Strain IFM Research Articles

Subtractive genomics integrated with deep learning, molecular docking, and MD simulation decipher therapeutic drug targets and their potential inhibitors against Nocardia farcinica strain IFM 10152

Subtractive genomics integrated with deep learning, molecular docking, and MD simulation decipher therapeutic drug targets and their potential inhibitors against Nocardia farcinica strain IFM 10152

Activation of Secondary Metabolism and Protease Activity Mechanisms in the Black Koji Mold Aspergillus luchuensis through Coculture with Animal Cells.

The activation of secondary metabolism plays a pivotal role in the discovery of novel natural products. We recently developed a coculture method involving actinomycetes and mouse macrophage-like cells to stimulate the production of bioactive compounds. A black koji mold, Aspergillus luchuensis IFM 61405, markedly enhanced the production of (3S,8R)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (1a), (3S,8S)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (1b), and (3S)-9-hydroxy-3-carboxy-2-methylenenonanoic acid (2) when coincubated with J774.1 mouse macrophage cells. The production of 1 and 2 increased by at least 3.5-fold and 2.7-fold, respectively, compared to monoculture after 7 days. A mechanistic investigation revealed that a protease from strain IFM 61405 plays a key role in enhancing the production of 1 and 2. This enhancement was not replicated in A. niger IFM 59706, a nonkoji mold, despite the presence of biosynthetic genes for 1 and 2 in A. niger IFM 59706. Furthermore, the addition of protease inhibitors suppressed the production of 1 and 2, suggesting that proteins secreted from animal cells, likely degraded by proteases secreted by strain IFM 61405, serve as precursors for 1 and 2. The results show that the strategy of coculturing koji mold with animal cells has the potential to enhance the production of natural products.

Nocardia sputorum sp. nov., an actinobacterium isolated from clinical specimens in Japan.

Two novel actinobacteria, designated IFM 12276T and IFM 12275, were isolated from clinical specimens in Japan, and their taxonomic positions were investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strains IFM 12276 T and IFM 12275 have completely identical 16S rRNA gene sequences and were closely related to members of the genus Nocardia. The highest 16S rRNA gene sequence similarity was observed to Nocardia beijingensis (99.6 %) and Nocarida sputi (99.6 %), followed by Nocardia niwae (99.3 %) and Nocardia araoensis (99.3 %). The whole-cell hydrolysates of strains IFM 12276T and IFM 12275 contained meso-diaminopimelic acid, arabinose and galactose. The acyl type of muramic acid was N-glycolyl. The predominant isoprenoid quinone was MK-8(H4, ω-cycl.) and the principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. Strains IFM 12276T and IFM 12275 contained mycolic acids that co-migrated with those from the type strain of N. niwae. These chemotaxonomic features corresponded to those of the genus Nocardia. Meanwhile, the differences in some phenotypic characteristics, along with the results of average nucleotide identity and digital DNA-DNA hybridization analyses, indicated that strains IFM 12276 T and IFM 12275 should be distinguished from the recognized species of the genus Nocardia. Therefore, these strains represent a novel species of the genus Nocardia, for which the name Nocardia sputorum sp. nov. is proposed. The type strain is IFM 12276T (=NBRC 115477T=TBRC 17096T).

Pulmonary Nocardia concava infection in a patient with mantle cell lymphoma receiving bortezomib and rituximab

