Diagnostic Usefulness of Liquid Culture Medium for Mycobacterium avium-intracellulare Complex Lung Disease: A Single-Centre, Retrospective Study.

Background The diagnosis of Mycobacterium avium-intracellulare complex lung disease (MAC-LD) requires two or more positive sputum cultures. Few reports have examined the usefulness of adding liquid culture to conventional solid culture for diagnosing MAC-LD. Methods A retrospective, cohort study of patients examined at Kurashiki Central Hospital in Japan with a confirmed diagnosis of MAC-LD between January 1, 2002, and June 20, 2021, was conducted. The primary endpoint was the culture positivity rate, which was compared between the liquid and Ogawa culture media in patients who underwent sputum culture using both methods. Secondary endpoints were the culture positivity rate in smear-positive specimens and the positivity rate by radiological type. Results The study, which involved 351 patients and 702 specimens, showed a higher positivity rate for liquid culture (n=690, 98.3%) than Ogawa culture (n=315, 44.9%). Overall, 265 patients (75.5%) would have had delayed MAC-LD diagnosis without liquid medium being used. Of the 95 smear-positive specimens, 71 (74.7%) were positive on both cultures, whereas 24 (25.3%) were positive only on liquid culture. The positivity rate of Ogawa culture varied by radiological type. Conclusions Liquid culture is more valuable for the early diagnosis of MAC-LD than Ogawa culture.

Conservation of Endangered Cordyceps sinensis Through Artificial Cultivation Strategies of C. militaris, an Alternate.

Cordyceps, an entomopathogenic fungus belonging to the Ascomycota phylum, is a familiar remedial mushroom that is extensively used in the traditional medicinal system, especially in South Asian nations. The significance of this genus' members in a range of therapeutic and biotechnological applications has long been acknowledged. The exceedingly valuable fungus Ophiocordyceps sinensis (Cordyceps sinensis) is found in the alpine meadows of Bhutan, Nepal, Tibet, and India, where it is severely harvested. Driven by market demand and ecological concerns, the study highlights challenges in natural C. sinensis collection and emphasizes the shift towards sustainable artificial cultivation methods. This in-depth review navigates Cordyceps cultivation strategies, focusing on C. sinensis and the viable alternative, C. militaris. The escalating demand for Cordyceps fruiting bodies and bioactive compounds prompts a shift toward sustainable artificial cultivation. While solid-state fermentation on brown rice remains a traditional method, liquid culture, especially submerged and surface/static techniques, emerges as a key industrial approach, offering shorter cultivation periods and enhanced cordycepin production. The review accentuates the adaptability and scalability of liquid culture, providing valuable insights for large-scale Cordyceps production. The future prospects of Cordyceps cultivation require a holistic approach, combining scientific understanding, technological innovation, and sustainable practices to meet the demand for bioactive metabolites while ensuring the conservation of natural Cordyceps populations.

Direct Shoot From Root and True-to-Type Micropropagation of Limonium "Misty Blue" in Partially Immersed Culture on an Aluminum Mesh Raft.

Commercial plant tissue culture now primarily serves the ornamental horticulture industry. The main pillars of the commercial tissue culture business are scalability of production, cost reduction, limited labor involvement, high quality, and genetic homogeneity of propagated plants. Based on these requirements, the current protocol employs a partially immersed liquid culture medium supported by a flexible aluminum mesh raft with a wire stand to facilitate shoot organogenesis from the horizontally placed root explants and hold the plants upright for shoot multiplication and rooting of Limonium Misty Blue. It is a florist crop that is in high demand as both dried and fresh flower fillers in various floral decorations. The majority of cultivated Limonium or statice cultivars are heterozygous in nature and propagate commercially through in vitro propagation to cater to the huge demand for planting materials needed for flower production. This is the first protocol to describe direct shoot organogenesis from the roots in a liquid half-component of Murashige and Skoog's (1962) (MS) basal medium supplemented with 1.6 µM NAA and 1.1 µM BA. The regenerated shoots are multiplied and rooted at the same time on the raft in a MS-based liquid culture medium that included 0.44 µM BA and 1.07 µM NAA. In comparison to agar-gelled medium, plants cultured in liquid medium grow more quickly without any signs of hyperhydricity. In liquid medium, a clump of 4-5 shoots is formed from a single shoot explant within 4 weeks and are rooted simultaneously within 6 weeks. On average, seven explants may fit on each raft, so on average, 25 healthy plants are produced from a single bottle. The regenerated plants are easily hardened in the greenhouse, and using ISSR-based molecular markers, the genetic homogeneity of the randomly selected hardened plants can be determined.

