Draft genome sequences of 53 Lactococcus and Leuconostoc strains isolated from two undefined DL-starter cultures.

This study presents the complete genomes of 53 strains of Lactococcus and Leuconostoc isolated from two undefined DL-starter cultures originating from Denmark, Tistrup, and P. The genomes were reconstructed using long-read, nanopore-based DNA sequencing, delivering comprehensive data set for comparative genomics and taxonomic classification, with potential utility in dairy fermentation processes.

Host genetic requirements for DNA release of lactococcal phage TP901-1.

The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram-positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901-1 due to mutations that affect TP901-1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three-component glycosylation system (TGS) was shown to be required for TP901-1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope-associated component that triggers TP901-1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram-positive infecting phage.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5.

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

Isolation and characterization of bacteriophages active against methicillin-resistant Staphylococcus pseudintermedius.

We aimed to isolate and characterize bacteriophages (phages) with preferential activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP), a multidrug-resistant canine pathogen. Four phages were isolated from canine faeces using two MRSP strains as initial hosts. Phage host range was evaluated by the spot test on 17 MRSP, 43 methicillin-susceptible S. pseudintermedius (MSSP), and six other staphylococci isolated from dogs. Transmission electron microscopy was used for presumptive identification followed by whole genome sequencing (WGS). All phages lysed all MRSP isolates whereas only 16-28% of MSSP were lysed. Their lytic activity was limited to S. pseudintermedius and S. schleiferi. All phages had similar morphology and belonged to the Siphoviridae family. WGS indicated that the phages were 93.8-99.7% identical to each other, and exhibited the highest similarity (87%) to the temperate S. aureus phage 187. Confirmatory lytic activity tests showed that phages were able to produce clear plaques on lysogens, which was enabled by recombination of the lysogeny modules as shown by WGS of the phages after propagation and plaque formation. This study provides insight into the genetic diversity and biology of S. pseudintermedius temperate phages, which could be further developed for topical therapy of MRSP skin and wound infections.

Extraction and Purification of Viruses from Fecal Samples for Metagenome and Morphology Analyses.

The human enteric virome consists of endogenous retro elements and viruses that infect the host and members of the gut microbiome (GM). Mounting evidence suggests that the gut virome plays a central role in maintaining homeostasis and via the GM influences immunology of the host. To thoroughly characterize the gut virome, it is often very useful to first separate and concentrate extracellular viral-like particles (eVLPs) enabling an integrative characterization of them. Here, we describe a detailed protocol for extraction and concentration of the viral fraction from fecal samples based on a polyethylene glycol precipitation (PEG) approach. These procedures maximize the yields of eVLPs (and their DNA) with high purity well suited for down-stream analysis such as quantification and morphological assessment, determination of phage-host pairs as well as virome sequencing.

Gut microbiota recovery and immune response in ampicillin-treated mice.

Ampicillin is applied in rodents to induce a temporarily depleted microbiota. To elucidate whether bacteria are just temporarily suppressed or fully eliminated, and how this affects the re-colonisation process, we compared the microbiota and immune system in conventionally housed untreated mice with newly weaned ampicillin treated mice subsequently housed in either a microbe containing environment or in an isolator with only host associated suppressed bacteria to recolonize the gut. Two weeks ampicillin treatment induced a seemingly germ-free state with no bacterial DNA to reveal. Four weeks after treatment caeca were still significantly enlarged in both treated groups, but bacteria re-appeared even in isolator housed mice. While some suppressed bacteria were able to recover and even dominate the community, the abundances and composition were far from the untreated mice and differed between isolator and conventional housing. The treatment reduced the innate cytokine expressions at least for three weeks after treatment, and had a non-lasting reducing impact on the regulatory T cells, and a more lasting impact on the natural killer T cells. We conclude that temporary ampicillin treatment suppresses the majority but does not eliminate all the gut microbiota members. The re-colonisation process is as such influenced by both suppressed host associated bacteria and by environmental bacteria. Treated mice do not re-obtain a complex gut microbiota comparable to untreated mice, and the immune response and gut morphology reflect this. This is a concern when comparing host parameters sensitive to microbial regulation after an antibiotic-induced temporarily "germ-free" state.

