Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

IMPORTANCE: Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

Effect of chronic and acute enterotoxigenic E. coli challenge on growth performance, intestinal inflammation, microbiome, and metabolome of weaned piglets.

Post-weaning enteropathies in swine caused by pathogenic E. coli, such as post-weaning diarrhea (PWD) or edema disease (ED), remain a significant problem for the swine industry. Reduction in the use of antibiotics over concerns of antibiotic resistance and public health concerns, necessitate the evaluation of effective antibiotic alternatives to prevent significant loss of livestock and/or reductions in swine growth performance. For this purpose, an appropriate piglet model of pathogenic E. coli enteropathy is required. In this study, we attempted to induce clinical signs of post-weaning disease in a piglet model using a one-time acute or lower daily chronic dose of a pathogenic E. coli strain containing genes for both heat stable and labile toxins, as well as Shiga toxin. The induced disease state was monitored by determining fecal shedding and colonization of the challenge strain, animal growth performance, cytokine levels, fecal calprotectin, histology, fecal metabolomics, and fecal microbiome shifts. The most informative analyses were colonization and shedding of the pathogen, serum cytokines, metabolomics, and targeted metagenomics to determine dysbiosis. Histopathological changes of the gastrointestinal (GI) tract and tight junction leakage as measured by fecal calprotectin concentrations were not observed. Chronic dosing was similar to the acute regimen suggesting that a high dose of pathogen, as used in many studies, may not be necessary. The piglet disease model presented here can be used to evaluate alternative PWD treatment options.

Sheep in wolves' clothing: Temperate T7-like bacteriophages and the origins of the Autographiviridae.

Bacteriophage T7 is an extensively studied virulent phage, and its taxonomic family, the Autographiviridae, is broadly synonymous with a strictly virulent lifestyle. It is difficult to imagine how a T7-like phage could function in a "domesticated" temperate lifestyle, in which it is incorporated into the host's genome. Here we describe two temperate T7-like bacteriophages: ProddE, a Desulfovibrio phage, and Pasto, an Agrobacterium phage. Each contains recognizable T7-like proteins in the canonical T7-like gene order, but with the addition of lysogeny gene modules. While ProddE contains a phage-like repressor, Pasto lysogeny appears to be controlled by a novel MarR-like transcriptional regulator. In addition, we identify similar T7-like prophage elements in a wide variety of Gram-negative bacterial genomes and a small number of Gram-positive genomes. Identification of these elements in diverse bacterial species raises interesting evolutionary questions about the origins of T7-like phages and which lifestyle, temperate or virulent, is the ancestral form.

Comparative Genomics of Three Novel Jumbo Bacteriophages Infecting Staphylococcus aureus.

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is ∼269 kb with each genome encoding ∼263 predicted protein-coding genes. Phage genome organization and content are similar to those of known jumbo phages of Bacillus sp., including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete virion and nonvirion RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates. IMPORTANCE This study describes the comparative genomics of the following three novel S. aureus jumbo phages: MarsHill, Madawaska, and Machias. These three S. aureus myophages represent an emerging class of S. aureus phage. These genomes contain abundant introns which show a pattern consistent with repeated acquisition rather than vertical inheritance, suggesting intron acquisition and loss are active processes in the evolution of these phages. These phages have presumably hypermodified DNA which inhibits sequencing by several different common platforms. Therefore, these phages also represent potential genomic diversity that has been missed due to the limitations of standard sequencing techniques. In particular, such hypermodified genomes may be missed by metagenomic studies due to their resistance to standard sequencing techniques. Phage MarsHill was found to be able to transduce host DNA at levels comparable to that found for other transducing S. aureus phages, making it a potential vector for horizontal gene transfer in the environment.

Coding-Complete Genome Sequence of Staphylococcus aureus Podophage Portland.

Staphylococcus aureus bacteria, especially the multidrug resistance strains, are responsible for a wide range of clinical infections. Here, we announce the genome sequence of S. aureus podophage Portland, which is closely related to a group of phi29-like S. aureus podophages, including phages phi44AHJD and phiP68. The exact genome sequence ends of phage Portland were not determined and may be obscured by terminal proteins.

Complete Genome Sequence of Staphylococcus aureus Myophage Maine.

Multidrug-resistant strains of Staphylococcus aureus cause serious human disease worldwide. Bacteriophages offer a promising alternative to traditional antibiotics. Here, we announce the 141,712-bp genome of S. aureus phage Maine. A myophage with 9,019-bp predicted terminal repeats and high similarity to other Staphylococcus phages, Maine falls into the Twort-like group.

Complete Genome Sequence of Staphylococcus aureus Siphophage Sebago.

Here, we introduce the genome of Sebago, a 43,878-bp siphophage that infects Staphylococcus aureus Sebago carries 70 proteins and is most closely related to StauST398, a Phietavirus.