Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov., Two Multidrug-Resistant Psychrotrophic Species Isolated From Antarctica.

Despite unfavorable Antarctic conditions, such as cold temperatures, freeze-thaw cycles, high ultraviolet radiation, dryness and lack of nutrients, microorganisms were able to adapt and surprisingly thrive in this environment. In this study, eight cold-adapted Flavobacterium strains isolated from a remote Antarctic island, James Ross Island, were studied using a polyphasic taxonomic approach to determine their taxonomic position. Phylogenetic analyses based on the 16S rRNA gene and 92 core genes clearly showed that these strains formed two distinct phylogenetic clusters comprising three and five strains, with average nucleotide identities significantly below 90% between both proposed species as well as between their closest phylogenetic relatives. Phenotyping revealed a unique pattern of biochemical and physiological characteristics enabling differentiation from the closest phylogenetically related Flavobacterium spp. Chemotaxonomic analyses showed that type strains P4023T and P7388T were characterized by the major polyamine sym-homospermidine and a quinone system containing predominantly menaquinone MK-6. In the polar lipid profile phosphatidylethanolamine, an ornithine lipid and two unidentified lipids lacking a functional group were detected as major lipids. These characteristics along with fatty acid profiles confirmed that these species belong to the genus Flavobacterium. Thorough genomic analysis revealed the presence of numerous cold-inducible or cold-adaptation associated genes, such as cold-shock proteins, proteorhodopsin, carotenoid biosynthetic genes or oxidative-stress response genes. Genomes of type strains surprisingly harbored multiple prophages, with many of them predicted to be active. Genome-mining identified biosynthetic gene clusters in type strain genomes with a majority not matching any known clusters which supports further exploratory research possibilities involving these psychrotrophic bacteria. Antibiotic susceptibility testing revealed a pattern of multidrug-resistant phenotypes that were correlated with in silico antibiotic resistance prediction. Interestingly, while typical resistance finder tools failed to detect genes responsible for antibiotic resistance, genomic prediction confirmed a multidrug-resistant profile and suggested even broader resistance than tested. Results of this study confirmed and thoroughly characterized two novel psychrotrophic Flavobacterium species, for which the names Flavobacterium flabelliforme sp. nov. and Flavobacterium geliluteum sp. nov. are proposed.

Streptomyces tardus sp. nov.: A Slow-Growing Actinobacterium Producing Candicidin, Isolated From Sediments of the Trondheim Fjord.

Marine environments are home to an extensive number of microorganisms, many of which remain unexplored for taxonomic novelty and functional capabilities. In this study, a slow-growing Streptomyces strain expressing unique genomic and phenotypic characteristics, P38-E01 T , was described using a polyphasic taxonomic approach. This strain is part of a collection of over 8,000 marine Actinobacteria isolates collected in the Trondheim fjord of Norway by SINTEF Industry (Trondheim, Norway) and the Norwegian University of Science and Technology (NTNU, Trondheim, Norway). Strain P38-E01 T was isolated from the sediments of the Trondheim fjord, and phylogenetic analyses affiliated this strain with the genus Streptomyces, but it was not closely affiliated with other described species. The closest related type strains were Streptomyces daliensis YIM 31724 T (98.6%), Streptomyces rimosus subsp. rimosus ATCC 10970 T (98.4%), and Streptomyces sclerotialus NRRL ISP-5269 T (98.3%). Predominant fatty acids were C16:0 iso, C16:0, and Summed Feature 3, and the predominant respiratory quinones were MK-10(H6), MK-10(H4), and MK9(H4). The main polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphoglycolipid. The whole-cell sugars were glucose, ribose, and in minor amounts, mannose. The cell wall peptidoglycan contained LL-diaminopimelic acid. The draft genome has a size of 6.16 Mb, with a %G + C content of 71.4% and is predicted to contain at least 19 biosynthetic gene clusters encoding diverse secondary metabolites. Strain P38-E01 T was found to inhibit the growth of the pathogenic yeast Candida albicans ATCC 90028 and a number of Gram-positive bacterial human and plant pathogens. Metabolites extracted from cultures of P38-E01 T were analyzed by mass spectrometry, and it was found that the isolate produced the antifungal compound candicidin. Phenotypic and chemotaxonomic signatures, along with phylogenetic analyses, distinguished isolate P38-E01 T from its closest neighbors; thus, this isolate represents a novel species of the genus Streptomyces for which the name Streptomyces tardus sp. nov. (P38-E01 T = CCM 9049 T = DSM 111582 T ) is proposed.