Investigation of the bacteriophage community in induced lysates of undefined mesophilic mixed-strain DL-cultures using classical and metagenomic approaches.

To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis.

Metagenomic Analysis of Dairy Bacteriophages: Extraction Method and Pilot Study on Whey Samples Derived from Using Undefined and Defined Mesophilic Starter Cultures.

Despite being potentially highly useful for characterizing the biodiversity of phages, metagenomic studies are currently not available for dairy bacteriophages, partly due to the lack of a standard procedure for phage extraction. We optimized an extraction method that allows the removal of the bulk protein from whey and milk samples with losses of less than 50% of spiked phages. The protocol was applied to extract phages from whey in order to test the notion that members of Lactococcus lactis 936 (now Sk1virus), P335, c2 (now C2virus) and Leuconostoc phage groups are the most frequently encountered in the dairy environment. The relative abundance and diversity of phages in eight and four whey mixtures from dairies using undefined mesophilic mixed-strain cultures containing Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc species (i.e., DL starter cultures) and defined cultures, respectively, were assessed. Results obtained from transmission electron microscopy and high-throughput sequence analyses revealed the dominance of Lc. lactis 936 phages (order Caudovirales, family Siphoviridae) in dairies using undefined DL starter cultures and Lc. lactis c2 phages (order Caudovirales, family Siphoviridae) in dairies using defined cultures. The 936 and Leuconostoc phages demonstrated limited diversity. Possible coinduction of temperate P335 prophages and satellite phages in one of the whey mixtures was also observed.IMPORTANCE The method optimized in this study could provide an important basis for understanding the dynamics of the phage community (abundance, development, diversity, evolution, etc.) in dairies with different sizes, locations, and production strategies. It may also enable the discovery of previously unknown phages, which is crucial for the development of rapid molecular biology-based methods for phage burden surveillance systems. The dominance of only a few phage groups in the dairy environment signifies the depth of knowledge gained over the past decades, which served as the basis for designing current phage control strategies. The presence of a correlation between phages and the type of starter cultures being used in dairies might help to improve the selection and/or design of suitable, custom, and cost-efficient phage control strategies.

A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages.

Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from ~1.1 x 105 to ~1.1 x 101 phage genomes per reaction, which corresponds to ~9 x 107 to ~9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother-bulk-cheese vat system. High levels of 936 and P335 phages were detected in the mother culture and the bulk starter, but also in the whey samples. Low levels of phages were detected in the cheese milk samples.

Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures.

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1-10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

Clear Plaque Mutants of Lactococcal Phage TP901-1.

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1.

Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage communities in the human gut.