A 75-year-old man presented to our hospital with a high fever, 39·5°C. 8 months previously, he had visited the haematology clinic for generalised lymphadenopathy. Whole body CT showed enlarged axillary and abdominal lymph nodes and splenomegaly, and a bone marrow biopsy showed lymphoma cell infiltration. He was diagnosed with metastatic mantle cell lymphoma and treated with rituximab and bortezomib. Treatment regimen consisted of rituximab 375 mg/m2 intravenously on day 1 and bortezomib 1·3 mg/m2 intravenously on days 1, 4, 8, and 11 for the first course in month 1, followed by five more courses with the same schedule for the next 5 months. At the end of treatment, CT showed that the lymphadenopathy had disappeared, and the lymphoma was in remission. 3 days before this scan, the patient developed a high fever (39·5°C). Blood tests showed an elevated white blood cell count (19 400 per μL vs 3300–8600 per μL expected range), with neutrophil predominance (95% vs 38·3–71·1% expected range) and high concentration of C-reactive protein (19·2 mg/dL vs <0·15 mg/dL expected range). Chest CT showed multiple nodules with bilateral pulmonary infiltrates (figure). Gram staining and modified Ziehl-Neelsen staining of the sputum showed branching gram-positive filaments. 16S rRNA gene sequencing identified the presence of a microorganism. A Basic Local Alignment Search Tool (BLAST) search of the GenBank database showed that the sequence was most similar to that of Nocardia concava type strains IFM 0576 and IFM 0354 (99% identity). On the basis of these results, the patient was diagnosed with pulmonary nocardiosis. Antibiotic susceptibility testing showed that the isolate was sensitive to minocycline and co-trimoxazole. Because the patient had a history of co-trimoxazole-induced renal dysfunction, he was treated with minocycline (100 mg twice a day orally), and his symptoms improved. Chest CT 4 months after initial CT showed disappearance of the pulmonary nodules. Minocycline was discontinued 1 year later, and he has not relapsed for 5 years to date. Multiple nodular consolidations with infiltration in both lungs.

Molecular Characterization of Lactic Acid Bacteria Isolated from Food Samples with Potentials for Bacteriocin Production

Lactic acid bacteria (LAB) are naturally found as part of fermented food products and other foodstuff. These organisms are used in the fermentation of vegetables to improve their nutritional value, acceptability, palatability, as well as microbiological qualityand shelf life. Standard microbiological techniques were used to examine 85 different food products for the presence of LAB. The crowded plate approach was used to screen distinct isolates for bacteriocin producing capacity. PCR-based techniques (16S rRNA) were used to identify and characterize lactic acid bacteria associated with food products that have the potential to produce bacteriocins. A total of five hundred and sixty-one (561) lactic acid bacteria were isolated from the food samples of which only 8 (1.42 %) were positive for bacteriocin production. The best five isolates from the 14 with an inhibition zone of 20 mm and more were identified as Lactobacillus plantarum strain PMM09-2528L, Lactobacillus acidophilus strain NK-S10, Lactococcus lactis subsp. lactis strain IFM 63517, Leuconostoc mesenteroides strain M6 and Pediococcus cellicola strain PMM-25. These bacteriocinogenic LAB can be used in the formulation of starter cultures and for commercial use in the biopreservation of vegetables and other food products.

CRISPR/Cas12a-Based Diagnostic Platform Accurately Detects Nocardia farcinica Targeting a Novel Species-Specific Gene.

Under the COVID-19 pandemic background, nucleic acid detection has become the gold standard to rapidly diagnose the infectious disease. A rapid, low cost, reliable nucleic acid detection platform will be the key to control next potential pandemic. In this study, a nucleic acid detection platform, which combined CRISPR/Cas12a-based detection with loop-mediated isothermal amplification (LAMP), was developed and termed CRISPR-CLA. In the CRISPR-CLA system, LAMP preamplification was employed, and CRISPR/Cas12a-based detection was used to monitor the preamplicons. The forward inner primer (FIP) was engineered with a protospacer adjacent motif (PAM) site TTTA of Cas12a effector at the linker region; thus, the CRISPR-CLA platform can detect any sequence as long as the primer design meets the requirement of LAMP. To demonstrate the validity of the CRISPR-CLA system, it was applied for the molecular diagnosis of nocardiosis caused by Nocardia farcinica (N. farcinica). A highly conserved and species-specific gene pbr1 of N. farcinica, which was first reported in this study, was used as the target of detection. A set of LAMP primers targeting a fragment of pbr1 of the N. farcinica reference strain IFM 10152 was designed according to the principle of CRISPR-CLA. Three CRISPR RNAs (crRNAs) with different lengths were designed, and the most efficient crRNA was screened out. Additionally, three single-strand DNA (ssDNA) probes were tested to further optimize the detection system. As a result, the N. farcinica CRISPR-CLA assay was established, and the whole detection process, including DNA extraction (20 min), LAMP preamplification (70°C, 40 min), and CRISPR/Cas12a-mediated detection (37°C, 8 min), can be completed within 70 min. A fluorescence reader (for fluorescence CRISPR-CLA) or a lateral flow biosensor (for lateral-flow CRISPR-CLA) can be the media of the result readout. Up to 132 strains were used to examine the specificity of N. farcinica CRISPR-CLA assay, and no cross-reaction was observed with non-N. farcinica templates. The limit of detection (LoD) of the N. farcinica CRISPR-CLA assay was 100 fg double-strand DNA per reaction. N. farcinica was detected accurately in 41 sputum specimens using the N. farcinica CRISPR-CLA assay, which showed higher specificity than a real-time qPCR method. Hence, the N. farcinica CRISPR-CLA assay is a rapid, economic and accurate method to diagnose N. farcinica infection.