Brain tuberculoma: a 52-year-old woman case report.

Introduction: One of the most serious extrapulmonary type of tuberculosis that affects people under the age of 40 is brain tuberculoma. They are space-occupying masses of granulomatous tissue that result from hematogenous spread from a distant focus of tuberculous infection by Mycobacterium tuberculosis . Symptoms and radiologic features being nonspecific usually leads to misdiagnosis and mimics a variety of other infectious diseases. Anti-tubercular drugs are essential for the successful treatment of cerebral tuberculomas. Case Report: The authors present a case report of a 52-year-old diabetic woman, who presented to the Emergency Department of a tertiary care hospital and was diagnosed with brain tuberculomas with a brain biopsy. Brain tuberculomas are rare and could be overlooked. Therefore, this is an important consideration in cases with higher suspicions, given the rapid decline in patient condition. Conclusion: Due to their rarity, ambiguous symptoms, and radiographic characteristics, intracranial tuberculomas continue to provide a clinical challenge and must always be considered in the differential diagnosis of cerebral space occupying lesions. As CSF may not yield positivity for both CBNAAT and smear examination, a brain biopsy specimen for culture should always be kept in mind for detecting tuberculoma and initiating anti-tubercular treatment at the earliest.

Storage of Mycobacterium tuberculosis culture isolates in MicrobankTM beads at a South African laboratory.

Background: Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at -70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated. Objective: The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. Methods: In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at -70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. Results: Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. Conclusion: MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. What this study adds: This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at -70 °C and provided a suitable option for medium-term storage of MTBC.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Bioreactor systems for micropropagation of plants: present scenario and future prospects.

Plant micropropagation has been adapted in the fields of agriculture, horticulture, forestry, and other related fields for large-scale production of elite plants. The use of liquid media and adoption of bioreactors have escalated the production of healthy plants. Several liquid-phase, gas-phase, temporary immersion, and other modified bioreactors have been used for plant propagation. The design, principle, operational mode, merits, and demerits of various bioreactors used for the regeneration of propagules, such as bulblets, cormlets, rhizomes, microtubers, shoots (subsequent rooting), and somatic embryos, are discussed here. In addition, various parameters that affect plant regeneration are discussed with suitable examples.

Metabolic Discrimination between Adventitious Roots and Standard Medicinal Part of Atractylodes macrocephala Koidz. Using FT-IR Spectroscopy.

This study aims to examine the metabolic discrimination between in vitro grown adventitious roots and the standard medicinal parts of Atractylodes macrocephala. To achieve this goal, firstly, in vitro culture conditions of adventitious roots such as indole-3-butyric acid (IBA) concentrations, types of media, inorganic salt strength of culture medium, and elicitor types and concentrations were optimized. The optimal culture conditions for proliferation of adventitious roots was found to consist of Murashige and Skoog (MS) medium containing 5 mg L-1 IBA. Whole cell extracts from adventitious roots and the standard medicinal parts of A. macrocephala were subjected to Fourier transform infrared spectroscopy (FT-IR). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from FT-IR spectral data showed that adventitious roots and standard medicinal parts were clearly distinguished in the PCA and PLS-DA score plot. Furthermore, the overall metabolite pattern from adventitious roots was changed depending on the dose-dependent manner of chemicals. These results suggest that FT-IR spectroscopy can be applied as an alternative tool for the screening of higher metabolic root lines and for discriminating metabolic similarity between in vitro grown adventitious roots and the standard medicinal parts. In addition, the adventitious roots proliferation system established in this study can be directly applied as an alternative means for the commercial production of A. macrocephala.

Effect of time, temperature and pH on Mycobacterium tuberculosis culture positivity of gastric aspirate: An experimental study.

