Complete genome sequence of the novel virulent phage PMBT24 infecting Enterocloster bolteae from the human gut.

PMBT24, the first reported virulent bacteriophage infecting the anaerobic human gut bacterium Enterocloster bolteae strain MBT-21, was isolated from a municipal sewage sample and its genome was sequenced and analysed. Transmission electron microscopy revealed a phage with an icosahedral head and a long, non-contractile tail. The circularly permutated, 99,962-bp dsDNA genome of the pac-type phage has a mol% G + C content of 32.1 and comprises 173 putative ORFs. Using amino acid sequence-based phylogeny, phage PMBT24 showed similarity to other, hitherto non-published phage genomes in the databases. Our data suggested phage PMBT24 to present the type phage of a novel genus (proposed name Kielvirus) and novel family of phages (proposed name Kielviridae).

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice.

Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.

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.

Novel Siphoviridae phage PMBT4 belonging to the group b Lactobacillus delbrueckii subsp. bulgaricus phages.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

Characterization of the first Pseudomonas grimontii bacteriophage, PMBT3.

The complete genome sequence of the virulent bacteriophage PMBT3, isolated on the proteolytic Pseudomonas grimontii strain MBTL2-21, showed no significant similarity to other known phage genome sequences, making this phage the first reported to infect a strain of P. grimontii. Electron microscopy revealed PMBT3 to be a member of the family Siphoviridae, with notably long and flexible whiskers. The linear, double-stranded genome of 87,196 bp has a mol% G+C content of 60.4 and contains 116 predicted protein-encoding genes. A putative tellurite resistance (terB) gene, originally reported to occur in the genome of a bacterium, was detected in the genome of phage PMBT3.

First Molecular Characterization of Siphoviridae-Like Bacteriophages Infecting Staphylococcus hyicus in a Case of Exudative Epidermitis.

Exudative epidermitis (EE), also known as greasy pig disease, is one of the most frequent skin diseases affecting piglets. Zoonotic infections in human occur. EE is primarily caused by virulent strains of Staphylococcus (S.) hyicus. Generally, antibiotic treatment of this pathogen is prone to decreasing success, due to the incremental development of multiple resistances of bacteria against antibiotics. Once approved, bacteriophages might offer interesting alternatives for environmental sanitation or individualized treatment, subject to the absence of virulence and antimicrobial resistance genes. However, genetic characterization of bacteriophages for S. hyicus has, so far, been missing. Therefore, we investigated a piglet raising farm with a stock problem due to EE. We isolated eleven phages from the environment and wash water of piglets diagnosed with the causative agent of EE, i.e., S. hyicus. The phages were morphologically characterized by electron microscopy, where they appeared Siphoviridae-like. The genomes of two phages were sequenced on a MiSeq instrument (Illumina), resulting in the identification of a new virulent phage, PITT-1 (PMBT8), and a temperate phage, PITT-5 (PMBT9). Sequencing of three host bacteria (S. hyicus) from one single farm revealed the presence of two different strains with genes coding for two different exfoliative toxin genes, i.e., exhA (2 strains) and exhC (1 strain). The exhC-positive S. hyicus strain was only weakly lysed by most lytic phages. The occurrence of different virulent S. hyicus strains in the same outbreak limits the prospects for successful phage treatment and argues for the simultaneous use of multiple and different phages attacking the same host.

Complete Genome Sequence of a Shiga Toxin-Producing Escherichia coli O26:H11 Strain (Sequence Type 21) and Two Draft Genome Sequences of Listeria monocytogenes Strains (Clonal Complex 1 [CC1] and CC59) Isolated from Fresh Produce in Germany.

The complete genome sequence of a Shiga toxin-producing Escherichia coli (STEC) O26:H11 strain, MBT-5 (sequence type 21 [ST21], stx 1a, stx 2a, eae, ehxA), and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1 (ST1, clonal complex 1 [CC1]) and 59 (ST59, CC59), respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.

Fast and Easy Phage-Tagging and Live/Dead Analysis for the Rapid Monitoring of Bacteriophage Infection.

Use of bacteriophages, which are viruses that kill bacteria, for biocontrol of pathogens and antimicrobial resistant bacteria has become increasingly important in recent years. As traditional culture-based methods are laborious and time-consuming, practicable use of bacteriophages will hinge on development of rapid and high throughput methods to analyze, characterize and screen large bacteriophage libraries. We thus established a novel method to fluorescently tag bacteriophages for virus screening and interaction studies, without the need for complicated and laborious purification procedures or genetic engineering of viruses to express fluorescent proteins. Bacteriophage PMBT14 was tagged using DNA dye Syto 13. Simply by using a membrane filter, tagged bacteriophages can be separated from non-sequestered excess dye rapidly, effortlessly, and cheaply. The procedure takes less than 30 min and makes use of simple laboratory consumables that are already commonly used for bacteriophage preparations. As proof of concept, we present here flow cytometric methods to analyze bacteriophage binding, infection and killing that are very accessible for high throughput analysis. We show that the resulting fluorescently tagged bacteriophage can be used to specifically stain its host bacterium Pseudomonas fluorescens DSM 50090. Individual fluorescent bacteriophages, their binding to and initial infection of bacteria could also be observed using confocal microscopy. The infection process was halted by the metabolic inhibitor sodium azide, suggesting a requirement of host metabolic processes for penetration by PMBT14. Flow cytometric live/dead assays was used as a complementary method to determine bacteriophage infection of its host. We made preliminary efforts to adapt the tagging method to two other bacteriophages and discuss potential pitfalls and solutions in the use of tagged phages. Fluorescent phage tagging has previously been demonstrated to facilitate analysis of bacteriophage-host interactions. The method adopted in this study makes it fast, easy as well as cost effective.

Genome analysis of the temperate bacteriophage PMBT6 residing in the genome of Bifidobacterium thermophilum MBT94004.

The Siphoviridae phage PMBT6 was identified by transmission electron microscopy in the supernatant of Bifidobacterium thermophilum MBT94004 bioreactor fermentation culture, where it occurred at a moderately high titer. Genome analysis of the bacterial DNA confirmed the presence of this prophage within the genome of the lysogenic host. Under laboratory conditions, the prophage could not be induced by mitomycin C, ultraviolet C irradiation or hydrogen peroxide, suggesting that the prophage was released by spontaneous induction under (yet unknown) bioreactor conditions. Genome sequencing of the virion resulted in a linear, double-stranded DNA molecule of 36,561 bp with a mol% G + C content of 61.7 and 61 predicted open reading frames with low similarity to other Bifidobacterium spp. genomes, confirming that PMBT6 represents a novel temperate phage for this genus.