Draft Genome Sequences of the Bap-Producing Strain Staphylococcus aureus V329 and Its Derived Phage-Resistant Mutant BIM-1

This study reports the draft genome sequences of Staphylococcus aureus V329, a Bap-producing strain isolated from a case of subclinical bovine mastitis in Spain, and a derived mutant (BIM-1) resistant to phage phiIPLA-RODI. Comparison of the two genomes revealed that the mutant strain has a point mutation in the gene tagO .

Phenotypic and Genotypic Characterization of Novel Polyvalent Bacteriophages With Potent In Vitro Activity Against an International Collection of Genetically Diverse Staphylococcus aureus

Phage therapy recently passed a key milestone with success of the first regulated clinical trial using systemic administration. In this single-arm non-comparative safety study, phages were administered intravenously to patients with invasive Staphylococcus aureus infections with no adverse reactions reported. Here, we examined features of 78 lytic S. aureus phages, most of which were propagated using a S. carnosus host modified to be broadly susceptible to staphylococcal phage infection. Use of this host eliminates the threat of contamination with staphylococcal prophage — the main vector of S. aureus horizontal gene transfer. We determined the host range of these phages against an international collection of 185 S. aureus isolates with 56 different multilocus sequence types that included multiple representatives of all epidemic MRSA and MSSA clonal complexes. Forty of our 78 phages were able to infect > 90% of study isolates, 15 were able to infect > 95%, and two could infect all 184 clinical isolates, but not a phage-resistant mutant generated in a previous study. We selected the 10 phages with the widest host range for in vitro characterization by planktonic culture time-kill analysis against four isolates:- modified S. carnosus strain TM300H, methicillin-sensitive isolates D329 and 15981, and MRSA isolate 252. Six of these 10 phages were able to rapidly kill, reducing cell numbers of at least three isolates. The four best-performing phages, in this assay, were further shown to be highly effective in reducing 48 h biofilms on polystyrene formed by eight ST22 and eight ST36 MRSA isolates. Genomes of 22 of the widest host-range phages showed they belonged to the Twortvirinae subfamily of the order Caudovirales in three main groups corresponding to Silviavirus, and two distinct groups of Kayvirus. These genomes assembled as single-linear dsDNAs with an average length of 140 kb and a GC content of c. 30%. Phages that could infect > 96% of S. aureus isolates were found in all three groups, and these have great potential as therapeutic candidates if, in future studies, they can be formulated to maximize their efficacy and eliminate emergence of phage resistance by using appropriate combinations.

Homburgvirus LP-018 Has a Unique Ability to Infect Phage-Resistant Listeria monocytogenes

Listeria phage LP-018 is the only phage from a diverse collection of 120 phages able to form plaques on a phage-resistant Listeria monocytogenes strain lacking rhamnose in its cell wall teichoic acids. The aim of this study was to characterize phage LP-018 and to identify what types of mutations can confer resistance to LP-018. Whole genome sequencing and transmission electron microscopy revealed LP-018 to be a member of the Homburgvirus genus. One-step-growth curve analysis of LP-018 revealed an eclipse period of ~60–90 min and a burst size of ~2 PFU per infected cell. Despite slow growth and small burst size, LP-018 can inhibit the growth of Listeria monocytogenes at a high multiplicity of infection. Ten distinct LP-018-resistant mutants were isolated from infected Listeria monocytogenes 10403S and characterized by whole genome sequencing. In each mutant, a single mutation was identified in either the LMRG_00278 or LMRG_01613 encoding genes. Interesting, LP-018 was able to bind to a representative phage-resistant mutant with a mutation in each gene, suggesting these mutations confer resistance through a mechanism independent of adsorption inhibition. Despite forming plaques on the rhamnose deficient 10403S mutant, LP-018 showed reduced binding efficiency, and we did not observe inhibition of the strain under the conditions tested. Two mutants of LP-018 were also isolated and characterized, one with a single SNP in a gene encoding a BppU domain protein that likely alters its host range. LP-018 is shown to be a unique Listeria phage that, with additional evaluation, may be useful in biocontrol applications that aim to reduce the emergence of phage resistance.

Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

ABSTRACTListeria-infecting phages are readily isolated fromListeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface ofListeria monocytogenesstrain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n= 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds toN-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminalN-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possessN-acetylglucosamine in their WTA.

Isolation of FC3-11, a bacteriophage specific for theKlebsiella pneumoniaeporin OmpK36, and its use for the isolation of porin-deficient mutants

FC3-11, a bacteriophage specific for the Klebsiella pneumoniae porin OmpK36, was isolated by its ability to infect Escherichia coli strains expressing the cloned OmpK36 porin. Porin OmpK36 was shown to be the receptor for phage FC3-11 by the observations that K. pneumoniae and E. coli strains that do not express OmpK36 were resistant to phage FC3-11, the purified porin inactivated the phage, and mutants selected for FC3-11 resistance had lost OmpK36. The outer membrane protein OmpK35 was isolated from a K. pneumoniae phage-resistant mutant by using porin isolation methods and was shown to contain an N-terminal sequence typical of enterobacterial porins. Bacteriophage FC3-11, alone or in combination with previously described lipopolysaccharide-specific phages, is a valuable tool to obtain OmpK36-porinless mutants.Key words: Klebsiella pneumoniae, porins, bacteriophage.

Characteristics of Erwinia amylovora bacteriophage and its possible role in the epidemiology of fire blight

Bacteriophage (S1) of Erwinia amylovora, isolated from soil at the base of fire-blight-infected trees, was characterized by small, clear plaques on E. amylovora strain PR1 and hazy plaques of the same size on a closely related, yellow, saprophytic bacterium, Y, which was isolated together with PR1 from the diseased trees. Phage S1 plated with optimal efficiency at 10C on PR1 and at 28C on Y was relatively unstable to storage at temperatures typical of summer and to ultraviolet (UV) irradiation, and lysogenized Y but not PR1. Pathogenicity tests in pear slices demonstrated that symptom development was (i) delayed when mixtures of either PR1 and phage S1 or PR1 and Y were inoculated, (ii) delayed when a culture of a phage-resistant mutant of PR1 was inoculated, and (iii) prevented from appearing at all when PR1 and the lysogenic form of Y, Y (S1), were inoculated together. Strain Y was easily lysogenized and Y (S1) released phage spontaneously and after UV irradiation. It is suggested that the yellow saprophyte, which is invariably isolated from fruit trees with E. amylovora, may frequently occur in its lysogenic form in nature and serve as a reservoir of phage which may exert some influence on the occurrence and severity of fire-blight disease.