Liquid Culture Medium Research Articles

Blood Supplementation Enhances Bartonella henselae Growth and Molecular Detection of Bacterial DNA in Liquid Culture.

Bacteria of the genus Bartonella, a member of the Alphaproteobacteria, are fastidious, Gram-negative, aerobic bacilli that comprise numerous species, subspecies, and genotypes. Bartonella henselae, with a worldwide distribution, infects cats, dogs, horses, humans, and other mammals. Diagnostically, direct detection of Bartonella henselae in patient blood specimens by culture or molecular methods is required to confirm infection with this bacterium. Enrichment blood culture combined with quantitative PCR (qPCR) or ddPCR enhances the sensitivity of direct detection. The addition of sheep blood to liquid culture media increased the Bartonella henselae DNA concentration compared to controls, additionally improving PCR direct detection sensitivity. IMPORTANCE This study aims to improve diagnostic detection of Bartonella henselae. Patient samples are combined with enriched bacterial cultures aimed at growing Bartonella henselae for the best possible chance at detection. However, current Bartonella growth methods could be improved. The DNA extraction method used by most laboratories should also be optimized. Sheep blood was added to increase the growth of Bartonella henselae and multiple DNA extraction methods were to be compared to each other.

Detection of “Non-culturable” <i>Mycobacterium tuberculosis</i> Cells by Culture Methods

The specific features of the reactivation of “non-culturable” (NC) Mycobacterium tuberculosis (MTB) cells for the following propagation in liquid medium were defined, as well as the approach for the quantification of these cells by culture methods was suggested. When NC cells obtained in vitro were inoculated into standard Sauton’s liquid medium, a prolonged (up to 20 days) lag-phase is observed, in which no cell propagation is occurred. During the lag-phase, dormant cells secrete substances of unknown nature that inhibit or completely arrest the reactivation and growth of cultures when initial dormant cells concentration is above 107–108 cells/mL. Addition of meat-peptone broth (3.25 g/L) into a standard Sauton’s medium with a 10-fold reduced concentration of glycerol significantly stimulated the reactivation and propagation of the initially dormant cells inoculated at a concentration above the threshold. This modification of the medium composition made it possible to detect up to 103 times more cells in the MTB population by the method of the Most Probable Number (MPN) of cells. Increased number of the detected dormant cells in the population (up to an average of 2.5 orders of magnitude) was also achieved by adding to the reactivation medium recombinant protein RpfB (5 ng/mL), a protein of the Rpf family – resuscitation promoting factor of dormant bacteria. Perhaps the action of a Rpf enzyme is related to the products of its enzymatic activity, since an increased MPN value in the dormant cells population was also observed when the products of mycobacteria peptidoglycan hydrolysis obtained by the coaction of RpfB and endopeptidase RipA were added. The addition of sonicated peptidoglycan fragments at a concentration up to 1 μg/mL had a similar effect. The obtained results may be used as approaches to optimize liquid media composition and culture conditions aiming to identify in clinical samples the pathogen of tuberculosis remain in “non-culturable” state.

Jasmonic and Salicylic Acids Enhance Biomass, Total Phenolic Content, and Antioxidant Activity of Adventitious Roots of Acmella radicans (Jacq.) R.K. Jansen Cultured in Shake Flasks.

