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Flagellotropic bacteriophages are interesting candidates as therapeutics against pathogenic bacteria dependent on flagellar motility for colonization and causing disease. Yet, phage resistance other than loss of motility has been scarcely studied. Here we developed a soft agar assay to study flagellotropic phage F341 resistance in motile Campylobacter jejuni. We found that phage adsorption was prevented by diverse genetic mutations in the lipooligosaccharides forming the secondary receptor of phage F341. Genome sequencing showed phage F341 belongs to the Fletchervirus genus otherwise comprising capsular-dependent C. jejuni phages. Interestingly, phage F341 encodes a hybrid receptor binding protein (RBP) predicted as a short tail fiber showing partial similarity to RBP1 encoded by capsular-dependent Fletchervirus, but with a receptor binding domain similar to tail fiber protein H of C. jejuni CJIE1 prophages. Thus, C. jejuni prophages may represent a genetic pool from where lytic Fletchervirus phages can acquire new traits like recognition of new receptors.

Deazaguanine DNA modifications are widespread in phages, particularly in those with pathogenic hosts. Pseudomonas phage iggy substitutes ∼16.5% of its genomic 2'-deoxyguanosine (G) with dPreQ0, and the iggy deazaguanine transglycosylase (DpdA) is unique in having a strict GA target motif, not observed previously. The iggy PreQ0 modification is shown to provide protection against both restriction endonucleases and Cas9 (when present in PAM), thus expanding our understanding of the deazaguanine modification system, its potential, and diversity. Phage iggy represents a new genus of Pseudomonas phages within the Queuovirinae subfamily; which have very little in common with other published phage genomes in terms of nucleotide similarity (<10%) and common proteins (<2%). Interestingly, shared similarity is concentrated in dpdA and preQ0 biosynthesis genes. TEM imaging confirmed a siphovirus morphology with a prolate icosahedral head and a non-contractile flexible tail with one long central tail spike. The observed protective effect of the deazaguanine modification on the iggy DNA may contribute to its broad within-species host range. Phage iggy was isolated on Pseudomonas aeruginosa PAO1, but also infects PDO300, PAK, PA14, as well as 10 of 27 tested environmental isolates and 13 of 20 tested clinical isolates of P. aeruginosa from patients with cystic fibrosis.

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application.

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.

Ultraviolet C (UVC) radiation is a widely used technology for the disinfection of surfaces, air flows, water and other liquids. Although extensive research has been conducted on the UV tolerance of bacteriophages used as surrogates for waterborne viruses, limited information is available on phages relevant to food processing. Phages of dairy starters may reach high numbers in dairy facilities and cause fermentation failure with great economic losses for the dairy industry. Here, the UV tolerance of virulent phages, belonging to the 936-group (Skunavirus) of Lactococcus lactis subsp. diacetylactis F7/2, was assessed, employing both host infectivity loss and qPCR assays. A highly heat-tolerant phage (P680) and a less heat-tolerant phage (P008) were exposed to UV radiation at 265 nm (UVC), 285 nm (UVB) and 365 nm (UVA), respectively, in an aqueous suspension, using UV Light-Emitting-Diodes (LEDs) in a static set-up. UVC at 265 nm achieved the highest total inactivation, leading to a 4 log10 reduction of the phage titer at a UV dose of 327 and 164 mJ/cm2 for P680 and P008, respectively. UVB at 285 nm achieved similar inactivation levels, while UVA at 365 nm did not cause major reductions. Phages were also suspended in yoghurt serum of pH 5.5 and pH 7.0 and exposed to UVC radiation at 265 nm. The heat-tolerant phage P680 was more UV tolerant for all wavelengths, matrices and pH values tested. A higher aggregation degree together with less DNA damage was observed for both phages at pH 5.5, especially for phage P680, indicating a UV light-shielding effect. Interestingly, there were indications of some phage survivors exhibiting higher UV tolerance on re-exposure, pointing out a need for further investigation. Our results show that UV LEDs emitting at 265 nm and 285 nm are efficient in reducing the phage population significantly, but also underline that 936-type phages are relatively UV resistant. A further understanding of the main factors influenc

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.

Phase variation is a common mechanism for creating phenotypic heterogeneity of surface structures in bacteria important for niche adaptation. In Campylobacter, phase variation occurs by random variation in hypermutable homonucleotide 7–11 G (polyG) tracts. To elucidate how phages adapt to phase-variable hosts, we study Fletchervirus phages infecting Campylobacter dependent on a phase-variable receptor. Our data demonstrate that Fletcherviruses mimic their host and encode hypermutable polyG tracts, leading to phase-variable expression of two of four receptor-binding proteins. This creates phenotypically diverse phage populations, including a sub-population that infects the bacterial host when the phase-variable receptor is not expressed. Such population dynamics of both phage and host promote co-existence in a shared niche. Strikingly, we identify polyG tracts in more than 100 phage genera, infecting more than 70 bacterial species. Future experimental work may confirm phase variation as a widespread strategy for creating phenotypically diverse phage populations.

Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.

Acinetobacter baumanniiis a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistantA. baumanniistrain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates ofA. baumanniifrom a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages g