Application of MALDI-TOF mass spectrometry for identification of Nocardia species.

BACKGROUND: Nocardiosis, despite its rarity and underreporting, is significant due to its severe impact, characterized by high morbidity and mortality rates. The development of a precise, reliable, rapid, and straightforward technique for identifying the pathogenic agent in clinical specimens is crucial to reduce fatality rates and facilitate timely antimicrobial treatment. In this study, we aimed to identify Nocardia spp. in clinical isolates, using MALDI-TOF MS as the primary method, with molecular methods as the gold standard. Clinical Nocardia isolates were identified using 16S rRNA/hsp65/gyrB/secA1/rpoB gene sequencing. Identification performance of the Bruker MALDI Biotyper 3.1 (V09.0.0.0_8468) and MBT Compass 4.1 (V11.0.0.0_10833) for Nocardia identification was evaluated. RESULTS: Seventy-six Nocardia isolates were classified into 12 species through gene sequencing. The MALDI Biotyper 3.1 (V09.0.0.0_8468) achieved 100% genus-level accuracy and 84.2% species accuracy (64/76). The MBT Compass 4.1 with the BDAL Database (V11.0.0.0_10833) improved species identification to 98.7% (75/76). The updated database enhanced species level identification with scores > 1.7, increasing from 77.6% (59/76) to 94.7% (72/76), a significant improvement (P = 0.001). The new and simplified extraction increased the proportion of strains identified to the species level with scores > 1.7 from 62.0% (18/29) to 86.2% (25/29) (P = 0.016). An in-house library construction ensured accurate species identification for all isolates. CONCLUSIONS: The Bruker mass spectrometer can accurately identify Nocardia species, albeit with some variations observed between different database versions. The MALDI Biotyper 3.1 (V09.0.0.0_8468) has limitations in identifying Nocardia brasiliensis, with some strains only identifiable to the genus level. MBT Compass 4.1 (V11.0.0.0_10833) effectively addresses this shortfall, improving species identification accuracy to 98.7%, and offering quick and reliable identification of Nocardia. Both database versions incorrectly identified the clinically less common Nocardia sputorum as Nocardia araoensis. For laboratories that have not upgraded their databases and are unable to achieve satisfactory identification results for Nocardia, employing the new and simplified extraction method can provide a degree of improvement in identification outcomes.

Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Nocardia coffeae sp. nov., an endophytic actinobacterium isolated from the root of Coffea arabica (L.).

The polyphasic taxonomic study of a novel endophytic actinobacterium strain (CA2R105T) was carried out. The strain formed fragmented substrate mycelium and showed chemotaxonomic properties typical of members of the genus Nocardia, i.e. the presence of mycolic acid and MK-8 (H4ω-cycl) in its cells. Strain CA2R105T exhibited the highest 16S rRNA gene sequence similarity to Nocardia jiangxiensis NBRC 101359T (99.2%). The genome-based taxonomic analysis revealed low average nucleotide identity-blast and digital DNA-DNA hybridization values (<93.7, and <65.2%, respectively) to its closest relative. Moreover, many different phenotypic characteristics were observed between strain CA2R105T and all related Nocardia-type strains. This taxonomic evidence suggested that strain CA2R105T should be judged as representing a novel species of the genus Nocardia and the name, Nocardia coffeae sp. nov. is proposed. The type strain is CA2R105T (=TBRC 11247T=NBRC 114292T).

MLSA phylogeny and antimicrobial susceptibility of clinical Nocardia isolates: a multicenter retrospective study in China.

BACKGROUND: With the increase of detection rate and long treatment period, nocardiosis has become a noticeable problem in China. However, there are limited large-scale studies on the epidemiology and antimicrobial susceptibility profiles of clinical Nocardia spp. in China. The present study aimed to explore the species distribution and drug susceptibility pattern of 82 clinical Nocardia isolates from three tertiary hospitals in China by multilocus sequence analysis (MLSA) and broth microdilution (BMD) method. RESULTS: Pulmonary nocardiosis (90.2%) was the most common clinical presentation of infection. N. cyriacigeorgica (n = 33; 40.2%) and N. farcinica (n = 20; 24.4%) were the most frequently encountered Nocardia species, followed by N. otitidiscaviarum (n = 7; 8.5%), N. abscessus (n = 5; 6.1%), N. asiatica (n = 4; 4.9%), and N. wallacei (n = 4; 4.9%). Trimethoprim/sulfamethoxazole (SXT) remained high activity against all Nocardia isolates (susceptibility rate: 98.8%). Linezolid and amikacin were also highly active; 100 and 95.1% of all isolates demonstrated susceptibility, respectively. Except for N. otitidiscaviarum, all the Nocardia isolates exhibited high susceptibility rates to imipenem. The resistance rates of all isolates to clarithromycin and ciprofloxacin were 92.7 and 73.2%, respectively, but the resistance rate of N. farcinica to ciprofloxacin was only 25%. CONCLUSIONS: The clinically isolated Nocardia spp. had diverse antimicrobial susceptibility patterns, which were similar to the reports by other groups elsewhere, but some differences were also observed, mainly including imipenem and ciprofloxacin. According to this study, SXT still can be the first choice for empirical therapy due to the low resistance rate. Linezolid can be chosen when a patient is allergic to SXT, and amikacin and imipenem can be the choice in a combination regimen.

Nocardia terrae sp. nov., an actinomycete isolated from soil in Thailand.

A novel actinomycete strain ET3-3T isolated from soil collected from Chachoengsao province, Thailand was taxonomic evaluated using a polyphasic approach. Chemotaxonomic analysis indicated that meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. Mycolic acids were present. Ribose, arabinose, and galactose were detected in its whole-cell hydrolysates. The strain comprised C16:0 and TBSA 10-methyl C18:0 as the main fatty acids and MK-8(H4ω-cycl) as the predominant menaquinone. Major polar lipids were phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylethanolamine, and diphosphatidylglycerol. The results of 16S rRNA gene sequence analysis showed that strain ET3-3T was closely related to Nocardia seriolae JCM 30082T (99.2%), Nocardia yunnanensis JCM 30082T (98.4%), and Nocardia concava JCM 12351T (98.2%). The draft genome of ET3-3T was 9.31 Mb with 8826 coding sequences with an average G + C content of 68.0%. The comparison of the draft genome of strain ET3-3T and N. seriolae NBRC 15557T showed the ANIb, ANIm and the digital DNA-DNA hybridization values of 86.3%, 88.5% and 32.9%, respectively. The results of the taxonomic analysis suggested that strain ET3-3T represented a novel species of the genus Nocardia for which the name Nocardia terrae sp. nov. is proposed. The type strain is ET3-3T (= JCM 33776T = TISTR 2837T).

Nocardia acididurans sp. nov., an acid-tolerant actinobacterium isolated from bio-fertilizer of Musa species.

A novel acid-tolerant actinobacterium (strain LPG 2T), which formed fragmented substrate mycelia, was isolated from bio-fertiliser of Musa spp. collected from Lampang Province, Thailand. Its morphological and chemotaxonomic properties, e.g., the presence of mycolic acid and MK-8 (H4ω-cycl) in the cells, showed that strain LPG 2T was a member of the genus Nocardia. 16S rRNA gene sequence analysis revealed that this strain was closely related to Nocardia otitidiscaviarum NBRC 14405T (98.7 %). The low average nucleotide identity-blast and digital DNA-DNA hybridization values (<78.6 and <24.0 %, respectively), and several phenotypic differences between strain LPG 2T and its related Nocardia type strains, indicated that the strain merits classification as representing a novel species of the genus Nocardia, for which we propose the name Nocardia acididurans sp. nov. The type strain is LPG 2T (=TBRC 11242T=NBRC 114293T).