BACKGROUND: The culture of gastric aspirate (GA) has been used for bacteriological confirmation of pulmonary tuberculosis in children and patients who are unable to expectorate. Sodium bicarbonate neutralization of gastric aspirates is commonly recommended to increase culture positivity. We aim to study Mycobacterium tuberculosis (MTB) culture positivity of GA collected from confirmed case of pulmonary tuberculosis after storing it at different temperature, pH & time. METHODS: GA specimens from 865 patients of either sex predominately non-expectorating children/adults with suspected pulmonary TB were collected. Gastric lavage was performed in the morning after an overnight fasting (at least 6hrs fasting). The GA specimens were tested by CBNAAT (GeneXpert) and AFB microscopy & those who were positive on CBNAAT were further processed with MTB culture on Growth Indicator Tube (MGIT™) culture. pH neutralized and non-neutralized CBNAAT positive GA specimens were culture within 2 hours of collection and 24 hours after storage at 4 °C & room temperature. RESULTS: MTB was detected in 6.8% of collected GA specimens by CBNAAT. Culture positivity of neutralized GA specimens when processed within 2 hours of collection, was higher compared to paired non-neutralized GA specimens. Neutralized GA specimens had higher contamination rate than non-neutralized GA specimens. Storage of GA specimens at $Deg C had better culture yield than those stored at room temperature. CONCLUSION: Early neutralization of acid in Gastric aspirate (GA) is essential for better culture positivity of M. tuberculosis (MTB). If there is a delay in processing GA, it should be kept at 4 °C after neutralization; however, positivity decreases with time.

Antibiofilm activity from endophyte bacteria, Vibrio cholerae strains, and actinomycetes isolates in liquid and solid culture.

BACKGROUND: Biofilm-associated infections are a global threat to our economy and human health; as such, development of antibiofilm compounds is an urgent need. Our previous study identified eleven environmental isolates of endophyte bacteria, actinomycetes, and two strains of Vibrio cholerae as having strong antibiofilm activity, but only tested crude extracts from liquid culture. Here we grew the same bacteria in solid culture to induce the formation of colony biofilms and the expression of genes that may ultimately produce antibiofilm compounds. This research aimed to compare antibiofilm inhibition and destruction activities between liquid and solid cultures of these eleven environmental isolates against the biofilms of representative pathogenic bacteria. RESULTS: We measured antibiofilm activity using the static antibiofilm assay and crystal violet staining. The majority of our isolates exhibited higher inhibitory antibiofilm activity in liquid media, including all endophyte bacteria, V. cholerae V15a, and actinomycetes strains (CW01, SW03, CW17). However, for V. cholerae strain B32 and two actinomycetes bacteria (TB12 and SW12), the solid crude extracts showed higher inhibitory activity. Regarding destructive antibiofilm activity, many endophyte isolates and V. cholerae strains showed no significant difference between culture methods; the exceptions were endophyte bacteria isolate JerF4 and V. cholerae B32. The liquid extract of isolate JerF4 showed higher destructive activity relative to the corresponding solid culture extract, while for V. cholerae strain B32 the solid extract showed higher activity against some biofilms of pathogenic bacteria. CONCLUSIONS: Culture conditions, namely solid or liquid culture, can influence the activity of culture extracts against biofilms of pathogenic bacteria. We compared the antibiofilm activity and presented the data that majority of isolates showed a higher antibiofilm activity in liquid culture. Interestingly, solid extracts from three isolates (B32, TB12, and SW12) have a better inhibition or/and destruction antibiofilm activity compared to their liquid culture. Further research is needed to characterize the activities of specific metabolites in solid and liquid culture extracts and to determine the mechanisms of their antibiofilm actions.

Production of Escovopsis weberi (Ascomycota: Hypocreales) Mycelial Pellets and Their Effects on Leaf-Cutting Ant Fungal Gardens.

The maintenance of the symbiosis between leaf-cutting ants and their mutualistic fungus Leucoagaricus gongylophorus Singer (Moller) is vital for the survival of both species. The specialist fungal parasite Escovopsis weberi Muchovej & Della Lucia is a threat to this symbiosis, causing severe damage to the fungal garden. Mycelial pellets are resistant fungal structures that can be produced under laboratory conditions. These structures were studied for use in biological pest control, but the production of mycelial pellets has not previously been documented in Escovopsis. One of the aims of this study was to induce Escovopsis weberi to produce mycelial pellets and investigate the potential of these pellets for the control of leaf-cutting ants. We compared the pathogenicity of Escovopsis weberi mycelial pellets and conidia against mini-colonies of Acromyrmex subterraneus subterraneus Forel when applied in the form of baits. Worker ants were able to distinguish mycelial pellets from conidia, as baits with mycelial pellets were more attractive to workers than those with conidia, causing a greater negative impact on colony health. All types of baits containing Escovopsis weberi influenced the foraging activity but only treatments with viable fungal propagules resulted in an increase in the quantity of waste material, with a significant negative impact on the fungal garden biomass. The results provided novel information regarding Escovopsis recognition by worker ants and differences between conidia and mycelial pellet dynamics in leaf-cutting ant colonies, with new perspectives for the biological control of these important pests.

The Influence of Solid and Liquid Systems In Vitro on the Growth and Biosynthetic Characteristics of Microshoot Culture of Spiraea betulifolia ssp. aemiliana.