Acmella radicans (Asteraceae) is a plant native to America. Despite it having medicinal attributes, studies on its phytochemical properties are scarce, and biotechnological studies do not exist for this species. In this study, we established an adventitious root culture from A. radicans internodal segments in shake flasks with indole-3-butyric acid (IBA), and then elicited it with jasmonic acid (JA) and salicylic acid (SA). The total phenolic content and antioxidant activity were evaluated, and a comparison was made using in vitro plantlets and wild plants. Internodal segments with 0.1 mg/L IBA showed 100% root induction and exhibited better growth after transfer to shake flasks with MS liquid culture medium. JA had a significant effect on biomass increase compared to unelicited roots, mainly with 50 µM JA (28%), while SA did not show significant results. Root elicited with 100 µM (SA and JA) showed a 0.34- and 3.9-fold increase, respectively, in total phenolic content (TPC) compared to the control. The antioxidant activity was also significant, and a lower half-maximal inhibitory concentration (IC50) was observed as the AJ concentration increased. Roots elicited with AJ (100 µM) exhibited high antioxidant activity with DPPH (IC50 = 9.4 µg/mL) and ABTS (IC50 = 3.3 µg/mL) assays; these values were close to those for vitamin C (IC50 = 2.0 µg/mL). The TPC and antioxidant activity of in vitro plants and root cultured in shake flasks showed the lowest values in most cases; even the root cultures without elicitation were better than those of a wild plant. In this study, we demonstrated that A. radicans root culture is capable of producing secondary metabolites, while its production and antioxidant activity can be enhanced using jasmonic acid.

In vitro cloning of Bambusa vulgaris Schrad. ex J. C. Wendl.: Effect of culture systems, sucrose and activated charcoal supplementation

Fast-growing forest species with multiple uses, like bamboo, have aroused interest for their silvicultural applications. Bamboo species are a valuable source of renewable raw material, and Bambusa vulgaris is an economically important species. However, there are limitations to large-scale cloning of adult-selected genotypes. This study aimed to evaluate the in vitro cloning of Bambusa vulgaris in different culture systems, sucrose and activated charcoal supplementation by the inter-simple-sequence repeat (ISSR) molecular markers. In vitro bud multiplication and shoot elongation were evaluated in three cultivation systems: semi-solid and liquid culture media, and temporary immersion bioreactor (TIB). The sucrose concentrations, 0 and 30 g L−1 were evaluated in the stages. Both the culture media were supplemented with 2.0 mg L−1 benzylaminopurine (BAP) and 0.5 mg L−1 α-naphthalene acetic acid (NAA). The absence and presence of activated charcoal (100 mg L−1) were evaluated in the in vitro rooting. MS culture medium was supplemented with 2 mg L−1 indole-3-butyric acid (IBA), 1.0 mg L−1 NAA, and 0.5 mg L−1 BAP. Semi-solid culture medium supplemented with 30 g L−1 of sucrose presented superior emission of bud per explant. Liquid culture medium supplemented with 30 g L−1 of sucrose presented the most elongated shoots. Activated charcoal in the culture medium did not influence the adventitious rooting. Micropropagated plants showed genetic fidelity and were clones of the adult selected plant.

On-farm Production of Microbial Entomopathogens for use in Agriculture: Brazil as a Case Study.

In Brazil, the production of beneficial microorganisms by growers exclusively for their own use is a practice known as "on-farm production". Regarding on-farm bioinsecticides, they were initially deployed for pests of perennial and semi-perennial crops in the 1970s but, since 2013, their use has extended to pests of annual crops such as maize, cotton, and soybean. Millions of hectares are currently being treated with these on-farm preparations. Local production reduces costs, meets local needs, and reduces inputs of environmentally damaging chemical pesticides, facilitating establishment of more sustainable agroecosystems. Critics argue that without implementation of stringent quality control measures there is the risk that the on-farm preparations: (1) are contaminated with microbes which may include human pathogens or (2) contain very little active ingredient, impacting on field efficacy. The on-farm fermentation of bacterial insecticides predominates, especially that of Bacillus thuringiensis targeting lepidopteran pests. However, there has been a rapid growth in the past 5years in the production of entomopathogenic fungi, mostly for the control of sap-sucking insects such as whitefly (Bemisia tabaci (Gennadius)) and the corn leafhopper (Dalbulus maidis (DeLong and Wolcott)). In contrast, on-farm production of insect viruses has seen limited growth. Most of the ca. 5 million rural producers in Brazil own small or medium size properties and, although the vast majority still do not practice on-farm production of biopesticides, the topic has aroused interest among them. Many growers who adopt this practice usually use non-sterile containers as fermenters, resulting in poor-quality preparations, and cases of failure have been reported. On the other hand, some informal reports suggest on-farm preparations may be efficacious even when contaminated, what could be explained, at least partially, by the insecticidal secondary metabolites secreted by the pool of microorganisms in the liquid culture media. Indeed, there is insufficient information on efficacy and mode of action of these microbial biopesticides. It is usually the large farms, some with > 20,000ha of continuouscultivated lands, that produce biopesticides with low levels of contamination, as many of them possess advanced production facilities and have access to specialized knowledge and trained staff. Uptake of on-farm biopesticides is expected to continue but the rate of adoption will depend on factors such as the selection of safe, virulent microbial strains and implementation of sound quality control measures (compliance with emerging Brazilian regulations and international standards). The challenges and opportunities of on-farm bioinsecticidesare presented and discussed.

