Improved bactericidal efficacy and thermostability of Staphylococcus aureus-specific bacteriophage SA3821 by repeated sodium pyrophosphate challenges

As antibiotic resistance is being a threat to public health worldwide, bacteriophages are re-highlighted as alternative antimicrobials to fight with pathogens. Various wild-type phages isolated from diverse sources have been tested, but potential mutant phages generated by genome engineering or random mutagenesis are drawing increasing attention. Here, we applied a chelating agent, sodium pyrophosphate, to the staphylococcal temperate Siphoviridae phage SA3821 to introduce random mutations. Through 30 sequential sodium pyrophosphate challenges and random selections, the suspected mutant phage SA3821M was isolated. SA3821M maintained an intact virion morphology, but exhibited better bactericidal activity against its host Staphylococcous aureus CCARM 3821 for up to 17 h and thermostability than its parent, SA3821. Sodium pyrophosphate-mediated mutations in SA3821M were absent in lysogenic development genes but concentrated (83.9%) in genes related to the phage tail, particularly in the tail tape measure protein, indicating that changes in the tail module might have been responsible for the altered traits. This intentional random mutagenesis through controlled treatments with sodium pyrophosphate could be applied to other phages as a simple but potent method to improve their traits as alternative antimicrobials.

The Novel Halovirus Hardycor1, and the Presence of Active (Induced) Proviruses in Four Haloarchaea

The virus Hardycor1 was isolated in 1998 and infects the haloarchaeon Halorubrum coriense. DNA from a frozen stock (HC1) was sequenced and the viral genome found to be 45,142 bp of dsDNA, probably having redundant, circularly permuted termini. The genome showed little similarity (BLASTn) to known viruses. Only twenty-two of the 53 (41%) predicted proteins were significantly similar to sequences in the NCBI nr protein database (E-value ≤ 10−15). Six caudovirus-like proteins were encoded, including large subunit terminase (TerL), major capsid protein (Mcp) and tape measure protein (Tmp). Hardycor1 was predicted to be a siphovirus (VIRFAM). No close relationship to other viruses was found using phylogenetic tree reconstructions based on TerL and Mcp. Unexpectedly, the sequenced virus stock HC1 also revealed two induced proviruses of the host: a siphovirus (Humcor1) and a pleolipovirus (Humcor2). A re-examination of other similarly sequenced, archival virus stocks revealed induced proviruses of Haloferax volcanii, Haloferax gibbonsii and Haloarcula hispanica, three of which were pleolipoviruses. One provirus (Halfvol2) of Hfx. volcanii showed little similarity (BLASTn) to known viruses and probably represents a novel virus group. The attP sequences of many pleolipoproviruses were found to be embedded in a newly detected coding sequence, split in the provirus state, that spans between genes for integrase and a downstream CxxC-motif protein. This gene might play an important role in regulation of the temperate state.

First Report of aacC5-aadA7Δ4 Gene Cassette Array and Phage Tail Tape Measure Protein on Class 1 Integrons of Campylobacter Species Isolated from Animal and Human Sources in Egypt

Campylobacter species are common commensals in the gastrointestinal tract of livestock animals; thus, animal-to-human transmission occurs frequently. We investigated for the first time, class 1 integrons and associated gene cassettes among pan drug-resistant (PDR), extensively drug-resistant (XDR), and multidrug-resistant (MDR) Campylobacter species isolated from livestock animals and humans in Egypt. Campylobacter species were detected in 58.11% of the analyzed chicken samples represented as 67.53% Campylobacter jejuni(C. jejuni) and 32.47% Campylobacter coli (C. coli). C. jejuni isolates were reported in 51.42%, 74.28%, and 66.67% of examined minced meat, raw milk, and human stool samples, respectively. Variable antimicrobial resistance phenotypes; PDR (2.55%), XDR (68.94%), and MDR (28.5%) campylobacters were reported. Molecular analysis revealed that 97.36% of examined campylobacters were integrase gene-positive; all harbored the class 1 integrons, except one possessed an empty integron structure. DNA sequence analysis revealed the predominance of aadA (81.08%) and dfrA (67.56%) alleles accounting for resistance to aminoglycosides and trimethoprim, respectively. This is the first report of aacC5-aadA7Δ4 gene cassette array and a putative phage tail tape measure protein on class 1 integrons of Campylobacter isolates. Evidence from this study showed the possibility of Campylobacter–bacteriophage interactions and treatment failure in animals and humans due to horizontal gene transfer mediated by class 1 integrons.

Conserved and Diverse Traits of Adhesion Devices from Siphoviridae Recognizing Proteinaceous or Saccharidic Receptors

Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails’ distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host’s surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae.

Lactococcus Ceduovirus Phages Isolated from Industrial Dairy Plants—From Physiological to Genomic Analyses

Lactococcus Ceduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new Lactococcus lactis Ceduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.

Different genetic and morphological outcomes for phages targeted by single or multiple CRISPR-Cas spacers

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

The Tape Measure Protein Is Involved in the Heat Stability ofLactococcus lactisPhages

ABSTRACTVirulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a fewLactococcus lactisphages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of theLactococcus lactis936 [nowSk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-typeL. lactisphages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of theSk1virusgroup.IMPORTANCEVirulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a fewLactococcus lactisphages, members of theSk1virusgroup, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulentSk1virusphage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.

Genome Sequences of Gordonia Phages Hotorobo, Woes, and Monty

Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date.