The paper focuses on the growth dynamics and biosynthetic characteristics of the microshoot culture of Spiraea betulifolia ssp. aemiliana obtained in vitro in agar-solidified and liquid media. Microshoots cultured in either type of media showed similar growth dynamics. The most active culture growth was observed from day 35 to day 60. A comparative analysis of the contents of flavonoids and phenol carboxylic acids showed a higher level of phenol carboxylic acids (5.3-6.84%) and a stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (half-maximal inhibitory concentration: 341 µg/mL) in S. betulifolia ssp. aemiliana microshoots grown in the liquid medium compared to the microshoots cultured in the solid medium. The flavonoid content of the cultured microshoot did not depend on the consistency of the medium. High-performance liquid chromatography (HPLC) was employed to study the profile and levels of phenolic compounds in microshoots, intact plants, and ex vitro-acclimated S. betulifolia ssp. aemiliana plants. The concentration of kaempferol glycosides was found to be higher in microshoots (1.33% in the solid medium, 1.06% in the liquid medium) compared to intact plants and ex vitro-acclimated plants. Thus, the microshoots of S. betulifolia ssp. aemiliana cultured in the liquid medium rapidly increase their biomass and are an inexpensive promising source of biologically active antioxidant substances, mainly phenol carboxylic acids and kaempferol glycosides.

Effect of culture method on storage, plasma, and dry heat treatment resistance of Salmonella enterica serovar Typhimurium on black pepper.

The purpose of this study was to determine the effect of the culture method on the resistance of Salmonella Typhimurium in low water activity foods to storage, plasma, and dry heat. Whole black peppers were used as the model food. S. Typhimurium cultured in liquid broth (tryptic soy broth) or solid agar (tryptic soy agar) and inoculated on whole black pepper was stored or treated with cold plasma or dry heat. Inactivation of S. Typhimurium cultured in liquid medium was higher in all the treatments. Liquid-cultured S. Typhimurium showed higher DPPP = O (diphenyl-1-pyrenylphosphine oxide) values compared to the solid-cultured S. Typhimurium after plasma or dry heat treatment. Furthermore, the unsaturated fatty acid and saturated fatty acid ratio (USFA/SFA) was significantly (P < 0.05) reduced from 0.41 to 0.29 when S. Typhimurium was cultured on solid agar. These results suggested that the use of food-borne pathogens cultured on solid agar is more suitable for low water activity food pasteurization studies.

Comparative transcriptomics of growth metabolism and virulence reveal distinct morphogenic profiles of yeast-like cells and hyphae of the fungus Metarhizium rileyi.

Metarhizium rileyiis an entomopathogenic fungus with a narrow host range which distinguishes it from other Metarhiziumspecies with broad host ranges. This species is also unique because the initial yeast-like growth on solid media is only observed in liquid culture in other Metharizium species. A lack of knowledge about the metabolism and genetic signatures of M. rileyiduring this yeast-like phase on solid and in liquid media is a bottleneck for its large-scale production as a commercial biocontrol agent.In this study wefound that M. rileyiyeast-like cells produced on solid medium infected and killed the important insect pest Spodoptera frugiperda with comparable efficiency as yeast-like cells grown in liquid medium. Secondly, we used comparative transcriptomic analysis to investigate theactive genes and genomic signatures of the M. rileyi yeast-like morphotypes produced on solid and in liquid media. Yeast-like cells grown in liquid medium had upregulated genes relating specifically to signal transduction andparticular membrane transporters. Thirdly, we compared the transcriptomic profiles of yeast-like phases of M. rileyi with those of M. anisopliae. The yeast-like phase of M. rileyi grown on solid medium upregulated unique genes not found in otherMetarhiziumspecies including specific membrane proteins and several virulence factors. Orthologous genes associated with heat shock protein, iron permease, membrane proteins and key virulence traits (e.g. collagen-like protein Mcl1) were upregulated in both species. Comparative transcriptome analyses of gene expression showed more differences than similarities between M. anisopliae and M. rileyi yeast-like cells.

Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi.

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism's genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

A multiple shoot induction system for peptide-mediated gene delivery into plastids in Arabidopsis thaliana, Nicotiana benthamiana, and Fragaria×ananassa.

The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.

Evaluation of a Liquid Media MALDI-TOF MS Protocol for the Identification of Dermatophytes Isolated from Tinea capitis Infections.

