Candidate Strains Research Articles

Alterations in Faecal Microbiota and Elevated Levels of Intestinal IgA Following Oral Administration of Lacticaseibacillus casei in mice.

The intestinal microbiota has been identified as a crucial regulator of the overall health, with studies describing its influence in a variety of disorders and developmental processes throughout the body. A widely accepted approach of influencing the microbiota and regulating its functionality in health or disease is the consumption of probiotics. In this study, we aimed to identify the impact of probiotic Lacticaseibacillus casei ATCC393 on the intestinal microbiota of mice and circulating soluble products of microbial origin or the immune system. Investigation of the gut microflora using next-generation sequencing analysis revealed alterations in the microbial populations following consumption of the probiotic. Abundance of taxa classified as Muribaculaceae was increased in lactobacilli-fed animals, while abundance of taxa classified as Lachnospiraceae and Oscillospiraceae was decreased. In addition, the composition of the intestinal microbiota was modified by the administration of L. casei, as evident by the clustering of test subjects when inspecting beta diversity, without however any significant effect on the alpha diversity of the animals. Finally, production of IgA in the intestinal lumen of mice that had received the microorganism was significantly increased, as was the concentration of lactic acid, while levels of acetic acid were noticeably lower in the L. casei group. The findings suggest that L. casei can be considered a potential candidate strain for the modulation of intestinal homeostasis and a component of dietary interventions aiming to improve overall health.

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.

Interactions between Indigenous Endophyte Bacillus subtilis L1-21 and Nutrients inside Citrus in Reducing Huanglongbing Pathogen Candidatus Liberibacter Asiaticus.

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay.

In Silico Prediction of Novel Probiotic Species Limiting Pathogenic Vibrio Growth Using Constraint-Based Genome Scale Metabolic Modeling.

The prevalence of bacterial diseases and the application of probiotics to prevent them is a common practice in shrimp aquaculture. A wide range of bacterial species/strains is utilized in probiotic formulations, with proven beneficial effects. However, knowledge of their role in inhibiting the growth of a specific pathogen is restricted. In this study, we employed constraint-based genome-scale metabolic modeling approach to screen and identify the beneficial bacteria capable of limiting the growth of V. harveyi, a common pathogen in shrimp culture. Genome-scale models were built for 194 species (including strains from the genera Bacillus, Lactobacillus, and Lactococcus and the pathogenic strain V. harveyi) to explore the metabolic potential of these strains under different nutrient conditions in a consortium. In silico-based phenotypic analysis on 193 paired models predicted six candidate strains with growth enhancement and pathogen suppression. Growth simulations reveal that mannitol and glucoronate environments mediate parasitic interactions in a pairwise community. Furthermore, in a mannitol environment, the shortlisted six strains were purely metabolite consumers without donating metabolites to V. harveyi. The production of acetate by the screened species in a paired community suggests the natural metabolic end product’s role in limiting pathogen survival. Our study employing in silico approach successfully predicted three novel candidate strains for probiotic applications, namely, Bacillus sp 1 (identified as B. licheniformis in this study), Bacillus weihaiensis Alg07, and Lactobacillus lindneri TMW 1.1993. The study is the first to apply genomic-scale metabolic models for aquaculture applications to detect bacterial species limiting Vibrio harveyi growth.

Responses of soil bacterial and fungal communities to the long-term monoculture of grapevine.

Soil microorganisms are essential for the long-term sustainability of agricultural ecosystems. However, continuous grapevine replanting can disrupt the stability of soil microbial communities. We investigated the bacterial and fungal abundance, diversity, and community composition in rhizosphere soils with continuous grapevine replanting for 5, 6, 7 (Y5, Y6, and Y7; short-term), and 20 (Y20; long-term) years with high-throughput sequencing. Results showed that diversities and abundances of bacterial and fungal communities in Y20 were significantly lower than in other samples. The bacterial and fungal community compositions were markedly affected by the replanting time and planting year. After short-term grapevine replanting, relative abundances of potential beneficial bacteria and harmful fungi in rhizosphere soils were higher compared to long-term planting. Bacterial and fungal communities were significantly correlated with available nitrogen (AN), available phosphorus, available potassium (AK), and pH. AK and AN were the primary soil factors related to the shift of bacterial and fungal communities. Bacterial and fungal co-occurrence patterns were remarkably affected by replanting time, showing that fallow land harbored co-occurrence networks more complex than those in other groups, with the Y20 group showing the lowest complexity. Then, we isolated the dominant fungi in grapevine rhizosphere soil after continuous replanting and verified the harmful effects of three candidate strains through pot experiments. The results showed that 12days post-treating the soil with fungal spore suspensions significantly inhibited grapevine seedlings' growth, whereas Fusarium solani inhibited plant growth. Overall, we showed that F. solani might be a potentially harmful fungus related to grapevine replant diseases. KEY POINTS: • Continuous grapevine planting reduced soil microbe diversities/abundances. • Beneficial bacteria and harmful fungi increased after short-term replanting. • F. solani may be a harmful fungus related to grapevine replant diseases.

Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

Prevalence and Evolution of Noroviruses between 1966 and 2019, Implications for Vaccine Design.

Noroviruses (NoVs), a group of single-stranded RNA viruses causing epidemic acute gastroenteritis in humans, are highly diverse, consisting of multiple genogroups with >30 genotypes. Their continual evolutions make NoV vaccine design and development difficult. Here, we report a study of NoV sequences obtained from a population-based diarrhea surveillance in Zhengding County of Hebei Province spanning from 2001 to 2019 and those available in the GenBank database from 1966 to 2019. NoV genotypes and/or variants that may evade immunity were screened and identified based on primary and conformational structures for vaccine design. We selected 366, 301, 139, 74 and 495 complete VP1-coding nucleotide sequences representing the predominant genotypes of GII.4, GII.2, GII.3, GII.6 and GII.17, respectively. A total of 16 distinct GII.4 variants were identified, showing a typical linear evolutionary pattern of variant replacement, while only 1–4 variants of the other genotypes were found to co-circulate over the 40–50-year period without typical variant replacement. The vaccine strain GII.4c is close to variant Sydney_2012 (0.053) in their primary structure, but they are distinct at epitopes A and E in conformations. Our data suggested GII.4 variant Sydney_2012, GII.2 variant A, a GII.3 strain, GII.6 variants B and C and GII.17 variant D are primary candidate strains for NoV vaccine development.

Animal health and food safety risk assessments.

Animal health risk assessment is one of the key tasks of Veterinary Services. There are well-established protocols created by the World Organisation for Animal Health and Codex Alimentarius Commission for assessing risk. They cover terrestrial and aquatic animals and zoonotic infectious diseases, food safety, and the environment, taking into consideration the connections between them. Significant effort has been made in developing methods to estimate the probability, and consequences, of infectious disease incursion in diseasefree countries through legal or illegal trade or via the movements of insects and wildlife. Additional efforts have been made in the design of prevention strategies and contingency plans. Concerns about possible pandemics of avian influenza continue to be important motivation for monitoring viruses for selection of vaccine candidate strains. The recent COVID-19 pandemic was zoonotic in nature and caused extensive disruption throughout the world. Tools are becoming available for quantitative food safety risk assessments for bacteria, toxins, viruses, and antimicrobial resistance genes, including tools that allow simulations for the selection of effective control options. Applying participatory techniques facilitates the conduct of risk analysis in low- and middle-income countries. In internationally established frameworks, risk assessment is the first step towards elimination of important infectious diseases in endemic countries and it is an important contributor to the reduction of disease risks. Quantitative and qualitative socio-economic and behavioural studies have been developed to design risk management options that are acceptable and sustainable for actors throughout value chains.

Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study

The present study aimed to carry out DNA extraction and polymerase chain reaction (PCR) amplification to isolate the physiological and phenotypic traits of Candida albicans and Candida dubliniensis in denture wearer patients with and without denture-induced stomatitis. A total sample size of 160 participants were divided into two equal groups (80 each), patients in the study group having 40 males and 40 females with Newton type II denture stomatitis, and in the control group, healthy 40 males and 40 females those who wear complete denture were selected. All the samples were collected from the hard palate with a sterile swab and inoculated on CHROM agar plate; samples that displayed dark green colored colonies were selected for DNA extraction. DNA isolation was done on agarose gel using electrophoresis. Biorad gene identification was used. Strands depicting the presence of DNA in particular samples were identified, and further standardization of the procedure was done. PCR amplification was done using Candida species-specific primer, preset to the hyphal wall of the protein 1 gene with the CRR forward and reverse primers, under strict standard conditions with reverse transcriptase technique. Results showed that prevalence of C. albicans was more in females with denture stomatitis which was 67.50% than in males, i.e., 52.50%, and prevalence of C. dubliniensis was found in one female and in one male who were having denture stomatitis and it was not isolated from patients without denture stomatitis. Statistical analyses were performed using the Chi-square test. Denture stomatitis is the most common problem faced by long-term denture wearers, with C. albicans as one of the causative organisms. However, recent findings show an emerging pathogenic yeast species, C. dubliniensis, which was isolated from denture-induced stomatitis candidates in the present study, which is closely related to the C. albicans species. The identification of candidal strains causing denture stomatitis with DNA extraction and PCR amplification and its management by determination of its susceptibility to antifungals may improve the treatment outcome of the same. Candidiasis is the most frequently seen mucocutaneous infection of the oral cavity especially in denture wearers. It is caused mainly by the genus Candida. C. dubliniensis is phenotypically similar but genotypically different from C. albicans. This affects the treatment outcome drastically as there is enough literature suggesting resistance to the common antifungal drugs. Hence, drugs like fungus-specific calcineurin inhibitors should also be considered in resistant patients. Therefore, DNA identification of Candida genus plays a major role in deciding the treatment outcome. How to cite this article: Patil A, Boparai NK, Shankargouda SB, et al. Candida dubliniensis: The New Culprit on the Block Causing Denture Stomatitis? An In Vivo Study. J Contemp Dent Pract 2021;22(5):517-521.