Transcriptomic responses of the fast-growing bacterium Vibrio natriegens during cold-induced loss of culturability.

Vibrio natriegens has massive biotechnological potential owing to its fast growth rate. However, this bacterium rapidly loses its culturability during low-temperature preservation (LTP), the reason for which is still unknown. To reveal the metabolic responses of V. natriegens during LTP, we analyzed and compared the transcriptome before and after 8 days of preservation at 4 or 25 °C (room-temperature preservation (RTP)) in liquid culture medium. Most genes exhibited significant transcriptional responses to LTP. Using gene set enrichment analysis, we compared the transcriptional responses of different V. natriegens Gene Ontology (GO) sets during LTP or RTP. The enrichment of the GO set "SOS response" during LTP, but not RTP, indicated the occurrence of DNA damage during LTP. The GO set "respiratory electron transport chain" was suppressed during LTP and RTP. Although the GO set "response to oxidative stress" was not significantly altered, we observed an increase in reactive oxygen species (ROS) during LTP, suggesting a relationship between ROS and cold-induced loss of culturability (CILC) in V. natriegens. The faster loss of culturability and accumulation of ROS in 20 mL compared to 100 mL of liquid culture medium further suggested a relationship between CILC and oxygen availability. Furthermore, we showed that the deletion of Na+-translocating NADH-ubiquinone oxidoreductase, but not type-II NADH dehydrogenase, accelerated CILC and increased intracellular ROS levels in V. natriegens. These findings will help to understand the cause of CILC which may lead to improving the stability of V. natriegens at low temperatures.

The effect of plant growth regulators on the antioxidant enzyme activity and secondary metabolite production in the cell suspension cultures of Melia azedarach L.

ABSTRACT In the present study, we investigated the effect of plant growth regulators and explant type on callus induction, cell suspension culture growth, antioxidant enzyme activity, and accumulation of rutin, quercetin, and kaempferol in M. azedarach L. cell cultures. Our studies showed that the inflorescence and petiole explants had a high percentage of callus induction compared to the leaf explants, whereas the highest callus growth was obtained from the leaf explants cultured on the MS medium containing 5 mg l−1 NAA and 5 mg l−1 BAP and 5 mg l−1 NAA and 3 mg l−1 kinetin Kin. Furthermore, the highest cell growth was obtained from the leaf and inflorescence calli transferred to liquid MS medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)+1 mg l−1 Kin/BAP. The highest activity of antioxidant enzymes and the lowest protein content was related to the leaf cell suspension cultures in liquid MS medium containing 3 mg l−1 NAA and 3 mg l−1 BAP. The inflorescence cell suspension cultures in the MS medium containing 3 mg l−1 NAA+1 mg l−1 BAP exhibited the highest rutin accumulation (47.536 mg g−1 FW). However, the highest amount of quercetin (8.570 mg g−1 FW) and kaempferol (5.420 mg g−1 FW) accumulation were obtained from the petiole cell suspension cultures in the MS medium with 1 mg l−1 2,4-D +1 mg l−1 Kin.