BACKGROUND: The human gut is densely populated with archaea, eukaryotes, bacteria, and their viruses, such as bacteriophages. Advances in high-throughput sequencing (HTS) as well as bioinformatics have opened new opportunities for characterizing the viral communities harbored in our gut. However, limited attention has been given to the efficiency of protocols dealing with extraction of phages from fecal communities prior to HTS and their impact on the metagenomic dataset. RESULTS: We describe two optimized methods for extraction of phages from fecal samples based on tangential-flow filtration (TFF) and polyethylene glycol precipitation (PEG) approaches using an adapted method from a published protocol as control (literature-adapted protocol (LIT)). To quantify phage recovery, samples were spiked with low numbers of c2, ϕ29, and T4 phages (representatives of the Siphoviridae, Podoviridae, and Myoviridae families, respectively) and their concentration (plaque-forming units) followed at every step during the extraction procedure. Compared with LIT, TFF and PEG had higher recovery of all spiked phages, yielding up to 16 times more phage particles (PPs) and up to 68 times more phage DNA per volume, increasing thus the chances of extracting low abundant phages. TFF- and PEG-derived metaviromes showed 10% increase in relative abundance of Caudovirales and unclassified phages infecting gut-associated bacteria (>92% for TFF and PEG, 82.4% for LIT). Our methods obtained lower relative abundance of the Myoviridae family (<16%) as compared to the reference protocol (22%). This decline, however, was not considered a true loss of Myoviridae phages but rather a greater level of extraction of Siphoviridae phages (TFF and PEG >32.5%, LIT 22.6%), which was achieved with the enhanced conditions of our procedures (e.g., reduced filter clogging). A high degree of phage diversity in samples extracted using TFF and PEG was documented by transmission electron microscopy. CONCLUSIONS: Two procedures (TFF and PEG) for extraction of bacteriophages from fecal samples were optimized using a set of spiked bacteriophages as process control. These protocols are highly efficient tools for extraction and purification of PPs prior to HTS in phage-metavirome studies. Our methods can be easily modified, being thus applicable and adjustable for in principle any solid environmental material in dissolution.

Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice.

Transferring gut microbiota from one individual to another may enable researchers to "humanize" the gut of animal models and transfer phenotypes between species. To date, most studies of gut microbiota transfer are performed in germ-free mice. In the studies presented, it was tested whether an antibiotic treatment approach could be used instead. C57BL/6 mice were treated with ampicillin prior to inoculation at weaning or eight weeks of age with gut microbiota from lean or obese donors. The gut microbiota and clinical parameters of the recipients was characterized one and six weeks after inoculation. The results demonstrate, that the donor gut microbiota was introduced, established, and changed the gut microbiota of the recipients. Six weeks after inoculation, the differences persisted, however alteration of the gut microbiota occurred with time within the groups. The clinical parameters of the donor phenotype were partly transmissible from obese to lean mice, in particularly ß cell hyperactivity in the obese recipients. Thus, a successful inoculation of gut microbiota was not age dependent in order for the microbes to colonize, and transferring different microbial compositions to conventional antibiotic-treated mice was possible at least for a time period during which the microbiota may permanently modulate important host functions.

Complete Genome Sequences of Four Novel Lactococcus lactis Phages Distantly Related to the Rare 1706 Phage Species.

Lactoccocus lactis is a Gram-positive bacterium widely used in the dairy industry in the production of an array of cheeses and other fermented milk products. Here, we describe the sequencing and genome annotations of a set of four phages virulent to L. lactis and exhibiting similarities to phage 1706.

Bacteriophages of leuconostoc, oenococcus, and weissella.

Leuconostoc (Ln.), Weissella, and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat, and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore, bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to, e.g., lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that (i) lysogeny seems to be abundant in Oenococcus strains, and (ii) several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus Lactobacillus.

Sequence and comparative analysis of Leuconostoc dairy bacteriophages.

Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4 kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages.

DPS - a rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque.

Bacteriophages (phages) coexist with bacteria in all environments and influence microbial diversity, evolution and industrial production processes. As a result of this major impact of phages on microbes, tools that allow rapid characterization of phages are needed. Today, one of the most powerful methods for characterization of phages is determination of the whole genome using high throughput sequencing approaches. Here a direct plaque sequencing (DPS) is described, which is a rapid method that allows easy full genome sequencing of DNA-containing phages using the Nextera XT™ kit. A combination of host-DNA removal followed by purification and concentration of the viral DNA, allowed the construction of Illumina-compatible sequencing libraries using the Nextera™ XT technology directly from single phage plaques without any whole genome amplification step. This method was tested on three Caudovirales phages; ϕ29 Podoviridae, P113g Siphoviridae and T4 Myovirdae, which are representative of >96% of all known phages, and were sequenced using the Illumina MiSeq platform. Successful de novo assembly of the viral genomes was possible.