Nocardia huaxiensis sp. nov., an actinomycete isolated from human skin.

A novel actinomycete, designated as strain WCH-YHL-001T, was isolated from skin biopsy specimens of a patient at West China Hospital, Chengdu, Sichuan Province, PR China. The cells were Gram-positive, aerobic, heterotrophic and non-motile. They formed an extensive substrate with short aerial mycelia, whose branches fragmented into rod-shaped elements. Growth occurred at 10-40 °C, pH 5.0-12.0 and with NaCl concentrations of 0-4.0 % (w/v). The major cellular fatty acids of strain WCH-YHL-001T were C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl and summed feature 3. The predominant respiratory quinone was MK-8 (H4ω-cycl). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol mannoside, unknown phospholipids and unidentified glycolipids. The diagnostic diamino acid of peptidoglycan was meso-diaminopimelic acid. The whole-cell sugar pattern consisted of arabinose and glucose. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain WCH-YHL-001T belonged to the genus Nocardia. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain WCH-YHL-001T and type strains of Nocardia species were lower than the cut-offs (≥95-96 % for ANI and ≥70 % for isDDH) required to define a bacterial species. The genomic DNA G+C content was 67.8 mol%. Phylogenetic, physiological and chemotaxonomic data suggested that strain WCH-YHL-001T represented a novel species of the genus Nocardia, for which the name Nocardia huaxiensis sp. nov. is proposed, with the type strain WCH-YHL-001T (=GDMCC 4.181T=JCM 34475 T=NBRC 114973T).

First evidence of fish nocardiosis in Mexico caused by Nocardia seriolae in farmed red drum (Sciaenops ocellatus, Linnaeus).

Between August and December 2013, the offshore cages of a commercial marine farm culturing red drum Sciaenops ocellatus in Campeche Bay Mexico were affected by an outbreak of an ulcerative granulomatous disease with up to 70% cumulative mortality. Thirty-one adults displaying open ulcers on the skin were submitted for diagnosis. At necropsy, multiple white-yellowish nodules (0.1-0.5 cm in diameter) were present in all internal organs, where the kidney and the spleen were the most severely affected. Histopathology evinced typical systemic granulomatous formations. Gram and Ziehl-Neelsen stains on tissue imprints, bacterial swabs and tissue sections revealed Gram-positive, acid-fast, branching beaded long rod filamentous bacteria. Tissue samples resulted positive for nocardiosis with a Nocardia genus-specific nested PCR. Definite identification at the species level and taxonomic positioning of the fastidious pathogen were achieved through a specific Nocardia seriolae PCR and by sequencing the gyrB gene of pure isolates. After administration of antibiotics during fry production, a posterior follow-up monitoring (from 2014 to 2017) detected mild but recurrent outbreaks of the bacteria with no seasonality pattern. To the extent of our knowledge, this is the first report of piscine nocardiosis in Mexico and the first time this disease is detected in red drum.

Retrospective Analysis of Antimicrobial Susceptibility Profiles of Nocardia Species from a Tertiary Hospital and Reference Laboratory, 2011 to 2017.

Nocardia species are found worldwide and are opportunistic pathogens of both immunocompromised and immunocompetent hosts. Recent updates to the taxonomy of this genus have indicated that there are more than 90 recognized species of Nocardia with 54 species reported to be clinically relevant. In this paper, we report the species distribution, specimen source distribution, and antimicrobial susceptibility profiles of 2,091 clinical isolates recovered for the years 2011 to 2017 using the updated taxonomy. The most commonly isolated species included Nocardia nova complex, Nocardia cyriacigeorgica, and Nocardia farcinica complex, with an additional 25 species or species complexes recovered from clinical specimens. The antimicrobial susceptibility profile was highly variable between the species, but in general, amikacin, linezolid, and trimethoprim-sulfamethoxazole demonstrated good in vitro activity against most species.