Streptomyces poriferorum sp. nov., a novel marine sponge-derived Actinobacteria species expressing anti-MRSA activity.

Marine sponges represent a rich source of uncharacterized microbial diversity, and many are host to microorganisms that produce biologically active specialized metabolites. Here, a polyphasic approach was used to characterize two Actinobacteria strains, P01-B04T and P01-F02, that were isolated from the marine sponges Geodia barretti (Bowerbank, 1858) and Antho dichotoma (Esper, 1794), respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains P01-B04T and P01-F02 are closely related to Streptomyces beijiangensis DSM 41794T, Streptomyces laculatispora NRRL B-24909T, and Streptomyces brevispora NRRL B-24910T. The two strains showed nearly identical 16S rRNA gene sequences (99.93%), and the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values were 99.96% and 99.6%, respectively, suggesting that these strains are affiliated with the same species. Chemotaxonomic and culture characteristics of both strains were also consistent with the genus Streptomyces, while phenotypic properties, genome-based comparisons, and phylogenomic analyses distinguished strains P01-B04T and P01-F02 from their closest phylogenetic relatives. In silico analysis predicted that the 8.9 Mb genome of P01-B04T contains at least 41 biosynthetic gene clusters (BGCs) encoding secondary metabolites, indicating that this strain could express diverse bioactive metabolites; in support of this prediction, this strain expressed antibacterial activity against Gram-positive bacteria including a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) EAMC30. Based on these results, the marine sponge-associated isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces poriferorum sp. nov. is proposed, with P01-B04T (=DSM 111306T = CCM 9048T) as the type strain.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.

Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity.

Ultraviolet germicidal irradiation of influenza-contaminated N95 filtering facepiece respirators.

BACKGROUND: Safe and effective decontamination and reuse of N95 filtering facepiece respirators (FFRs) has the potential to significantly extend FFR holdings, mitigating a potential shortage due to an influenza pandemic or other pandemic events. Ultraviolet germicidal irradiation (UVGI) has been shown to be effective for decontaminating influenza-contaminated FFRs. This study aims to build on past research by evaluating the UVGI decontamination efficiency of influenza-contaminated FFRs in the presence of soiling agents using an optimized UVGI dose. METHODS: Twelve samples each of 15 N95 FFR models were contaminated with H1N1 influenza (facepiece and strap), then covered with a soiling agent-artificial saliva or artificial skin oil. For each soiling agent, 3 contaminated FFRs were treated with 1 J/cm2 UVGI for approximately 1 minute, whereas 3 other contaminated FFRs remained untreated. All contaminated surfaces were cut out and virus extracted. Viable influenza was quantified using a median tissue culture infectious dose assay. RESULTS: Significant reductions (≥3 log) in influenza viability for both soiling conditions were observed on facepieces from 12 of 15 FFR models and straps from 7 of 15 FFR models. CONCLUSIONS: These data suggest that FFR decontamination and reuse using UVGI can be effective. Implementation of a UVGI method will require careful consideration of FFR model, material type, and design.

Assessment of half-mask elastomeric respirator and powered air-purifying respirator reprocessing for an influenza pandemic.

BACKGROUND: Health care facilities are considering the use of reusable respiratory protective devices (RPDs) to mitigate a potential N95 filtering facepiece respirator shortage caused by an influenza pandemic. US regulators are also considering stockpiling reusable RPDs for pandemic preparedness, but limited data exist on the effectiveness of cleaning and disinfection of these devices. This study defines reprocessing protocols and evaluates their effectiveness against a pandemic influenza strain in a laboratory setting. METHODS: Five half-mask elastomeric respirator models and 3 powered air-purifying respirator models were contaminated with influenza virus and artificial skin oil on multiple surfaces. RPDs were then manually treated with 1 of 2 methods: cleaned or cleaned and disinfected. Presence of viable influenza was determined via swab sampling and a median tissue culture infectious dose assay. RESULTS: Across 41 RPD surfaces, a mean log reduction in viable influenza of 4.54 ± 0.97 log10 median tissue culture infectious dose was achieved for all treated surfaces, which included both cleaned and cleaned and disinfected surfaces. CONCLUSIONS: The methods defined as part of this study are effective for eliminating viable influenza in the presence of artificial skin oil on most of the RPD surfaces tested. Material type and RPD design should be considered when implementing RPD reprocessing protocols.