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica.

To establish a CRISPR-based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. A CRISPR-based nucleic acid detection platform, termed CRISPR-CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre-amplification of target sequences and CRISPR-Cas12a-based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR-CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR-CPA. The whole detection process of N. farcinica CRISPR-CPA assay, including sample pre-treatment and DNA extraction (~20min), PCR pre-amplification (60min), CRISPR-based detection (10min), can be completed within 90min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR-CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR-CPA assay is 1pg DNA per reaction in pure cultures and 105 CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. The N. farcinica CRISPR-CPA assay is an economic and specific method to detect N. farcinica and provides a high-efficiency tool for screening of pathogens especially of some hard-to-culture and slow-growth infectious agents. In CRISPR-CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5' end of the engineered PCR primer for protecting PAM site, thus the CRISPR-CPA can detect any sequence. Also, we applied CRISPR-CPA to rapidly detect N. farcinica, which is slow-growing bacteria and is firstly detected by a CRISPR-based method.

Mechanism of Polyhexamethylene Biguanide Resistance in Purpureocillium lilacinum Strains.

Purpureocillium lilacinum has been recently found to contaminate a 20% (200,000 μg/mL) aqueous solution of polyhexamethylene biguanide hydrochloride (PHMB) . We aimed to elucidate the mechanism underlying the resistance of P. lilacinum to PHMB. First, we induced the PHMB-resistant (IR) strains IFM 67050 (IR) and IFM 65838 (IR) from the type strain P. lilacinum CBS 284.36T via cultivation in a medium containing high concentrations of PHMB. We then analyzed the DNA sequences via Illumina sequencing to evaluate the presence of genetic mutations in IFM 65838 (IR) . Further, we established an IFM 65838 (IR) uridine/uracil auxotrophic strain, and using the orotidine-5'-decarboxylase gene, pyrG as a selection marker, we tried to knockout a mutant gene in IFM 65838 (IR) using the CRISPR-Cas9 genome-editing technique. The growth rates of IFM 67050 (IR) and IFM 65838 (IR) in medium containing PHMB increased, and the minimum inhibitory concentrations (MICs) against PHMB also increased. Based on the DNA sequence analysis, we found a nonsynonymous point mutation in the gene PLI-008146 (G779A) in IFM 67050 (IR) and IFM 65838 (IR) . This point mutation leads to site combinations of splicing changes that cause partial sequences deletion (p.Y251_G281del) in the ΔPLI-008146 locus of IFM 65838 (IR) , and deletion sequences include partial adenosine/AMP deaminase motif (PF00962) orthologous to adenosine deaminase (ADA) (GeneBank: OAQ82383.1) . Furthermore, the mutant gene ΔPLI-008146 was successfully knocked out from the resistanceinduced strain using a novel CRISPR-Cas9 gene transformation method. A considerable reduction in growth rate and MIC against PHMB was observed in the absence of the mutant gene. Therefore, ADA may represent an important resistance factor in PHMB-resistant P. lilacinum.