Combining elicitor treatment of chitosan, methyl jasmonate, and cyclodextrin to induce the generation of immune response bioactive peptides in peanut hairy root culture

The endogenous peptides from peanut hairy root culture were induced upon elicitor treatment with chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT+MeJA+CD. The peptides secreted into the liquid culture medium play an important role in plant signaling and stress responses. By performing gene ontology (GO) analysis, a number of plant proteins involved in biotic and abiotic defense responses were identified, such as endochitinase, defensin, antifungal protein, cationic peroxidase and Bowman-Birk type protease inhibitor A-II. The bioactivity of 14 peptides synthesized from secretome analysis was determined. Peptide BBP1–4, derived from the diverse region of Bowman-Birk type protease inhibitor, displayed high antioxidant activity and mimicked the property of chitinase and β-1,3-glucanase enzymes. The antimicrobial activity against S. aureus, S. typhimurium, and E. coli was evidenced with different peptide concentrations. Additionally, peptide BBP1–4 has the potential to be a useful candidate for an immune response property, as it was found to increase the expression of some pathogenesis-related (PR) proteins and stilbene biosynthesis genes in peanut hairy root tissues. The findings indicate that secreted peptides may play a role in plant responses to both abiotic and biotic stresses. These peptides, which possess bioactive properties, could be considered as potential candidates for use in the pharmaceutical, agricultural, and food industries.

Simultaneous removal of nitrogen and arsenite by heterotrophic nitrification and aerobic denitrification bacterium Hydrogenophaga sp. H7.

Nitrogen and arsenic contaminants often coexist in groundwater, and microbes show the potential for simultaneous removal of nitrogen and arsenic. Here, we reported that Hydrogenophaga sp. H7 was heterotrophic nitrification and aerobic denitrification (HNAD) and arsenite [As(III)] oxidation bacterium. The appearance of nitrogen removal and As(III) oxidation of Hydrogenophaga sp. H7 in liquid culture medium was studied. The effect of carbon source, C/N ratio, temperature, pH values, and shaking speeds were analyzed. The impact of strains H7 treatment with FeCl3 on nitrogen and As(III) in wastewater was assessed. The key pathways that participate in simultaneous nitrogen removal and As(III) oxidation was analyzed by genome and proteomic analysis. Strain H7 presented efficient capacities for simultaneous NH4 +-N, NO3 --N, or NO2 --N removal with As(III) oxidation during aerobic cultivation. Strikingly, the bacterial ability to remove nitrogen and oxidize As(III) has remained high across a wide range of pH values, and shaking speeds, exceeding that of the most commonly reported HNAD bacteria. Additionally, the previous HNAD strains exhibited a high denitrification efficiency, but a suboptimal concentration of nitrogen remained in the wastewater. Here, strain H7 combined with FeCl3 efficiently removed 96.14% of NH4 +-N, 99.08% of NO3 --N, and 94.68% of total nitrogen (TN), and it oxidized 100% of As(III), even at a low nitrogen concentration (35 mg/L). The residues in the wastewater still met the V of Surface Water Environmental Quality Standard of China after five continuous wastewater treatment cycles. Furthermore, genome and proteomic analyses led us to propose that the shortcut nitrification-denitrification pathway and As(III) oxidase AioBA are the key pathways that participate in simultaneous nitrogen removal and As(III) oxidation.

