The impact of bacteriophage genomics

The discovery of (bacterio)phages revolutionised microbiology and genetics, while phage research has been integral to answering some of the most fundamental biological questions of the twentieth century. The susceptibility of bacteria to bacteriophage attack can be undesirable in some cases, especially in the dairy industry, but can be desirable in others, for example, the use of bacteriophage therapy to eliminate pathogenic bacteria. The relative ease with which entire bacteriophage genome sequences can now be elucidated has had a profound impact on the study of these bacterial parasites.     

Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence

In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 10(5) PFU ml(-1)).     

噬菌体在食品工业中的应用与危害防控

近年来,噬菌体由于其特异性侵染细菌的特性,在食品加工及保藏过程中有害微生物的控制和检测方面展现出良好的应用前景。例如在食品表面喷洒噬菌体或将噬菌体与食品包装材料结合,对食源性致病菌及腐败菌加以控制,以及利用基因工程手段构建报告噬菌体对食源性致病菌进行快速检测等。然而,噬菌体也是危害食品发酵的重要因素之一,轻则减产,重则引起整个发酵过程失败,造成巨大的经济损失。目前主要通过噬菌体消毒及灭活、发酵菌种变换等方式防止噬菌体污染。本文综述了食品工业中噬菌体应用及危害的研究现状,以期为拓宽噬菌体在食品工业中的应用途径及开发噬菌体污染防治的新技术提供理论依据。     

Bacteriophages of lactic acid bacteria and their impact on milk fermentations

Every biotechnology process that relies on the use of bacteria to make a product or to overproduce a molecule may, at some time, struggle with the presence of virulent phages. For example, phages are the primary cause of fermentation failure in the milk transformation industry. This review focuses on the recent scientific advances in the field of lactic acid bacteria phage research. Three specific topics, namely, the sources of contamination, the detection methods and the control procedures will be discussed.     

Phages in the global fruit and vegetable industry

From recent articles, we have learned that phages can constitute a promising alternative in the food industry to eliminate bacterial pathogens from seedlings in greenhouse and field environments, as well as from fresh‐cut food products. The fruit and vegetable industry requires quite a different approach than the meat or dairy industry. Several factors can inhibit efficacy of phage treatment such as plant watering or washing ready‐to‐eat products (water may dilute therapeutic doses), UV irradiation or extensive spreading of phytopathogens by wind, insects or even humans. Spontaneously occurring anomalous weather conditions in different parts of the world also may have an enormous impact on phage persistence in cultivations and on yields. Despite that, some phage preparations are commercially available and, without doubt, are much safer than chemical treatments. Along with increasing worldwide fruit and vegetable consumption, plant diseases and human foodborne illnesses are becoming a serious economic problem, resulting in a focus on optimization of phage treatment.     

The inhibitory effects of pyrocarbonic acid diethyl ester on lactobacillus casei and its phage Jl – A novel strategy for phage control in technical fermentation processes

The effects of pyrccarbonic acid diethyl ester (PADE) on Lactobacillus casei Sl and its phage Jl was investigated in relation to the control of phages in the dairy industry and other technica fermentation processes. PADE exhibited a bacteriostatic effect at 0.5 to 8 mM and a bactericidal effect at 10 mM or higher. It inhibited the growth of the phage at its bacteriostatic and bactericidal concentrations. The growth inhibition of the phage was reversible at the bacteriostatic concentrations but complete and irreversible at the bactericidal concentrations. PADE inactivated the free phage within several minutes; 10 and 30 mM of PADE inactivated 90 and 100%, respectively, of the phage. It completely decomposed into ineffective components in several minutes. The bacteria grew almost normally when they were inoculated after the complete decomposition of PADE. These four characteristics of PADE – its bactericidal effect, its inhibitory effect on phage growth, its phage-inactivating effect and its decomposition – suggest a novel strategy for phage control in technical fermentation processes, including the dairy industry.     

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.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Abstract Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

The Impact of Resource Availability on Bacterial Resistance to Phages in Soil

Resource availability can affect the coevolutionary dynamics between host and parasites, shaping communities and hence ecosystem function. A key finding from theoretical and in vitro studies is that host resistance evolves to greater levels with increased resources, but the relevance to natural communities is less clear. We took two complementary approaches to investigate the effect of resource availability on the evolution of bacterial resistance to phages in soil. First, we measured the resistance and infectivity of natural communities of soil bacteria and phage in the presence and absence of nutrient-providing plants. Second, we followed the real-time coevolution between defined bacteria and phage populations with resource availability manipulated by the addition or not of an artificial plant root exudate. Increased resource availability resulted in increases in bacterial resistance to phages, but without a concomitant increase in phage infectivity. These results suggest that phages may have a reduced impact on the control of bacterial densities and community composition in stable, high resource environments.     