Nocardia stercoris sp. nov., a novel actinomycete isolated from the cow dung.

A novel actinobacterial strain, designated NEAU-LL90T, was isolated from cow dung collected from Shangzhi, Heilongjiang Province, north-east PR China and characterized by using a polyphasic approach. Morphological and chemotaxonomic characteristics were consistent with those members of the genus Nocardia. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The predominant menaquinone detected was MK-8(H4, ω-cycl). Major fatty acids (>10 %) were identified as C16:0, C18:1ω9c, C18:0 and 10-methyl C19:0. Mycolic acids were present. The results of 16S rRNA gene sequence analysis showed that strain NEAU-LL90T belongs to the genus Nocardia with high sequence similarity to Nocardia niigatensis JCM11894T (98.1 %), similarities to other type strains of species of the genus Nocardia were found to be lower than 98.0 %. Furthermore, a combination of DNA-DNA hybridization results and some phenotypic characteristics demonstrated that strain NEAU-LL90T could be distinguished from its closest relative. Therefore, it is proposed that strain NEAU-LL90T represents a novel species of the genus Nocardia, for which the name Nocardia stercoris sp. nov. is proposed. The type strain is NEAU-LL90T (=CGMCC 4.7500T=JCM 32663T).

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA-DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia, such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia: Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).

Nocardia aurantiaca sp. nov., isolated from soil in Thailand.

A novel actinomycete strain, CT2-14T, belonging to the genus Nocardia, was isolated from a soil sample collected from Phichit Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic approach. The strain grew at 15-40 °C (optimum, 28-37 °C), pH 6-11 (optimum, pH 6-8) and on an International Streptomyces Project 2 with 4 % (w/v) NaCl agar plate. Meso-diaminopimelic acid was detected in the cell-wall peptidoglycan. Ribose, arabinose and galactose were detected in its whole-cell hydrolysates. Mycolic acids were present. The strain contained C16 : 0, summed feature 3, C17 : 0 10-methyl and C18 : 1 ω9c as the major fatty acids and MK-8(H4ω-cycl) as the major menaquinone. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol mannosides. Strain CT2-14T showed the highest 16S rRNA gene similarity to Nocardia veterana JCM 11307T (98.4 %), Nocardia africana JCM 11438T (98.2 %) and Nocardia kruczakiae JCM 13032T (98.0 %). The draft genome of strain CT2-14T was 7.37 Mb with 6685 coding sequences with an average G+C content of 67.9 mol %. Based on the phylogenomic tree analysis, the strain was closely related to Nocardia niigatensis NBRC 100131T. On the basis of polyphasic and genome analyses, strain CT2-14T represented a novel species of the genus Nocardia for which the name Nocardia aurantiaca sp. nov. is proposed. The type strain is CT2-14T (=JCM 33775T=TISTR 2838T).

Nocardia colli sp. nov., a new pathogen isolated from a patient with primary cutaneous nocardiosis.

A novel nocardioform strain, CICC 11023T, was isolated from a tissue biopsy of neck lesions of a patient with primary cutaneous nocardiosis and characterized to establish its taxonomic position. The morphological, biochemical, physiological and chemotaxonomic properties of strain CICC 11023T were consistent with classification in the genus Nocardia. Whole-cell hydrolysates were rich in meso-diaminopimelic acid, galactose, arabinose and fructose. Mycolic acids were present. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and two unidentified lipids, and the predominant menaquinone was cyclo MK-8 (H4, ω-cyclo). The main fatty acids (>5 %) were C18 : 0 10-methyl (TBSA), C16 : 0, summed feature 4 (C16 : 1 trans 9/C15 : 0 iso 2OH), C15 : 0 and C17 : 0 10-methyl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the isolate is most closely related (>98 % similarity) to the type strains Nocardia ninae OFN 02.72T, Nocardia iowensis UI 122540T and Nocardia alba YIM 30243T, and phylogenetic analysis of gyrB gene sequences showed similarity (89.1-92.2 %) to Nocardia vulneris NBRC 108936T, Nocardia brasiliensis IFM 0236T and Nocardia exalbida IFM 0803T. DNA-DNA hybridization results for strain CICC 11023T compared to Nocardia type strains ranged from 20.4 to 35.4 %. The genome of strain CICC 11023T was 8.78 Mbp with a G+C content of 67.4 mol% overall. The average nucleotide identity (ANI) values between strain CICC 11023T and N. alba YIM 30243T were low (OrthoANIu=77.47 %), and the ANI values between strain CICC 11023T and N. vulneris NBRC 108936 T were low (OrthoANIu=83.75 %). Consequently, strain CICC 11023T represents a novel Nocardia species on the basis of this polyphasic study, for which the name Nocardia colli sp. nov. is proposed. The type strain is CICC 11023T (=KCTC 39837T).

