Characterisation of the Novel Filamentous Phage PMBT54 Infecting the Milk Spoilage Bacteria Pseudomonas carnis and Pseudomonas lactis.

Filamentous bacteriophages are lysogenic and pseudo-lysogenic viruses that do not lyse their host but are often continuously secreted from the infected cell. They belong to the order Tubulavirales, which encompasses three families, with the Inoviridae being the largest. While the number of identified inoviral sequences has greatly increased in recent years due to metagenomic studies, morphological and physiological characterisation is still restricted to only a few members of the filamentous phages. Here, we describe the novel filamentous phage PMBT54, which infects the spoilage-relevant Pseudomonas species P. carnis and P. lactis. Its genome is 7320 bp in size, has a mol% GC content of 48.37, and codes for 13 open-reading frames, two of which are located on the (-) strand. The virion exhibits a typical filamentous morphology and is secreted from the host cell at various lengths. The phage was shown to promote biofilm formation in both host strains and, therefore, has potential implications for milk spoilage, as biofilms are a major concern in the dairy industry.

Pseudomonas rustica sp. nov., isolated from bulk tank raw milk at a German dairy farm.

Here we present the description of a novel Pseudomonas species, designated Pseudomonas rustica sp. nov., which was isolated from raw milk samples obtained from Germany. Results of initial 16S rRNA gene sequence analysis assigned the strain into the genus Pseudomonas and showed Pseudomonas helmanticensis, Pseudomonas neuropathica and Pseudomonas atagonensis to be its closest relatives. Further studies including sequence analysis of the rpoB gene, multi-gene phylogenetic tree reconstruction, whole-genome sequence comparisons, cellular fatty acid analysis and chemotaxonomic characterization showed a clear separation from the known Pseudomonas species. Isolate MBT-4T was closely related to Pseudomonas helmanticensis, 'Pseudomonas crudilactis' and Pseudomonas neuropathica with average nucleotide identities based on blast values of 88.8, 88.8 and 88.6%, respectively. Therefore, the strain can be classified into the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens group. The G+C content of strain MBT-4T was 58.9 mol%. The strain was catalase- and oxidase-positive, while the ß-galactosidase reaction was negative. Growth occurred between 4 and 30 °C and at pH values from pH 6.0 to 8.0. In conclusion, strain MBT-4T belongs to a novel species, for which the name Pseudomonas rustica sp. nov. is proposed. The type strain is MBT-4T (=DSM 112348T=LMG 32241T) and strain MBT-17 is also a representative of this species.

Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov., isolated from raw milk in Germany.

In this study, nine Gram-negative, motile and rod-shaped bacteria were isolated during a Germany-wide investigation of raw milk microbiota. The strains could be differentiated from their closest relatives by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. Strains MBT-1T, MBT-8, MBT-9, MBT-10, MBT-11 and MBT-12 were related to the Pseudomonas chlororaphis subgroup. Isolates MBT-2T, MBT-13 and MBT-14 were closely related to Pseudomonas rhizosphaerae DSM 16299T with an ANIb of 88.2 % and a genome-to-genome distance result of 36.0 %. The G+C content of the DNA of strains MBT-1T and MBT-2T was 60.84 and 62.48 mol%, respectively. The major fatty acids were C16 : 1 ω7c (summed feature 3), C16 : 0 and C18 : 1 ω7c (summed feature 8). The strains were catalase-positive, while production of urease, ß-galactosidase and indole were negative. Growth occurred at 4-30 °C and at pH values of pH 6.0-8.0. Based on these results, we conclude that the strains belong to two novel species, for which the names Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov. are proposed. The type strains are MBT-1T (=DSM 111668 T= LMG 31954T) and MBT-2T (=DSM 111761 T=LMG 31955T).

Genome sequence of the novel virulent bacteriophage PMBT14 with lytic activity against Pseudomonas fluorescens DSM 50090R.

Psychrotrophic gram-negative Pseudomonas spp. represent a serious problem in the dairy industry as they can cause spoilage of milk and dairy products. Bacteriophages have moved into focus as promising biocontrol agents for such food spoilage bacteria. The virulent Siphoviridae phage PMBT14 was isolated on a mutant variant of P. fluorescens DSM 50090 challenged with an unrelated virulent P. fluorescens DSM 50090 Podoviridae phage (i.e., mutant strain DSM 50090R). PMBT14 has a 47,820-bp dsDNA genome with 76 predicted open reading frames (ORFs). Its genome shows no significant sequence similarity to that of known phages, suggesting that PMBT14 represents a novel phage. Phage PMBT14 could be a promising biocontrol agent for P. fluorescens in milk or dairy foods.