Multilocus sequence typing scheme for the characterization of 936-like phages infecting Lactococcus lactis

Lactococcus lactis phage infections are costly for the dairy industry because they can slow down the fermentation process and adversely impact product safety and quality. Although many strategies have been developed to better control phage populations, new virulent phages continue to emerge. Thus, it is beneficial to develop an efficient method for the routine identification of new phages within a dairy plant to rapidly adapt antiphage tactics. Here, we present a multilocus sequence typing (MLST) scheme for the characterization of the 936-like phages, the most prevalent phage group infecting L. lactis strains worldwide. The proposed MLST system targets the internal portion of five highly conserved genomic sequences belonging to the packaging, morphogenesis, and lysis modules. Our MLST scheme was used to analyze 100 phages with different restriction fragment length polymorphism (RFLP) patterns isolated from 11 different countries between 1971 and 2010. PCR products were obtained for all the phages analyzed, and sequence analysis highlighted the high discriminatory power of the MLST system, detecting 93 different sequence types. A conserved locus within the lys gene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within the mcp gene (major capsid protein) was the most conserved (54 distinct alleles). Phylogenetic analyses of the concatenated sequences exhibited a strong concordance of the clusters with the phage host range, indicating the clonal evolution of these phages. A public database has been set up for the proposed MLST system, and it can be accessed at http://pubmlst.org/bacteriophages/.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages

AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.     

Use of phages to control Campylobacter spp.

The use of phages to control pathogenic bacteria has been investigated since they were first discovered in the beginning of the 1900s. Over the last century we have slowly gained an in-depth understanding of phage biology including which phage properties are desirable when considering phage as biocontrol agents and which phage characteristics to potentially avoid. Campylobacter infections are amongst the most frequently encountered foodborne bacterial infections around the world. Handling and consumption of raw or undercooked poultry products have been determined to be the main route of transmission. The ability to use phages to target these bacteria has been studied for more than a decade and although we have made progress towards deciphering how best to use phages to control Campylobacter associated with poultry production, there is still much work to be done. This review outlines methods to improve the isolation of these elusive phages, as well as methods to identify desirable characteristics needed for a successful outcome. It also highlights the body of research undertaken so far and what criteria to consider when doing in-vivo studies, especially because some in-vitro studies have not been found to translate into to phage efficacy in-vivo.     

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.     

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.     

Isolation of lactococcal prolate phage-phage recombinants by an enrichment strategy reveals two novel host range determinants

Virulent lactococcal prolate (or c2-like) phages are the second most common phage group that causes fermentation failure in the dairy industry. We have mapped two host range determinants in two lactococcal prolate phages, c2 and 923, for the host strains MG1363 and 112. Each phage replicates on only one of the two host strains: c2 on MG1363 and 923 on 112. Phage-phage recombinants that replicated on both strains were isolated by a new method that does not require direct selection but rather employs an enrichment protocol. After initial mixed infection of strain 112, two rotations, the first of which was carried out on strain MG1363 and the second on 112, permitted continuous amplification of double-plating recombinants while rendering one of the parent phages unamplified in each of the two rotations. Mapping of the recombination endpoints showed that the presence of the N-terminal two-thirds of the tail protein L10 of phage c2 and a 1,562-bp cosR-terminal fragment of phage 923 genome overcame blocks of infection in strains MG1363 and 112, respectively. Both infection inhibition mechanisms act at the stage of DNA entry; in strain MG1363, the infection block acts early, before phage DNA enters the cytoplasm, and in strain 112, it acts late, after most of the DNA has entered the cell but before it undergoes cos-end ligation. These are the first reported host range determinants in bacteriophage of lactic acid bacteria required for overcoming inhibition of infection at the stage of DNA entry and cos-end ligation.     

Importance of bacteriophages in fermentation processes

Any bacterial strain can be infected by virulent phages or harbour one or more prophages. Therefore, bacteria-phage interactions are to be regarded as fundamental properties of bacteria. In current industrial fermentation processes phages can be advantageously employed for the identification of bacterial production strains (phage typing). In some cases phages are involved in the production of enzymes and special substances. The fundamental importance of phages in any technical fermentation process, however, is based on the peculiarities of their obligately parasitic life cycle. The propagation of phages in fermentation processes can cause complete (or at least partial) lysis of the production strains and, consequently, serious disturbances in the production process and considerable economic losses. The phage problem in the fermentation industry has not yet been completely solved. For the protection of technical processes against virulent phages five measures are discussed: phage-protected sterile fermentation, employment of alternative cultures, employment of phage-resistant mutants, employment of phage inhibitors, and employment of immobilized bacterial cells. The problem of the protection of bacterial production strains from prophage induction is more difficult and practically unsolved. Two possibilities to minimize the process risk due to temperate phages, the elimination of inducing factors during the fermentation process, and the selection of production strains which are difficult to induce, are discussed.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Selection of bacteriophage-resistant mutants of Streptococcus thermophilus

Phage-resistant mutants have been isolated from Streptococcus thermophilus. Selection was carried out using anti-phage antibodies or Hoechst 33258-labelled phages. Two mutants out of eight tested displayed reduced acidifying capacity. Selection of the bacteria that extruded more rapidly the fluorochrome 5-6-carboxyfluorescein diacetate (CFDA) restored the acidifying capacity of these two mutants to the level of the parental strains. Mutants displaying phage resistance and good acidifying capacity were obtained in 4-5 days. New phages that are able to overcome the protection mechanisms of the existing bacteria arise continually in the dairy environment. The procedures described here permit to replace promptly the starter culture susceptible to newly emerged phages with a resistant one.     

Biofilm control with natural and genetically-modified phages

Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.