Optimizing bacteriophage treatment of resistant Pseudomonas.

Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of administering phages in clinical settings, preferred over monophage treatment because of their ability to eliminate multiple bacterial strains and reduce resistance formation. In our study, we compare monophage applications and phage cocktails to our chosen method of phage sequential treatments. To do so, we isolated four novel bacteriophages capable of infecting Pseudomonas alcaligenes T3, a close relative of P. aeruginosa, and characterized them using sequencing and transmission electron microscopy. While investigating monophage treatments, we observed that different phage concentrations had a strong impact on the timing and amount of resistance formation. When using phage cocktails, we observed that P. alcaligenes were capable of forming resistance in the same timespan it took them to become resistant to single phages. We isolated mutants resistant to each single phage as well as mutants exposed to phage cocktails, resulting in bacteria resistant to all four phages at once. Sequencing these mutants showed that different treatments yielded unique single nucleotide polymorphism mutation patterns. In order to combat resistance formation, we added phages one by one in intervals of 24 h, thus managing to delay resistance development and keeping bacterial growth significantly lower compared to phage cocktails.IMPORTANCEWHO declared antimicrobial resistance a top threat to global health; while antibiotics have stood at the forefront in the fight against bacterial infection, the increasing number of multidrug-resistant bacteria highlights a need to branch out in order to address the threat of antimicrobial resistance. Bacteriophages, viruses solely infecting bacteria, could present a solution due to their abundance, versatility, and adaptability. For this study, we isolated new phages infecting a fast-mutating Pseudomonas alcaligenes strain capable of forming resistance within 30 h. By using a sequential treatment approach of adding one phage after another, we were able to curb bacterial growth significantly more compared to state-of-the-art phage cocktails.

Maribacter halichondriae sp. nov., isolated from the marine sponge Halichondria panicea, displays features of a sponge-associated life style.

A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-ß-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).

Adaptive lifestyle of bacteria determines phage-bacteria interaction.

Bacteriophages and their interactions with microbes are not well understood. As a first step toward achieving a better understanding, we isolated and sequenced the Curvibacter phage PCA1 for the purpose of eliminating Curvibacter sp. AEP1.3, the main colonizer of Hydra vulgaris AEP. Our experiments showed that PCA1 phage caused a strong, virulent infection only in sessile Curvibacter sp. AEP1.3 but was unable to infect planktonic and host-associated bacterial cells of the same strain. In an effort to investigate this phenomenon, we compared sessile, planktonic, and host-associated bacteria via RNA sequencing and found that all three states differed significantly in their expression patterns. This finding led us to propose that the adaptive lifestyle of Curvibacter sp. AEP1.3 results in varying degrees of susceptibility to bacteriophage infection. This concept could be relevant for phage research and phage therapy in particular. Finally, we were able to induce phage infection in planktonic cells and pinpoint the infection process to a membrane protein. We further identified potential phage-binding protein candidates based on expression pattern analysis.

Micromonospora tarapacensis sp. nov., a bacterium isolated from a hypersaline lake.

Strain Llam7T was isolated from microbial mat samples from the hypersaline lake Salar de Llamará, located in Taracapá region in the hyper-arid core of the Atacama Desert (Chile). Phenotypic, chemotaxonomic and genomic traits were studied. Phylogenetic analyses based on 16S rRNA gene sequences assigned the strain to the family Micromonosporaceae with affiliation to the genera Micromonospora and Salinispora. Major fatty acids were C17 : 1ω8c, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The cell walls contained meso-diaminopimelic acid and ll-2,6 diaminopimelic acid (ll-DAP), while major whole-cell sugars were glucose, mannose, xylose and ribose. The major menaquinones were MK-9(H4) and MK-9(H6). As polar lipids phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and several unidentified lipids, i.e. two glycolipids, one aminolipid, three phospholipids, one aminoglycolipid and one phosphoglycolipid, were detected. Genome sequencing revealed a genome size of 6.894 Mb and a DNA G+C content of 71.4 mol%. Phylogenetic analyses with complete genome sequences positioned strain Llam7T within the family Micromonosporaceae forming a distinct cluster with Micromonospora (former Xiangella) phaseoli DSM 45730T. This cluster is related to Micromonospora pelagivivens KJ-029T, Micromonospora craterilacus NA12T, and Micromonospora craniellae LHW63014T as well as to all members of the former genera Verrucosispora and Jishengella, which were re-classified as members of the genus Micromonospora, forming a clade distinct from the genus Salinispora. Pairwise whole genome average nucleotide identity (ANI) values, digital DNA-DNA hybridization (dDDH) values, the presence of the diamino acid ll-DAP, and the composition of whole sugars and polar lipids indicate that Llam7T represents a novel species, for which the name Micromonospora tarapacensis sp. nov. is proposed, with Llam7T (=DSM 109510T,=LMG 31023T) as the type strain.