Thermorudis pharmacophila sp. nov., a novel member of the class Thermomicrobia isolated from geothermal soil, and emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus.

An aerobic, thermophilic and cellulolytic bacterium, designated strain WKT50.2T, was isolated from geothermal soil at Waikite, New Zealand. Strain WKT50.2T grew at 53-76 °C and at pH 5.9-8.2. The DNA G+C content was 58.4 mol%. The major fatty acids were 12-methyl C18 : 0 and C18 : 0. Polar lipids were all linked to long-chain 1,2-diols, and comprised 2-acylalkyldiol-1-O-phosphoinositol (diolPI), 2-acylalkyldiol-1-O-phosphoacylmannoside (diolP-acylMan), 2-acylalkyldiol-1-O-phosphoinositol acylmannoside (diolPI-acylMan) and 2-acylalkyldiol-1-O-phosphoinositol mannoside (diolPI-Man). Strain WKT50.2T utilized a range of cellulosic substrates, alcohols and organic acids for growth, but was unable to utilize monosaccharides. Robust growth of WKT50.2T was observed on protein derivatives. WKT50.2T was sensitive to ampicillin, chloramphenicol, kanamycin, neomycin, polymyxin B, streptomycin and vancomycin. Metronidazole, lasalocid A and trimethoprim stimulated growth. Phylogenetic analysis of 16S rRNA gene sequences showed that WKT50.2T belonged to the class Thermomicrobia within the phylum Chloroflexi, and was most closely related to Thermorudis peleae KI4T (99.6% similarity). DNA-DNA hybridization between WKT50.2T and Thermorudis peleae DSM 27169T was 18.0%. Physiological and biochemical tests confirmed the phenotypic and genotypic differentiation of strain WKT50.2T from Thermorudis peleae KI4T and other members of the Thermomicrobia. On the basis of its phylogenetic position and phenotypic characteristics, we propose that strain WKT50.2T represents a novel species, for which the name Thermorudis pharmacophila sp. nov. is proposed, with the type strain WKT50.2T ( = DSM 26011T = ICMP 20042T). Emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus are also proposed, and include the description of a novel respiratory quinone, MK-8 2,3-epoxide (23%), in Thermomicrobium roseum.

Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture.

Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na(+)/K(+) ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior.

Limisphaera ngatamarikiensis gen. nov., sp. nov., a thermophilic, pink-pigmented coccus isolated from subaqueous mud of a geothermal hotspring.

A novel bacterial strain, NGM72.4(T), was isolated from a hot spring in the Ngatamariki geothermal field, New Zealand. Phylogenetic analysis based on 16S rRNA gene sequences grouped it into the phylum Verrucomicrobia and class level group 3 (also known as OPB35 soil group). NGM72.4(T) stained Gram-negative, and was catalase- and oxidase-positive. Cells were small cocci, 0.5-0.8 µm in diameter, which were motile by means of single flagella. Transmission electron micrograph (TEM) imaging showed an unusual pirellulosome-like intracytoplasmic membrane. The peptidoglycan content was very small with only trace levels of diaminopimelic acid detected. No peptidoglycan structure was visible in TEM imaging. The predominant isoprenoid quinone was MK-7 (92%). The major fatty acids (>15%) were C(16 : 0), anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0). Major phospholipids were phosphatidylethanolamine (PE), phosphatidylmonomethylethanolamine (PMME) and cardiolipin (CL), and a novel analogous series of phospholipids where diacylglycerol was replaced with diacylserinol (sPE, sPMME, sCL). The DNA G+C content was 65.6 mol%. Cells displayed an oxidative chemoheterotrophic metabolism. NGM72.4(T) is a strictly aerobic thermophile (growth optimum 60-65 °C), has a slightly alkaliphilic pH growth optimum (optimum pH 8.1-8.4) and has a NaCl tolerance of up to 8 g l(-1). Colonies were small, circular and pigmented pale pink. The distinct phylogenetic position and phenotypic traits of strain NGM72.4(T) distinguish it from all other described species of the phylum Verrucomicrobia and, therefore, it is considered to represent a novel species in a new genus for which we propose the name Limisphaera ngatamarikiensis gen. nov., sp. nov. The type strain is NGM72.4(T) ( = ICMP 20182(T) = DSM 27329(T)).

Thermoflavifilum aggregans gen. nov., sp. nov., a thermophilic and slightly halophilic filamentous bacterium from the phylum Bacteroidetes.

A strictly aerobic, thermophilic, moderately acidophilic, non-spore-forming bacterium, strain P373(T), was isolated from geothermally heated soil at Waikite, New Zealand. Cells were filamentous rods, 0.2-0.4 µm in diameter and grew in chains up to 80 µm in length. On the basis of 16S rRNA gene sequence similarity, strain P373(T) was shown to belong to the family Chitinophagaceae (class Sphingobacteriia) of the phylum Bacteroidetes, with the most closely related cultivated strain, Chitinophaga pinensis UQM 2034(T), having 87.6 % sequence similarity. Cells stained Gram-negative, and were catalase- and oxidase-positive. The major fatty acids were i-15 : 0 (10.8 %), i-17 : 0 (24.5 %) and i-17 : 0 3-OH (35.2 %). Primary lipids were phosphatidylethanolamine, two unidentified aminolipids and three other unidentified polar lipids. The presence of sulfonolipids (N-acyl-capnines) was observed in the total lipid extract by mass spectrometry. The G+C content of the genomic DNA was 47.3 mol% and the primary respiratory quinone was MK-7. Strain P373(T) grew at 35-63 °C with an optimum temperature of 60 °C, and at pH 5.5-8.7 with an optimum growth pH of 7.3-7.4. NaCl tolerance was up to 5 % (w/v) with an optimum of 0.1-0.25 % (w/v). Cell colonies were non-translucent and pigmented vivid yellow-orange. Cells displayed an oxidative chemoheterotrophic metabolism. The distinct phylogenetic position and the phenotypic characteristics separate strain P373(T) from all other members of the phylum Bacteroidetes and indicate that it represents a novel species in a new genus, for which the name Thermoflavifilum aggregans gen. nov., sp. nov. is proposed. The type strain of the type species is P373(T) ( = ICMP 20041(T) = DSM 27268(T)).

Triketones active against antibiotic-resistant bacteria: synthesis, structure-activity relationships, and mode of action.

A series of acylated phloroglucinols and triketones was synthesized and tested for activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and multi-drug-resistant Mycobacterium tuberculosis (MDR-TB). A tetra-methylated triketone with a C12 side chain was the most active compound (MIC of around 1.0 microg/ml against MRSA) and was shown to stimulate oxygen consumption by resting cell suspensions, suggesting that the primary target was the cytoplasmic membrane.