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Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [ Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum . The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin ( mrsA ), amylocyclicin ( acnA ), plipastatin ( ppsA ), and surfactin ( srfAA ) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea., Competing Interests: The authors declare this study received funding from Tea Gallery Group (Thailand) Co., Ltd. and Amazing Tea Limited Partnership. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. All authors declare no other competing interests., (Copyright © 2021 Rungsirivanich, Parlindungan, O’Connor, Field, Mahony, Thongwai and van Sinderen.)

Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the tra R gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery., Competing Interests: HS, ET, and PW were employed by the company DSM. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ortiz Charneco, Kelleher, Buivydas, Streekstra, van Themaat, de Waal, Mahony and van Sinderen.)

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as "critical" for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no "gold standard" for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows "phage synergy" to inform cocktail design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Haines, Hodges, Nale, Mahony, van Sinderen, Kaczorowska, Alrashid, Akter, Brown, Sauvageau, Sicheritz-Pontén, Thanki, Millard, Galyov and Clokie.)

Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis . Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups., (Copyright © 2019 Feyereisen, Mahony, Neve, Franz, Noben, O’Sullivan, Boer and van Sinderen.)

Plasmids are autonomous, self-replicating, extrachromosomal genetic elements that are typically not essential for growth of their host. They may encode metabolic capabilities, which promote the maintenance of these genetic elements, and may allow adaption to specific ecological niches and consequently enhance survival. Genome sequencing of 16 Lactococcus lactis strains revealed the presence of 83 plasmids, including two megaplasmids. The limitations of Pacific Biosciences SMRT sequencing in detecting the total plasmid complement of lactococcal strains is examined, while a combined Illumina/SMRT sequencing approach is proposed to combat these issues. Comparative genome analysis of these plasmid sequences combined with other publicly available plasmid sequence data allowed the definition of the lactococcal plasmidome, and facilitated an investigation into (bio) technologically important plasmid-encoded traits such as conjugation, bacteriocin production, exopolysaccharide (EPS) production, and (bacterio) phage resistance.

Despite the persistent and costly problem caused by (bacterio)phage predation of Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3' end of the lysis module)-through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor (responsible for the initial attachment of the phage to the host cell) of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium.

Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9.

Lactococcus lactis strains, being intensely used in the dairy industry, are particularly vulnerable to members of the so-called 936 group of phages. Sanitization and disinfection using purpose-made biocidal solutions is a critical step in controlling phage contamination in such dairy processing plants. The susceptibility of 36 936 group phages to biocidal treatments was examined using 14 biocides and commercially available sanitizers. The targets of a number of these biocides were investigated by means of electron microscopic and proteomic analyses. The results from this study highlight significant variations in phage resistance to biocides among 936 phages. Furthermore, rather than possessing resistance to specific biocides or biocide types, biocide-resistant phages tend to possess a broad tolerance to multiple classes of antimicrobial compounds.

Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp., and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species.

The so-called 936-type phages are the most frequently encountered lactococcal phage species in dairy fermentations, where they cause slow or even failed fermentations with concomitant economic losses. Several dairy phage population studies, performed in different geographical locations, have detailed their dominance in dairy phage populations, while various phage-resistance mechanisms have been assessed in a bid to protect against this virulent phage group. The impact of thermal and chemical treatments on 936 phages is an important aspect for dairy technologists and has been assessed in several studies, and has indicated that these phages have adapted to better resist such treatments. The abundance of 936 phage genome sequences has permitted a focused view on genomic content and regions of variation, and the role of such variable regions in the evolution of these phages. Here, we present an overview on detection and global prevalence of the 936 phages, together with their tolerance to industrial treatments and anti-phage strategies. Furthermore, we present a comprehensive review on the comparative genomic analyses of members of this fascinating phage species.