Regulation of Renin Expression by Β1-Integrin in As4.1 Juxtaglomerular Line Cells

(1) Background: Renal dysfunction and hypertension are mutually aggravating factors; however, the details of their interaction remain unclear. In a study using renal tissue from diabetic rats, we found that β1-integrin, a cell-substrate adhesion molecule, is specifically phosphorylated in juxtaglomerular cells that secrete renin, a blood pressure regulator. (2) Methods: A mouse juxtaglomerular cell line (As4.1 cells) was used for the following experiments: drug-induced promotion of β1-integrin phosphorylation/dephosphorylation; knockdown of β1-integrin and the cell adhesion molecule connexin-40 (a candidate for the main body of baroreceptor); and pressurization to atmospheric pressure + 100 mmHg. culture in hypotonic liquid medium. The expression of renin under these conditions was measured by qRT-PCR. (3) Results: Phosphorylation of β1-integrin suppressed the expression of renin, while dephosphorylation conversely promoted it. β1-integrin and connexin-40 knockdown both promoted the expression of renin. Pneumatic pressurization and hypotonic medium culture both decreased the expression of renin, which was restored by the knockdown of β1-integrin. (4) Conclusions: β1-integrin plays an inhibitory role in the regulation of the expression of renin, which may be controlled by phosphorylation and dephosphorylation. It is hypothesized that β1-integrin and other adhesion factors regulate the expression of renin by altering the sensitivity of baroreceptors on the plasma membrane.

Identification of seven types of pili in Mycobacterium tuberculosis: Using atomic force microscopy.

Pili are polymeric, hydrophobic, proteinaceous structures generally composed of a major repeating subunit called pilin and, in some cases, a minor tip-associated adhesin subunit. Pili are involved in many virulence-associated functions, such as biofilm formation, adherence, and colonization of mucosal surfaces. Mycobacterium tuberculosis (MTB) strains were isolated from clinically and laboratory-confirmed cases of tuberculosis (TB). The TB isolates were subjected to the Xpert MTB/rifampicin test and then, further susceptibility testing was performed on them against first- and second-line drugs using proportional methods. Thereafter, the selected isolates were subculture in Dubos Tween-albumin liquid culture medium, and at their exponential growth phase (OD600 = 0.05 (5 × 106 colony-forming unit/mL), cells were observed under atomic force microscopy (AFM). For each isolate, 15-20 steel sample packs were prepared and observed under AFM. Here, the data presented are the result of average observation. Under AFM, seven different types of pili were detected, out of which four types, i.e., Type III, Type IV secretion pili, and Type IV-like pili, curli-like pili (MTP) were similar to reported pili in Gram-negative and Gram-positive bacteria. Whereas the other three forms, i.e., Type V (relief funnel pili), Type VI (adhesion tapering), and Type VII (adhesion flap pili), were newly identified and named according to their appearance. Both Types of IV pili were detected in all clinical isolates irrespective of their susceptibility patterns, although significant differences were observed from the side of their protruding. Type Curli pili is similar in appearance in all clinical isolates. Types VI and VII were detected only in extensively drug-resistant and totally drug-resistant-TB isolates (100%). The Type III pili (secretion needle pili) was present in both susceptible- and drug-resistant bacilli, although in drug-resistant strains, we found a considerable difference in their length (50 μ ±10 nm in length) and sometimes, they also had tapering at end. The Type V pili was seen in susceptible isolates but it was at the resting stage (100%; lying aside of cell wall) whereas in drug-resistant isolates, they were getting apart from the cell wall of bacilli with a clear tapering or funnel shape structure. The results of this study highlight the importance of new types of pili expressions in respect of susceptibility patterns in TB. The identified new types of pili would be promising approaches for the treatment and prevention of drug-resistant TB, which needs further investigation.

Rhamnogalacturonan-I as a nematode chemoattractant from Lotus corniculatus L. super-growing root culture.