One of the most common types of tinea is the superficial infection of the hair and scalp area known as tinea capitis. It is responsible for frequent outbreaks in nurseries and schools and represents a global health problem. Correct identification of the infection agent is essential in the determination of the infection source, epidemiological course, and treatment initiation. The conventional identification methods (direct exam, culture, DNA sequencing) are time-consuming, require experienced staff, are time-consuming, and the latter is expensive for routine identifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is gaining new ground for routine identification of filamentous fungi. The main advantages of MALDI-TOF MS are its rapid and accurate identification capability, relatively low cost, and easy integration into the laboratory routine. Its accuracy heavily depends on the quality of the reference spectra database. Identification of clinical isolates with MALDI-TOF MS protocol requires a sub-culturing step to ensure reliable identification. It can take days to weeks before fungal growth appears on solid medium. In this study, a unique MALDI-TOF MS protocol using liquid cultures of dermatophyte species was developed in order to shorten the turnaround time for the culture and identification of clinical isolates. Material and Method A standard MALDI-TOF MS protocol was adapted for liquid instead of solid cultures. Three different databases were tested. Results Using the liquid media MALDI-TOF MS protocol, a global rate of 62% correct identification (RCI) was obtained, compared with 87% for the protocol based on solid cultures. Trichophyton tonsurans was not correctly identified in all isolates using liquid cultures, with 88% of the isolates misidentified as Trichophyton interdigitale. The turnaround time for primary isolates for the solid and liquid protocols were respectively 11.7 and 11.6 days (no significant difference between both methods (p = 0.96)). Conclusions The newly designed liquid MALDI-TOF MS protocol did not lead to a significantly shorter turnaround time for the identification of dermatophytes isolated from tinea capitis infections. The turnaround time for the method with primary isolates was not significantly lower, and the rate of correct identification decreased remarkably, which emphasizes the need for a sub-culturing step. Using different database did not lead to improvement in turnaround time or rate of correct identification. This study highlights the importance of the medium and the reference database when performing MALDI-TOF MS.

Non-targeted metabonomics and transcriptomics revealed the mechanism of mulberry branch extracts promoting the growth of Sanghuangporus vaninii mycelium.

Sanghuangprous vaninii is a wood-inhabiting fungus, and its mycelium and fruiting body show excellent medicinal values. Mulberry is one of the major hosts of S. vaninii, however, the mechanism of mulberry affecting the growth of S. vaninii has not been reported. In the present study, a mulberry-inhabiting strain of S. vaninii was selected to explore the effects of mulberry branch extracts (MBE) on the growth of the strain. Results showed that MBE could significantly promote the growth of S. vaninii mycelium at the concentration of 0.2 g/l. After 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium was higher by three times compared with that in the control. The non-targeted metabonomic analysis of the culture medium at different culture times and concentrations was conducted to find the key components in MBE that promoted the growth of S. vaninii mycelium. Under the different concentrations of MBE culture for 10 and 16 days, 22 shared differential metabolites were identified. Next, in accordance with the peak value trend of these metabolites, HPLC-MS and liquid culture validation, four components derived from MBE (i.e., scopoletin, kynurenic acid, 3,5-dihydroxybenzoic acid and 2,4-dihydroxybenzoic acid) could significantly increase the growth rate of mycelium at the concentration of 2 mg/l. Transcriptomic and qRT-PCR analyzes showed that MBE could upregulate hydrolase-related genes, such as serine-glycine-asparaginate-histidine (SGNH) hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase, glycosyl hydrolase family 61, cerato-platanin protein and Fet3, which might enhance the nutrient absorption ability of S. vaninii. Importantly, MBE could significantly increase the content of harmine, androstenedione and vesamicol, which have been reported to possess various medicinal effects. Results suggested that MBE could be an excellent additive for liquid culture of S. vaninii mycelium, and these hydrolase-related genes also provided candidate genes for improving the nutrient absorption capacity of S. vaninii.

Theobroma cacao: Somatic Embryogenesis.

A two-step process combining direct and indirect somatic embryogenesis, on solid and liquid medium, respectively is described for Theobroma cacao L. Staminodes and petals from unopened bud flowers are used to induce primary direct embryos. Then, these primary embryos are cut to produce embryogenic calli which will develop secondary embryos. This step of indirect SE allows us to produce large quantities of embryos and to do mass propagation using liquid culture medium. Despite a very strong clone dependency and high batch-to-batch variability, about 80% of T. cacao cultivars respond to somatic embryogenesis and can be propagated by this method.

Rapid and Accurate Identification of Nontuberculous Mycobacteria Directly from Positive Primary MGIT Cultures by MALDI-TOF MS.

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture.