins were detected that could bind phage antibodies.     

Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese

AIMS: This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. METHODS AND RESULTS: The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. CONCLUSIONS: Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.     

Predictive formulae for goat cheese yield based on milk composition

Prediction of the yield and quality of different types of cheeses that could be produced from a given type and/or amount of goat milk is of great economic benefit to goat milk producers and goat cheese manufacturers. Bulk tank goat milk was used for manufacturing hard, semi-hard and soft cheeses (N = 25, 25 and 24, respectively) to develop predictive formulae of cheese yield based on milk composition. Fat, total solids, total protein and casein contents in milk and moisture-adjusted cheese yield were determined to establish relationships between milk composition and cheese yield. Soft, semi-hard and hard cheeses in this study had moisture contents of 66, 46 and 38%, respectively, which could be used as reference standards. In soft cheese, individual components of goat milk or a combination of two or three components predicted cheese yield with a reasonably high correlation coefficient (R² = 0.73–0.81). However, correlation coefficients of predictions were lower for both semi-hard and hard cheeses. Overall, total solids of goat milk was the strongest indicator of yield in all three types of cheeses, followed by fat and total protein, while casein was not a good predictor for both semi-hard and hard cheeses. When compared with moisture-adjusted cheese yield, there was no difference (P > 0.05) in predicting yield of semi-hard and hard goat milk cheeses between the developed yield formulae in this study and a standard formula (the Van Slyke formula) commonly used for cow cheese. Future research will include further validation of the yield predictive formulae for hard and semi-hard cheeses of goat milk using larger data sets over several lactations, because of variation in relationships between milk components due to breed, stage of lactation, season, feeding regime, somatic cell count and differences in casein variants.     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

An analysis of the volatile flavour compounds in a soft raw goat milk cheese

The major components of the musty cow rind cheeses were identified in a soft raw goat milk cheese as heptan-2-one, nonan-2-one, their corresponding secondary alcohols, some esters and sulfur compounds. Their production was associated with the manufacturing process and its influence on the microbial activity. However a specificity in goat cheese compounds was displayed concerning in particular limonene and some ketones, alcohols and aldehydes.     

Structural Similarity and Genetic Homology between Lactobacillus casei Bacteriophages Isolated in Japan and in Finland

Three Lactobacillus casei bacteriophages, LC-Nu, PL-1, and ?FSW, were compared. Phage LC-Nu, which has not been previously characterized, originated from a local cheese plant in Finland. Phages PL-1 and ?FSW (isolated in Japan) represent the most thoroughly studied L.casei phages so far. All three phages had similar morphotypes, but still had different patterns of structural proteins, as analyzed by SDS-PAGE. The phages differed also in types of genome organization: LC-Nu and PL-1 had cohesive ends in their DNAs, and the DNA of ?FSW was circularly permuted. The initiation site and orientation of packaging of ?FSW DNA were identified. The homologies between the phage genomes were analyzed by Southern hybridization. About one-third of each phage gem me was highly homologous with other phages (homology over 85%), and two-thirds were slightly homologous (homology between 65% and 76%). DNAs from five industrial L. casei strains were also tested for homology with phage LC-Nu DNA. Phage LC-Nu related sequences were present in all the L. casei strains tested.     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

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.     

Argentinean Lactococcus lactis bacteriophages: genetic characterization and adsorption studies

Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca²⁺, physiological cell state, pH and temperature on cell adsorption was also investigated. The double‐stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca²⁺ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C. Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.     

Molecular ecology of Streptococcus thermophilus bacteriophage infections in a cheese factory.

A mozzarella cheese factory using an undefined, milk-derived Streptococcus thermophilus starter system was monitored longitudinally for 2 years to determine whether the diversity of the resident bacteriophage population arose from environmental sources or from genetic changes in the resident phage in the factory. The two hypotheses led to different predictions about the genetic diversity of the phages. With respect to host range, 12 distinct phage types were observed. With two exceptions, phages belonging to different lytic groups showed clearly distinct restriction patterns and multiple isolates of phages showing the same host range exhibited identical or highly related restriction patterns. Sequencing studies in a conserved region of the phage genome revealed no point mutations in multiple isolates of the same phage type, while up to 12% nucleotide sequence diversity was observed between the different phage types. This diversity is as large as that between the most different sequences from phages in our collection. These observations make unlikely a model that postulates a single phage invasion event and diversification of the phage during its residence in the factory. In the second stage of our factory study, a defined starter system was introduced that could not propagate the resident factory phage population. Within a week, three new phage types were observed in the factory while the resident phage population was decreased but not eliminated. Raw milk was the most likely source of these new phages, as phages with identical host ranges and restriction patterns were isolated from raw milk delivered to the factory during the intervention trial. Apparently, all of the genetic diversity observed in the S. thermophilus phages isolated during our survey was already created in their natural environment. A better understanding of the raw-milk ecology of S. thermophilus phages is thus essential for successful practical phage control.     

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.     

Sex identification of northern ungulates using low quality and quantity DNA

We evaluated PCR primer sets to determine the most effective technique for identifying sex of northern ungulates. We sought markers that required only a single pair of primers to amplify both X- and Y-linked alleles; that amplified X- and Y-linked products that were easily distinguishable using agarose gel electrophoresis; and that produced short amplicons amenable to amplification using DNA of poor quality and low quantity, as is often found in non-invasively collected samples such as feces. Primer pairs KY1/KY2 and SE47/SE48, which amplify X- and Y-specific alleles of the amelogenin gene, met our criteria and were tested for moose (Alces alces), mountain goat (Oreamnos americanus), Sitka black-tailed deer (Odocoileus hemionus sitkensis), and caribou (Rangifer tarandus). KY primers amplified shorter PCR products than did SE primers; moreover, SE primers inconsistently amplified certain Y-chromosome products, creating potential for misidentification of sex. DNA fragments amplified using KY primers were sequenced for each species, allowing us to characterize a 45-bp deletion for Y-linked alleles (136-bp product) relative to X-linked alleles (181-bp product) in all species and a 9-bp deletion in the X-linked allele of moose relative to other species. This is the first sex-determination technique using PCR reported for several ungulate species of Alaska. Although other protocols exist for cervids and bovids, this is the first report of markers meeting the aforementioned criteria for Odocoileus, the most abundant and intensively managed genus of large mammals in North America.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.