The soil houses a tremendous amount of micro-organisms, many of which are plant parasites and pathogens by feeding off plant roots for sustenance. Such root pathogens and parasites often rely on plant-secreted signaling molecules in the rhizosphere as host guidance cues. Here we describe the isolation and characterization of a chemoattractant of plant-parasitic root-knot nematodes (Meloidogyne incognita, RKN). The Super-growing Root (SR) culture, consisting of excised roots from the legume species Lotus corniculatus L., was found to strongly attract infective RKN juveniles and actively secrete chemoattractants into the liquid culture media. The chemo-attractant in the culture media supernatant was purified using hydrophobicity and anion exchange chromatography, and found to be enriched in carbohydrates. Monosaccharide analyses suggest the chemo-attractant contains a wide array of sugars, but is enriched in arabinose, galactose and galacturonic acid. This purified chemoattractant was shown to contain pectin, specifically anti-rhamnogalacturonan-I and anti-arabinogalactan protein epitopes but not anti-homogalacturonan epitopes. More importantly, the arabinose and galactose sidechain groups were found to be essential for RKN-attracting activities. This chemo-attractant appears to be specific to M. incognita, as it wasn't effective in attracting other Meloidogyne species nor Caenorhabditis elegans. This is the first report to identify the nematode attractant purified from root exudate of L corniculatus L. Our findings re-enforce pectic carbohydrates as important chemicals mediating micro-organism chemotaxis in the soil, and also highlight the unexpected utilities of the SR culture system in root pathogen research.

A Maverick-like cluster in the genome of a pathogenic, moderately virulent strain of Gallibacterium anatis, ESV200, a transient biofilm producer.

Gallibacterium anatis causes gallibacteriosis in birds. These bacteria produce biofilms and secrete several fimbrial appendages as tools to cause disease in animals. G. anatis strains contain up to three types of fimbriae. Complete genome sequencing is the strategy currently used to determine variations in the gene content of G. anatis, although today only the completely circularized genome of G. anatis UMN179 is available. The appearance of growth of various strains of G. anatis in liquid culture medium was studied. Biofilm production and how the amount of biofilm was affected by DNase, Proteinase K, and Pronase E enzymes were analyzed. Fimbrial gene expression was performed by protein analysis and qRT-PCR. In an avian model, the pathogenesis generated by the strains G. anatis ESV200 and 12656-12 was investigated. Using bioinformatic tools, the complete genome of G. anatis ESV200 was comparatively studied to search for virulence factors that would help explain the pathogenic behavior of this strain. G. anatis ESV200 strain differs from the 12656-12 strain because it produces a biofilm at 20%. G. anatis ESV200 strain express fimbrial genes and produces biofilm but with a different structure than that observed for strain 12656-12. ESV200 and 12656-12 strains are pathogenic for chickens, although the latter is the most virulent. Here, we show that the complete genome of the ESV200 strain is similar to that of the UNM179 strain. However, these strains have evolved with many structural rearrangements; the most striking chromosomal arrangement is a Maverick-like element present in the ESV200 strain.

Real-Time Detection of the Bacterial Biofilm Formation Stages Using QCM-Based Sensors

Bacterial biofilms are a major cause of harm related to medical infections and biofouling. Thus, 80% of total infections are caused by biofilm-forming microorganisms. Consequently, knowledge of biofilm formation stages is crucial to develop effective treatments to prevent their formation in medical implants, tools, and devices. For this purpose, quartz crystal microbalance (QCM) sensors are becoming a good alternative to analytical methods for the real-time monitoring of bacterial growth in liquid media culture. In a previous paper, the authors described an affordable multi-channel measurement instrument based on QCM sensors. However, in order to validate its correct operation, complementary experimental measurements based on bacterial biofilm growth were performed. In this work, the experimental measurements that allow the identification of the different biofilm formation stages are described. The results obtained are discussed.

Association between the severity of histopathological lesions and Mycobacterium avium subspecies paratuberculosis (MAP) molecular diversity in cattle in southern Chile.