Assessment of VITEK® MS IVD database V3.0 for identification of Nocardia spp. using two culture media and comparing direct smear and protein extraction procedures.

We assessed the performance of the VITEK® MS IVD V3.0 matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-ToF MS) V3.0 database for the identification of Nocardia spp. as compared with targeted DNA sequencing. A collection of 222 DNA sequence-defined Nocardia spp. strains encompassing 18 different species present or not in the database was tested. Bromocresol purple agar (BCP) and Columbia agar +5% sheep's blood (COS) culture media were used together with two different preparation steps: direct smear and a "3 attempts" procedure that covered (1) spotting of an extract, (2) new spotting of the same extract, and (3) spotting of a new extract. The direct smear protocol yielded low correct identification rates (≤ 15% for both media) whereas protein extraction yielded correct identification results (> 67% regardless of the media used.). The use of 2 additional attempts using repeat or new extracts increased correct identification rates to 87% and 91% for BCP and COS, respectively. When using the 3 attempts procedure, the best identification results, independent of media types, were obtained for N. farcinica and N. cyriacigeorgica (100%). Identification attempts 2 and 3 allowed to increase the number of correct identifications (BCP, +20%; COS, +13%). The enhancement in performance during attempts 2 and 3 was remarkable for N. abscessus (81% for both media) and low prevalence species (BCP, 70%; COS, 85%). Up to 3.4% and 2.4% of the strains belonging to species present in the database were misidentified with BCP and COS media, respectively. In 1.9% of the cases for BCP and 1.4% for COS, these misidentifications concerned a species belonging to the same phylogenetic complex. Concerning strains that are not claimed in the V3.0 database, N. puris and N. goodfellowi generated "No identification" results and 100% of the strains belonging to N. arthritidis, N.cerradoensis, and N. altamirensis yielded a misidentification within the same phylogenetic complex. Vitek® MS IVD V3.0 is an accurate and useful tool for identification of Nocardia spp.

Molecular identification and structural characterization of marine endophytic actinomycetes Nocardiopsis sp. GRG 2 (KT 235641) and its antibacterial efficacy against isolated ESBL producing bacteria.

