Zhiheng Liu

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of ≈20,000 microbial extracts, 12 hits were identified with broad-spectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Phylogenetic study of the species within the family Streptomycetaceae

Species of the genus Streptomyces, which constitute the vast majority of taxa within the family Streptomycetaceae, are a predominant component of the microbial population in soils throughout the world and have been the subject of extensive isolation and screening efforts over the years because they are a major source of commercially and medically important secondary metabolites. Taxonomic characterization of Streptomyces strains has been a challenge due to the large number of described species, greater than any other microbial genus, resulting from academic and industrial activities. The methods used for characterization have evolved through several phases over the years from those based largely on morphological observations, to subsequent classifications based on numerical taxonomic analyses of standardized sets of phenotypic characters and, most recently, to the use of molecular phylogenetic analyses of gene sequences. The present phylogenetic study examines almost all described species (615 taxa) within the family Streptomycetaceae based on 16S rRNA gene sequences and illustrates the species diversity within this family, which is observed to contain 130 statistically supported clades, as well as many unsupported and single member clusters. Many of the observed clades are consistent with earlier morphological and numerical taxonomic studies, but it is apparent that insufficient variation is present in the 16S rRNA gene sequence within the species of this family to permit bootstrap-supported resolution of relationships between many of the individual clusters.

Saccharopolyspora spinosporotrichia sp. nov., a novel actinomycete from soil.

The generic position of an aerobic, Gram-positive, non-acid-alcohol-fast actinomycete was determined following isolation of the PCR-amplified 16S rRNA genes and alignment of the resultant sequence with corresponding sequences from representatives of the family Pseudonocardiaceae. The assignment of the organism to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strain was distinguished from representatives of validly described Saccharopolyspora species by a number of phenotypic properties. It is proposed that the organism, strain AS4.198T, be classified in the genus Saccharopolyspora as Saccharopolyspora spinosporotrichia sp. nov.

Proposal to combine the genera Actinobispora and Pseudonocardia in an emended genus Pseudonocardia, and description of Pseudonocardia zijingensis sp. nov..

The 16S rDNA sequences of four strains, i.e. three type strains of Actinobispora and strain 6330T, were determined and compared with those of representatives of the family Pseudonocardiaceae by using two tree-making algorithms. All the validly described species of the genera Actinobispora and Pseudonocardia were consistently recovered as a mixed group in phylogenetic trees, and were distinct from the other genera of the family Pseudonocardiaceae. Strain 6330T formed a distinct phyletic line in the 16S rDNA tree and was most closely associated with the type strain of Actinobispora aurantiaca. The use of specific PCR primers designed for differentiating the genus Pseudonocardia from other genera of the family Pseudonocardiaceae showed that all the Actinobispora species and strain 6330T have the same amplified 640 bp 16S rDNA fragment as members of the genus Pseudonocardia. The DNA-DNA relatedness, chemotaxonomic and phenotypic data also supported classification of these taxa in the genus Pseudonocardia, and distinguished each from the others. On the basis of these observations, it is proposed that the genera Actinobispora and Pseudonocardia be combined in an emended genus Pseudonocardia, and that strain 6330T be classified in the same genus as Pseudonocardia zijingensis sp. nov. The type strain is 6330T (= AS 4.1545T = JCM 11117T).

Nocardia beijingensis sp. nov., a novel isolate from soil.

The taxonomic status of a soil isolate, provisionally assigned to the genus Nocardia, was established following a polyphasic study. An almost complete sequence of the 16S rDNA of the strain was determined and aligned with available sequences for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organism was associated consistently with the type strains of Nocardia brasiliensis and Nocardia farcinica in the neighbour-joining analysis, albeit with low bootstrap values. It was also found to have chemotaxonomic and morphological properties consistent with its assignment to the genus Nocardia. The organism was readily separated from representatives of other Nocardia species by a broad range of phenotypic properties. The name proposed for the novel species is Nocardia beijingensis sp. nov. The type strain is AS4.1521T (= IFO 16342T = JCM 10666T).

BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the Genus streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus

The type strains of 451 validly described Streptomyces species were screened using the BOX-PCR fingerprint technique. Of the different primers tested, the BOX primer yielded the most robust and reproducible band patterns. The majority of the species (350) had an unique pattern each. A total number of 30 clusters were delineated comprising species with nearly identical patterns. Four of these clusters grouped synonymous Streptomyces species known from previous studies. A high correlation exists between BOX-PCR fingerprinting and DNA-DNA homology data. In the present study, the latter was confirmed for 7 additional BOX clusters and led to the following emended descriptions: S. cochleatus is a subjective synonym of S. cinereorectus, S. roseoflavus of S. fradiae, S. roseodiastaticus of S. tricolor, S. roseosporus of S. filamentosus, S. distallicus of S. colombiensis, S. arabicus of S. vinaceus and S. phaeoviridis of S. phaeopurpureus. Although not yet confirmed by DNA-DNA pairing studies, rep-PCR data suggested the presence of at least 33 additional junior synonyms.

Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., novel actinomycetes from soil

The generic position of two aerobic, Gram-positive, non-acid-alcohol-fast actinomycetes was established following the isolation of their PCR-amplified 16S rRNA genes and alignment of the resultant sequences with the corresponding sequences from representatives of the families Actinosynnemataceae and Pseudonocardiaceae. The assignment of the organisms to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strains were distinguished both from one another and from representatives of validly described Saccharopolyspora species on the basis of a number of phenotypic properties. It is proposed that the organisms, strains 07T (= AS4.1520T = IFO 16345T = JCM 10665T) and 216T (= AS4.1511T = IFO 16346T = JCM 10664T), be classified in the genus Saccharopolyspora as Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., respectively.

Amycolatopsis marina sp nov., an actinomycete isolated from an ocean sediment

A Gram-positive, aerobic, non-motile actinobacterium, designated strain Ms392A(T), was isolated from an ocean-sediment sample collected from the South China Sea. The isolate contained chemical markers that supported chemotaxonomic assignment to the genus Amycolatopsis. On the basis of an analysis of 16S rRNA gene sequence similarities, strain Ms392A(T) represents a novel subclade within the genus Amycolatopsis, with Amycolatopsis palatopharyngis 1BDZ(T) as its closest phylogenetic neighbour (99.4 % similarity). However, DNA-DNA hybridization demonstrated that strain Ms392A(T) was distinct from A. palatopharyngis AS 4.1729(T) (48.6 % relatedness). The polyphasic analysis demonstrated that the ocean isolate can be clearly distinguished from recognized species of the genus Amycolatopsis. Therefore, strain Ms392A(T) represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis marina sp. nov. is proposed. The type strain is Ms392A(T) (=CGMCC 4.3568(T) =NBRC 104263(T)).

The search for synonyms among streptomycetes by using SDS-PAGE of whole-cell proteins. Emendation of the species Streptomyces aurantiacus, Streptomyces cacaoi subsp. cacaoi, Streptomyces caeruleus and Streptomyces violaceus.

A collection of 93 Streptomyces reference strains were investigated using SDS-PAGE of whole-cell proteins. Computer-assisted numerical analysis revealed 24 clusters encompassing strains with very similar protein profiles. Five of them grouped several type strains with visually identical patterns. DNA-DNA hybridizations revealed homology values higher than 70% among these type strains. According to the current species concept, it is proposed that Streptomyces albosporeus subsp. albosporeus LMG 19403T is considered as a subjective synonym of Streptomyces aurantiacus LMG 19358T, that Streptomyces aminophilus LMG 19319T is considered as a subjective synonym of Streptomyces cacaoi subsp. cacaoi LMG 19320T, that Streptomyces niveus LMG 19395T and Streptomyces spheroides LMG 19392T are considered as subjective synonyms of Streptomyces caeruleus LMG 19399T, and that Streptomyces violatus LMG 19397T is considered as a subjective synonym of Streptomyces violaceus LMG 19360T.

Arthrobacter scleromae sp. nov. Isolated from Human Clinical Specimens

ABSTRACT A gram-positive, coryneform bacterium was isolated from swollen scleromata of a dermatosis patient. An analysis of its phenotypic, chemotaxonomic, and genotypic characteristics showed that this bacterium is closely associated with Arthrobacter oxydans and Arthrobacter polychromogenes but that it belongs to a distinct species, for which the name Arthrobacter scleromae sp. nov. is proposed.