The objective was to evaluate the association between the severity of histopathological lesions caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and the molecular diversity of this pathogen. Blood, ileum, and mesenteric lymph node samples were collected at slaughter, from 1,352 adult cattle [source population 1 (SP1)]. In addition, 42 dairy herds (n = 4,963 cows) were followed for 2 years, and samples from compatible paratuberculosis clinical cases [source population 2 (SP2)] were collected. MAP infection was confirmed using an ELISA test, liquid media culture, and PCR. Isolates were genotyped using five MIRU-VNTR markers. Tissues from confirmed samples were subjected to a histopathological examination. A histopathological severity score (HSS) system was developed and used to grade (0 to 5) the magnitude of lesions caused by MAP. In general, the HSS system assesses the number of foci and degree of macrophage infiltration, together with the presence of multinucleated giant cells (MGCs) and acid-fast bacilli (AFB), in addition to the fusion of the intestinal villi and hyperplasia of the crypts. Despite the large sampling effort, only 79 MAP isolates were successfully genotyped, where 19 different haplotypes were described. A mixed-effect Poisson regression model was used to assess the relationship between haplotypes and HSS values. The model was controlled by animal age, and the farm was used as a random effect. Haplotypes were grouped based on their relative frequency: the most frequent haplotype (group i, 49.4%), the second most frequent haplotype (group ii, 12.7%), and all other haplotypes (group iii, 37.9%). Model outputs indicated that group i had significantly higher HSS values than group iii. In addition, group i was also associated with higher optical density (OD) values of the ELISA test. These results support the existence of differences in pathogenicity between MAP haplotypes. However, results were based on a relatively small sample size; thus, these should be taken with caution. Despite this, study findings suggest that haplotypes would be associated with differences in disease progression, where the dominant haplotype tends to generate more severe lesions, which could be linked to a greater shed of MAP cells than non-dominant haplotypes, increasing their chances of transmission.

Role of line probe assay in detection of Mycobacterium tuberculosis in children with pulmonary tuberculosis

BackgroundIndia is the highest TB burden country in the world with almost 27 lakh cases reported in 2019. Pediatric tuberculosis in India accounts for almost 31% of global TB burden. Despite such high mortality and morbidity in children, diagnosis of pulmonary TB in children still remains very challenging. Material and methodsThis cross-sectional study was conducted in a tertiary care hospital in Delhi, India. Children between 1 and 12 months with clinical suspicion of pulmonary tuberculosis who had not previously taken ATT were included.Early morning gastric aspirate samples were collected after overnight fasting, on two days. Both days sputum sample were subjected to sputum smear microscopy and one of the two samples was subjected to line probe assay (LPA), cartridge based nucleic acid amplification (CBNAAT) and mycobacterium growth in tube (MGIT-960). Results84 children with pulmonary tuberculosis were enrolled. The most common presenting complaint was fever seen in 83 patients (98.8%). Only 17 (20.24%) were sputum smear positive by Ziehl- Neelsen (ZN) staining. LPA was positive in 47 (55.95%) samples and among these positive samples both INH and RIF resistance was detected in 2 (4.26%) samples. CBNAAT was positive in 53 patients (63%). Growth in liquid culture media (MGIT 960) was observed in 44 (52.38%) samples. Among 17 smear positive samples, LPA was detected in 14 (82.35%) samples and among 67 smear negative sample LPA was detected in 33 (49.25%) samples. LPA had 63.46% sensitivity, 100% specificity in detecting mycobacterium tuberculosis. DiscussionWHO's recommendation for using LPAs has been limited to culture isolates or smear-positive sputum specimens. New data has since been generated on the use of LPAs as newer versions of LPA have been developed over past few years.Previous studies conducted using LPA version 1.0 reported much lower detection rate of mycobacterium tuberculosis in smear negative specimens.With the availability of newer versions such as Hain GenoTypeMTBDRplusversion 2 and Nipro NTM + MDRTB detection kit 2, the diagnostic utility of LPA may be enhanced.