The present study was designed to identify the potential bioactive compound from endophytic actinomycetes (EA) Nocardiopsis sp. GRG 2 (KT 235641) against selected extended spectrum beta lactamase (ESBL) producing Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae). Initially, the multi drug resistance (MDR) effect of selected uropathogens was confirmed by respective UTI panel of Hexa antibiotics disc methods. The zone of inhibition ≤22 mm for ceftazidime, ≤ 27 mm for cefotaxime and ≤8 mm zone of MIC stripe against both the uropathogens of phenotypic methods confirmed, the selected strains were ESBL producer. Among the various EA extracts, GRG 2 extract showed excellent antibacterial activity against both ESBL producing P. aeruginosa and K. pneumonia by agar well diffution method. The molecular identification of selected GRG 2 strain was named as Nocardiopsis sp. GRG 2 (KT235641). The antibacterial metabolites present in the TLC elution was exhibited at 274 nm by UV visible spectrometer. The partial purification of preparative HPLC fraction 3 showed 14, 16 mm against P. aeruginosa and K. pneumoniae, respectively. Based on the antibacterial effect, the FT-IR, GC-MS and LC-MS analysis of fraction 3 was confirmed as 1, 4-diaza-2, 5-dioxo-3-isobutyl bicyclo[4.3.0]nonane (DDIBN). Further, the dose dependent inhibition of DDIBN against both ESBL producing pathogens was observed at 75 µg/mL by minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The increased cell death and disrupted cell membrane integrity were observed at MIC of DDIBN by confocal laser scanning electron microscope (CLSM) and scanning electron microscope (SEM). The results were proved that the DDIBN has potential antibacterial metabolites against ESBL producing pathogens and it can be applied for various other biomedical fields.

Nocardia yunnanensis sp. nov., an actinomycete isolated from a soil sample.

A novel actinomycete strain, designated CFH S0054T, was isolated from a soil sample collected from Xiaokongshan in Tengchong, Yunnan Province, in south-west PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain CFH S0054T belonged to the genus Nocardia. Based on the results of 16S rRNA gene sequence analysis, strain CFH S0054T was most closely related to Nocardia concava IFM 0354T (98.3 %), Nocardia inohanensis NBRC 100128T (97.9 %), Nocardia otitidiscaviarum NBRC 14405T (97.9 %) and Nocardia heshunensis CFH S0067T (97.8 %). Strain CFH S0054T contained C16 : 0, C18 : 1 ω9c, C18 : 0 10-methyl (TBSA) and summed feature 3 as major cellular fatty acids and MK-8 (H4ω-cycl) as the major respiratory quinone. The polar lipid profile included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and unknown phospholipids. The cell wall contained meso-diaminopimelic acid, and the whole-cell hydrolysates were arabinose, galactose, glucose, fructose, ribose and mannose. The genome of strain CFH S0054T was 7.9 Mbp with a G+C content of 68.4 mol%, the average nucleotide identity (ANI) values between strain CFH S0054T and N. concava IFM0354T were low (ANIm<87.7 % and ANIb=84.1 %). On the basis of phylogenetic, physiological and chemotaxonomic data, strain CFHS0054T is considered to represent a novel species of the genus Nocardia, for which the name Nocardiayunnanensis sp. nov. is proposed, The type strain is CFH S0054T (DSM 46763T=JCM 30082T).

Nocardia mangyaensis sp. nov., a novel actinomycete isolated from crude-oil-contaminated soil.

A Gram-stain-positive, aerobic, non-motile and mycolic-acid-containing strain, designated Y48T, was isolated from soil contaminated by crude oil located in the northern margin of the Qaidam Basin. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Y48T belongs to the genus Nocardia and is closely related to N. cummidelens DSM 44490T (99.0 % similarity), N. soli DSM 44488T (99.0 %), N. lasii 3C-HV12T (98.9 %), N. salmonicida NBRC 13393T (98.6 %), N. ignorata NBRC 108230T (98.6 %) and N. coubleae NBRC 108252T (98.6 %). The average nucleotide identity and DNA-DNA hybridization values between strain Y48T and the reference strains were 75.9-84.5 and 27.5-29.0 %, respectively, values that were below the thresholds for species delineation. Chemotaxonomic analysis indicated that the major fatty acids of strain Y48T were C16 : 0, summed feature 3 (C16 : 1ω6c/C16 : 1ω7c), C18 : 1ω9c and C18 : 0 10-methyl (TBSA). The respiratory quinone was MK-8(H4, ω-cycl). The polar lipid profile was composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two glycolipids and three unidentified lipids. The cell-wall hydrolysates contained meso-diaminopimelic acid, with ribose, arabinose, glucose and galactose as whole-cell sugars. A combination of 16S rRNA gene sequence analysis, and phenotypic and chemotaxonomic characterizations demonstrated that strain Y48T represents a novel species of the genus Nocardia, for which the name Nocardia mangyaensis sp. nov. is proposed. The type strain is Y48T (=JCM 32795T=CGMCC 4.7494T).

Nocardia aurea sp. nov., a novel actinobacterium isolated from a karstic subterranean environment.

A novel actinobacterium, designated strain SYSU K10002T, was isolated from a soil sample collected from a karst cave in Xingyi county, Guizhou province, south-western China. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of the strain were aerobic and Gram-stain-positive. On the basis of 16S rRNA gene sequence similarities and phylogenetic analysis, strain SYSU K10002T was most closely related to the type strains of Nocardiaaltamirensis NBRC 108246T (99.0 % sequence similarity) and Nocardiatenerifensis NBRC 101015T (98.8 %) and is therefore considered to represent a member of the genus Nocardia. DNA-DNA hybridization values between strain SYSU K10002T and the closely related type strains of the genus Nocardia were less than 70 %. In addition, meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell sugars were arabinose, ribose and galactose. The major isoprenoid quinone was MK-8(H4,ω-cycl), while the major fatty acids (>10 %) were C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl. The polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and an unidentified glycolipid. Mycolic acids were present. The genomic DNA G+C content of strain SYSU K10002T was 67.4 mol%. On the basis of phenotypic, genotypic and phylogenetic data, strain SYSU K10002T represents a novel species of the genus Nocardia, for which the name Nocardiaaurea sp. nov. is proposed. The type strain is SYSU K10002T (=KCTC 39849T=DSM 103986T).

Molecular identification and phylogenetic relationships of clinical Nocardia isolates.

Species identification of Nocardia is difficult because of a complex and rapidly evolving taxonomy. In this study, gene sequencing (16S rRNA, gyrB, secA1, hsp65, rpoB), single 16S rRNA gene sequence phylogenetic analysis, and MALDI-TOF analysis were used to accurately identify 46 clinical Nocardia isolates to the species level. This provided a basis for establishing a routine method of multi-locus sequence analysis (MLSA) for molecular identification of Nocardia species. Genetic polymorphism analysis showed that MLSA was a powerfully discriminating method compared with the 16S rRNA gene to identify clinical Nocardia isolates. However, five-locus (gyrB-16S rRNA-secA1-hsp65-rpoB) MLSA led to misidentifications of all of the five Nocardia abscessus, which were confirmed by digital DNA-DNA hybridization (DDH) analysis. Interestingly, four strains identified as Nocardia beijingensis by a 16S rRNA gene phylogenetic tree may be novel species as suggested by DDH studies. For the purpose of achieving both accuracy and discrimination, the data of MLSA were reanalyzed. A three-locus MLSA with concatenated gyrB-16S rRNA-secA1 sequences was used to construct the phylogenetic tree with high accuracy and powerful discrimination. Therefore, a routine method of MLSA was developed to identify clinical Nocardia species.

Bioactive molecules from Nocardia: diversity, bioactivities and biosynthesis.

Nocardia spp. are catalase positive, aerobic, and non-motile Gram-positive filamentous bacteria. Many Nocarida spp. have been reported as unusual causes of diverse clinical diseases in both humans and animals. Therefore, they have been studied for a long time, primarily focusing on strain characterization, taxonomic classification of new isolates, and host pathophysiology. Currently, there are emerging interests in isolating bioactive molecules from diverse actinobacteria including Nocardia spp. and studying their biosynthetic mechanisms. In addition, these species possess significant metabolic capacity, which has been utilized for generating diverse functionalized bioactive molecules by whole cell biotransformation. This review summarizes the structural diversity and biological activities of compounds biosynthesized or biotransformed by Nocardia spp. Furthermore, the recent advances on biosynthetic mechanisms and genetic engineering approaches for enhanced production or structural/functional modification are presented.