Inhibition Activity of Avibactam against Nocardia farcinica β-Lactamase FARIFM10152.

Nocardia farcinica, one of the most frequent pathogenic species responsible for nocardiosis, is characterized by frequent brain involvement and resistance to β-lactams mediated by a class A β-lactamase. Kinetic parameters for hydrolysis of various β-lactams by FARIFM10152 from strain IFM 10152 were determined by spectrophotometry revealing a high catalytic activity (kcat/Km ) for amoxicillin, aztreonam, and nitrocefin. For cephems, kcat/Km was lower but remained greater than 104 M-1 s-1 A low catalytic activity was observed for meropenem, imipenem, and ceftazidime hydrolysis. FARIFM10152 inhibition by avibactam and clavulanate was compared using nitrocefin as a reporter substrate. FARIFM10152 was efficaciously inhibited by avibactam with a carbamoylation rate constant (k2/Ki ) of (1.7 ± 0.3) × 104 M-1 s-1 The 50% effective concentrations (EC50s) of avibactam and clavulanate were 0.060 ± 0.007 μM and 0.28 ± 0.06 μM, respectively. Amoxicillin, cefotaxime, imipenem, and meropenem MICs were measured for ten clinical strains in the presence of avibactam and clavulanate. At 4 μg/ml, avibactam and clavulanate restored amoxicillin susceptibility in all but one of the tested strains but had no effect on the MICs of cefotaxime, imipenem, and meropenem. At 0.4 μg/ml, amoxicillin susceptibility (MIC ≤ 8 μg/ml) was restored for 9 out of 10 strains by avibactam but only for 4 out of 10 strains by clavulanate. Together, these results indicate that avibactam was at least as potent as clavulanate, suggesting that the amoxicillin-avibactam combination could be considered as an option for the rescue treatment of N. farcinica infections if clavulanate cannot be used.

Analysis of an Intrinsic Mycovirus Associated With Reduced Virulence of the Human Pathogenic Fungus Aspergillus fumigatus.

Aspergillus fumigatus is an airborne fungal pathogen that causes severe infections with invasive growth in immunocompromised patients. Several mycoviruses have recently been isolated from A. fumigatus strains, but there are presently no reports of mycoviral-mediated reduction or elimination of fungal pathogenicity in vertebrate models. Here, we report the biological features of a novel mycovirus, A. fumigatus chrysovirus 41362 (AfuCV41362), isolated from the hypovirulent A. fumigatus strain IFM 41362. The AfuCV41362 genome is comprised of four dsRNAs, each of which contains a single ORF (ORF1-4). ORF1 encodes a protein with sequence similarity to RNA-dependent RNA polymerases of viruses in the family Chrysoviridae, while ORF3 encodes a putative capsid protein. Viral RNAs are expressed primarily during the germination stage, and RNA-seq analysis of virus-infected A. fumigatus at the germination stage suggested that the virus suppressed expression of several pathogenicity-associated host genes, including hypoxia adaptation and nitric oxide detoxification genes. In vitro functional analysis revealed that the virus-infected strain had reduced tolerance to environmental stressors. Virus-infected A. fumigatus strain IFM 41362 had reduced virulence in vivo compared to the virus-free strain in a mouse infection model. Furthermore, introduction of the mycovirus to a natively virus-free KU A. fumigatus strain induced virus-infected phenotypes. To identify mycovirus genes responsible for the reduced virulence of A. fumigatus, each viral ORF was ectopically expressed in the virus-free KU strain. Ectopic expression of the individual ORFs only nominally reduced virulence of the host fungus in a mouse infection model. However, we found that ORF3 and ORF4 reduced tolerance to environmental stresses in in vitro analysis. Based on these results, we suggest that the AfuCV41362 mycovirus ORF3 and ORF4 reduce fungal virulence by suppressing stress tolerance together with other viral genes, rather than alone.

Pertumbuhan dan Uji Gula Reduksi 5 Isolat Jamur Asal Batang Kelapa Sawit ke Potongan Batang Kelapa Sawit

Fungi is a microorganism that has been widely used to accelerate the rate of decomposition of organic matter. This research was conducted in the soil biology laboratory of the Faculty of Agriculture, University of Sumatera Utara. This study was aimed to examine the potential of fungi isolates derived from palm oil trunks. This research begins by isolating the fungi contained in the cutting of oil palm trunk from the plant rejuvenation. The isolate is purified to obtain a pure culture. Selected isolates were identified by DNA sequencing using PCS 28s ITS and the results were identified as Rhizopus arrhizus Strain ATCC 111450 (A), Rhizopus microspores var rhozopodiformis Strain IFM 46417 (B) Syncephalastrum racemosum isolate VPCI 1857/11) (C), Trichoderma asperellum Strain ATCC 52438 (D), and Trichoderma asperellum Strain G (E). All isolates were applied to sterilized palm oil trunks. The results of this study were Rhizopus microspores var rhozopodiformis Strain IFM 46417 capable of growing well in pH 4, 6, and 8, Trichoderma asperellum Strain G (E) had the fastest growth rate of 64.58 mm / day. Test of cellulase enzyme production by using a method of Fehling A and B aiming at the fungus Trichoderma asperellum Strain G (E) produces the highest cellulase enzyme.

High-Quality Draft Genome Sequence of the Actinobacterium Nocardia terpenica IFM 0406, Producer of the Immunosuppressant Brasilicardins, Using Illumina and PacBio Technologies.

The bacterium Nocardia terpenica IFM 0406 is known as the producer of the immunosuppressant brasilicardin A. Here, we report the completely sequenced genome of strain IFM 0406, which facilitates the heterologous expression of the brasilicardin biosynthetic gene cluster but also unveils the intriguing biosynthetic capacity of the strain to produce secondary metabolites.

Nocardia shinanonensis sp. nov., isolated from a patient with endophthalmitis

A nocardioform strain IFM 11456T was isolated from the aqueous humor from a patient with endophthalmitis and was characterized to its taxonomic position. IFM 11456T contained arabinose, galactose and meso-diaminopimelic acid in whole-cell hydrolysates and mycolic acids that co-migrated with those from the type strain of Nocardiaasteroides. The acyl type of muramic acid was N-glycolyl. The diagnostic polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and two unidentified glycolipids and the predominant menaquinone was MK-8(H4, ω-cycl.). These characteristics are typical of members of the genus Nocardia. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate represented a novel species of the genus Nocardia and was most closely related to the type strains of Nocardia mikamii JCM 15508T (98.1 %) and Nocardiaaobensis IFM 0372T (98.1 %). However, analysis of partial gyrB sequences showed that strain IFM 11456T had 90.2 % similarity to Nocardia concava IFM 0354T and 90 % to Nocardianiigatensis IFM 0330T. The DNA-DNA relatedness values for strain IFM 11456T compared with N. mikamii JCM 15508T, N. aobensisIFM 0372T and N. concava IFM 0354T ranged from 24.4 to 39.9 %. Phenotypic characteristics that differentiated IFM 11456T from phylogenetically related species were growth at 45 °C, utilization of citrate and growth with inositol as a sole carbon source. On the basis of this polyphasic study, the isolate represents a novel species within the genus Nocardia, for which the name Nocardia shinanonensis sp. nov. is proposed. The type strain is IFM 11456T (=NBRC 109590T=TBRC 5149T).