High Frequencies Repetitive Shoots organogenesis of Potato (Solanum TuberosumL.) Under Partial Immersion Bioreactor Culture

Potato plays important role in nutritional security, poverty alleviation, and income generation in Indonesia. Potato production restricted and inconsistent by lower quality of seedlings application. Solid medium technique for long time has been using for producing pathogen free plants. This procedure has typically costly as labor intensive and longer durability of production. Liquid culture medium allows the close contact to explants which stimulates and facilitates the nutrients and hormones uptake leading to higher rate multiplication and shoot and root growth of higher quality seedling mother plants. The aim of study is to introduce partial immerse bioreactor system (PIB) for high frequencies of granola Kembang (GK) organogenesis and survival rate during weaning stage in nursery. A 20 shoots of GK were transferred to PIB containing MS medium aerated with 0.5, 1.0 and 1.5 vvm air atmosphere during 30 days of culture. High rate repetitive shoots organogenesis were observed from PIB culture, significantly different to semisolid culture. Averagely 500, 616 and 602 new shoots were harvested from 0.5, 1.0 and 1.5 vvm treatment respectively, which are 30, 37 and 36 folds compared to semisolid culture. Approximately 95-98% of regenerated shoots were survived in acclimatization chamber for both PIB and semisolid culture.

In vitro propagation, shoot regeneration, callus induction, and suspension from lamina explants of Sorbus caloneura.

Sorbus caloneura has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of S. caloneura were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.5 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of S. caloneura.

Biodegradation of the Pesticides Bifenthrin and Fipronil by Bacillus Isolated from Orange Leaves.

The pyrethroid bifenthrin and the phenylpyrazole fipronil are widely employed insecticides, and their extensive use became an environmental issue. Therefore, this study evaluated their biodegradation employing bacterial strains of Bacillus species isolated from leaves of orange trees, aiming at new biocatalysts with high efficiency for use singly and in consortium. Experiments were performed in liquid culture medium at controlled temperature and stirring (32°C, 130rpm). After 5days, residual quantificationby HPLC-UV/Vis showed that Bacillus amyloliquefaciens RFD1C presented 93% biodegradation of fipronil (10.0mg.L-1 initial concentration) and UPLC-HRMS analyses identified the metabolite fipronil sulfone. Moreover, Bacillus pseudomycoides 3RF2C showed a biodegradation of 88% bifenthrin (30.0mg.L-1 initial concentration). A consortium composed of the 8 isolated strains biodegraded 81% fipronil and 51% bifenthrin, showing that this approach did not promote better results than the most efficient strains employed singly, although high rates of biodegradation were observed. In conclusion, bacteria of the Bacillus genus isolated from leaves of citrus biodegraded these pesticides widely applied to crops, showing the importance of the plant microbiome for degradation of toxic xenobiotics.

Identification of Enolase 1 as a Potential Target for Magnaporthe oryzae: Integrated Proteomic and Molecular Dynamics Simulation

Rice blast is an essential factor affecting rice yield and quality, which is caused by Magnaporthe oryzae (M. oryzae). Isobavachalcone (IBC) is a botanical fungicide derived from the seed extract of the Leguminosae plant Psoralea corylifolia L. and has shown an excellent rice blast control effect in field applications. To explore the potential targets of rice blast control, the analysis of the differentially expressed proteins (DEPs) between the liquid culture medium of mycelium treated by 10 mg/L of IBC for 2 h and the control group indicated that Enolase 1 (ENO1) was the most significantly down-regulated DEP with a fold change value of 0.305. In vitro experiments showed that after treating liquid culture mycelium with 10 mg/L of IBC for 0.5, 1, 2, 4, and 8 h, the enzymatic activity of ENO1 in the IBC experimental groups was 0.97, 0.75, 0.52, 0.44, and 0.39 times as much as in the control groups, respectively. To further explore the molecular interaction and binding mode between IBC and ENO1, the three-dimensional structure of ENO1 was established based on homology modeling. Molecular docking and molecular dynamics simulation showed that IBC had a pi-pi stacking effect with the residue TYR_365, a hydrogen bond interaction with the residue ARG_393, and hydrophobic interactions with non-polar residues ALA_361, LYS_362, and VAL_371 of ENO1. These findings indicated that ENO1 is a potential target of M. oryzae, which would pave the way for screening novel effective fungicides targeting ENO1.