Gordonia iterans sp. nov., isolated from a patient with pneumonia

A second novel clinical actinobacterial strain, designated IFM 10348(T), was isolated from the sputum of the same Japanese patient with bacterial pneumonia from whom the type strain of Gordonia araii had been isolated. The strains differed in phylogenetic position and drug-resistance profiles. The taxonomic position of strain IFM 10348(T) was clarified by phenotypic, chemotaxonomic and phylogenetic studies. Phylogenetic analyses based on 16S rRNA gene sequences clearly demonstrated that strain IFM 10348(T) occupied a distinct clade within the genus Gordonia and was related closely to Gordonia malaquae DSM 45064(T) and Gordonia hirsuta DSM 44140(T) (97.3 and 97.1% similarities, respectively). Strain IFM 10348(T) was also clearly differentiated from G. malaquae DSM 45064(T) and G. hirsuta DSM 44140(T) based on gyrB and secA1 gene sequence similarity values. Strain IFM 10348(T) had MK-9(H2) as the predominant menaquonine, contained meso-diaminopimelic acid, arabinose, galactose and glucosamine as cell-wall components, and contained C18:1ω9c, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0 as the major cellular fatty acids. Mycolic acids were present. The DNA G+C content of strain IFM 10348(T) was 68.0 mol%. DNA-DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that strain IFM 10348(T) represents a novel species of the genus Gordonia, for which the name Gordonia iterans sp. nov. is proposed. The type strain is IFM 10348(T) ( = CCTCC M2011245(T) = NCCB 100436(T)).

Nanocellulose and Bioethanol Production from Orange Waste using Isolated Microorganisms

Citrus processing waste from oranges (CPWO) was explored for the production of nanocellulose and bioethanol. After the isolation of 20 microorganisms from CPWO, their fermentation abilities were screened and two microorganisms identified as Candida parapsilosis strains IFM 48375 and NRRL Y-12969 (ATCC 22019) were selected for a further fermentation. The CPWO was steam distilled for the isolation of essential oil (1.5% g g-1 of dry CPWO) and converted into a mixture of fermentable sugars (40% g g-1 of dry CPWO) using acid or enzymes hydrolyses. Hydrolyzates were fermented with three different yeast strains, the two Candida sp. and Saccharomyces cerevisiae. Candida parapsilosis strain IFM 48375 accomplished excellent results in ethanol production (21% g g-1 of dry CPWO) from CPWO, higher when compared to other strains. Nanocellulose (2.5% g g-1 dry CPWO) and nanofibers (0.5% g g-1 dry CPWO) were isolated from solid residues obtained from enzymatically treated and fermented CPWO. To the best of our knowledge, this work reports for the first time the nanocellulose production from CPWO.

Orange waste as a biomass for 2G-ethanol production using low cost enzymes and co-culture fermentation

The successful conversion of orange waste into a mixture of fermentable sugars was achieved using low cost enzymes obtained from the citrus-canker bacterium Xanthomonas axonopodis pv. citri strain 306 (IBSBF 1594). Then, fermentable sugars were converted into 2G-ethanol using Saccharomyces cerevisiae in combination with one of the two isolated yeast strains, Candida parapsilosis strains IFM 48375 and NRRL Y-12969 (ATCC 2219), in independent submerged co-culture fermentations. The co-culture fermentations enabled reducing fermentation time to 6 h. Pure ethanol in excellent yield with C-6 sugars conversion of 98.9% was obtained. Therefore, diminished costs of enzyme biomass processing and reduced fermentation time could make the 2G-ethanol production from orange waste even more feasible, cost-effective and an environmentally friendly process.

Homozygous Triplicate Mutations in Three 16S rRNA Genes Responsible for High-Level Aminoglycoside Resistance inNocardia farcinicaClinical Isolates from a Canada-Wide Bovine Mastitis Epizootic

Nocardia farcinica strains showing high-level resistance to amikacin were isolated from clinical cases in a Canada-wide bovine mastitis epizootic. Shotgun cloning of the resistance genes in the amikacin-resistant mastitis isolate N. farcinica IFM 10580 (W6220 [Centers for Disease Control and Prevention]) using a multicopy vector system revealed that the 16S rRNA gene with an A-to-G single-point mutation at position 1408 (in Escherichia coli numbering) conferred "moderate" cross-resistance to amikacin and other aminoglycosides to an originally susceptible N. farcinica strain IFM 10152. Subsequent DNA sequence analyses revealed that, in contrast to the susceptible strain, all three chromosomal 16S rRNA genes of IFM 10580, the epizootic clinical strain, contained the same A1408G point mutations. Mutant colonies showing high-level aminoglycoside resistance were obtained when the susceptible strain N. farcinica IFM 10152 was transformed with a multicopy plasmid carrying the A1408G mutant 16S rRNA gene and was cultured in the presence of aminoglycosides for 3 to 5 days. Of these transformants, at least two of the three chromosomal 16S rRNA genes contained A1408G mutations. A triple mutant was easily obtained from a strain carrying the two chromosomal A1408G mutant genes and one wild-type gene, even in the absence of the plasmid. The triple mutant showed the highest level of resistance to aminoglycosides, even in the absence of the plasmid carrying the mutant 16S rRNA gene. These results suggest that the homozygous mutations in the three 16S rRNA genes are responsible for the high-level aminoglycoside resistance found in N. farcinica isolates of the bovine mastitis epizootic.

Nocardithiocin, a novel thiopeptide antibiotic, produced by pathogenic Nocardia pseudobrasiliensis IFM 0757

Nocardithiocin is a novel thiopeptide compound produced by the pathogenic Nocardia pseudobrasiliensis strain IFM 0757. It shows a strong activity against acid-fast bacilli such as the Mycobacterium and Gordonia species. Nocardithiocin was highly active against rifampicin-resistant as well as -sensitive Mycobacterium tuberculosis strains, and most of the resistant strains were inhibited at concentrations ranging from 0.025 to 1.56 microg ml(-1). The structure of the thiopeptide antibiotic containing thiazole, natural and unnatural amino acids was elucidated by MS and NMR spectral analyses.

Nocardia terpenica sp. nov., isolated from Japanese patients with nocardiosis

Two bacterial strains isolated from different hospitals in Japan were subjected to a polyphasic analysis. Strains IFM 0406 and IFM 0706(T), producers of novel terpenoid antibiotics, were found to have morphological, biochemical, physiological and chemotaxonomic properties consistent with their classification in the genus Nocardia, except for the presence of MK-8(H(4)) as one of the predominant menaquinones in addition to the major menaquinone MK-8(H(4 omega-cyc)). The 16S rRNA gene sequence similarity of strains IFM 0406 and IFM 0706(T) was 99.9 %, and the closest members of Nocardia to these strains were the type strains of Nocardia nova and Nocardia mexicana, showing similarity of 97.5 and 97.1 %, respectively. Based on their characteristic phenotypic and phylogenetic properties, a novel species of the genus Nocardia, Nocardia terpenica sp. nov. is proposed for the two strains. The type strain is IFM 0706(T) (=JCM 13033(T)=DSM 44935(T)=NBRC 100888(T)).

Gordonia araii sp. nov. and Gordonia effusa sp. nov., isolated from patients in Japan

Two bacterial strains, IFM 10211(T) and IFM 10200(T), were isolated from the sputum of two Japanese patients, and were subjected to a polyphasic taxonomic study. The two strains were found to have morphological, physiological and chemotaxonomic properties that were consistent with their assignment to the genus Gordonia, except for a few chemotaxonomic characteristics. Almost complete 16S rRNA gene sequences of the two strains were determined; the data showed that they are related distantly to Gordonia amarae, Gordonia hirsuta, Gordonia hydrophobica and Gordonia sihwensis, showing 16S rRNA gene sequence similarities to the type strains of these species of 96.2-97.9 %. DNA-DNA relatedness data coupled with the combination of genotypic and phenotypic data indicated that the two strains are representatives of two novel, separate species. The names proposed to accommodate these two strains are Gordonia araii sp. nov. (type strain IFM 10211(T)=DSM 44811(T)=NBRC 100433(T)=JCM 12131(T)) and Gordonia effusa sp. nov. (type strain IFM 10200(T)=DSM 44810(T)=NBRC 100432(T)=JCM 12130(T)).