Screening of High 1,2-Propanediol Production by Lactobacillus buchneri Strains and Their Effects on Fermentation Characteristics and Aerobic Stability of Whole-Plant Corn Silage

The study was conducted to screen high 1,2-propanediol produced by Lactobacillus buchneri strains, isolated from baled silages stored for 1 or 2 years, and to evaluate their effects on fermentation quality and aerobic stability of whole-plant corn silage. In total, 31 L. buchneri strains were isolated from alfalfa, whole-plant corn and oat silages. Based on growth performance and 1,2-propanediol and acetic acid production, two strains, L. buchneri 9-2 and L. buchneri 10-1, from alfalfa silage, were further assessed in an ensiling trial on whole-plant corn. The corn silage inoculated with L. buchneri 9-2 or L. buchneri 10-1 had a higher concentration of 1,2-propanediol (34.7 or 34.6 g/kg dry matter (DM)) and acetic acid (47.2 or 45.9 g/kg DM) in comparison with L. buchneri 40788 (reference strain) treated silage (19.5 and 35.9 g/kg DM) after 90 d of fermentation. In addition, these two strains performed better in improving silage aerobic stability relative to control and L. buchneri 40788. The results above indicated that L. buchneri 9-2 and L. buchneri 10-1 could be candidate strains to increase 1,2-propanediol and acetic acid concentrations and improve the aerobic stability of whole-plant corn silage.

Bacillus velezensis WZ-37, a New Broad-Spectrum Biocontrol Strain, Promotes the Growth of Tomato Seedlings

A biological control agent is arguably an ideal alternative to chemical fungicide for the prevention and control of gray mold disease. During this process, a biological control produces low levels of pollution, generates few residues that pose no risk to the environment, and pathogens cannot gain resistance to it easily. A new antifungal strain isolated from plant rhizosphere exhibited high antifungal activity against the phytopathogens Botrytis cinerea, Fusarium oxysporum f. sp. cucumerinum, F. moniliforme, Sclerotinia sclerotiorum, Colletotrichum orbiculare, Alternaria nees, F. equiseti, and F. oxysporum f. sp. lycopersici. It was identified as Bacillus velezensis WZ-37 by morphological and physiological indices and comparisons of 16S rRNA and gyrB genes. WZ-37 can significantly inhibit the mycelia growth of B. cinerea by 96.97%. It can reduce a tomato fruit’s decay rate after 21 days of storage by 33.33% (13.34% less for the control) without significantly affecting its firmness and soluble solids. Plant height, stem diameter, and the fresh and dry weight of tomato seedlings were significantly increased when their seeds were soaked in a WZ-37 suspension (106 cfu/mL) for 3 h and grown for 21 days in soil. WZ-37 has broad-spectrum biocontrol and can prolong a tomato’s storage period and enhance its seedlings’ growth, making it a promising candidate strain for broad-spectrum biocontrol applications in agriculture.

Assessment of fitness of foot-and-mouth disease virus A IND 27/2011 as candidate vaccine strain.

Antigenic profiling of recent field outbreak strains of foot-and-mouth disease virus (FMDV) serotype A in India has revealed considerable antigenic drift from the vaccine strain, A IND 40/2000, necessitating the selection of a new strain. The complete genome sequence of A IND 27/2011 was analysed. Vaccine quality attributes of the new candidate strain including potency as an inactivated vaccine in cattle were evaluated. The capsid coding region of A IND 27/2011 showed variation at eight antigenically critical amino acid positions from that of A IND 40/2000. The strain suited well with traits required by a vaccine in terms of its adaptability to adherent and suspension cell line, its immunogenicity, and potency as an inactivated vaccine formulation in cattle. Complete protection was observed upon homologous virus challenge at 4weeks post-vaccination. Taken together, these data demonstrate the suitability of A IND 27/2011 as an effective vaccine strain of FMDV serotype A.

Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.

Natural Killer Cell Activation by Weissella cibaria JW15 Isolated from Kimchi

Natural killer (NK) cells are innate immune cells with the capacity to eliminate virus-infected and cancer cells. Therefore, enhancing NK cell activity remains a promising strategy to control viral infection and cancer development. Recent studies highlighted the immunostimulatory effect of lactic acid bacteria (LAB), especially via activation of NK cells. Among LAB, Weissella cibaria JW15, isolated from kimchi, activated splenocytes and enhanced immune responses, but whether JW15 enhanced NK cell activity was unclear. In this study, we investigated if JW15 affects NK cell activity and a possible role of exopolysaccharides (EPS) produced by JW15 in immune-stimulatory activity. Splenocytes or NK cells isolated from mice were treated with live JW15 or EPS, and then evaluated for cell surface levels of activation markers, intracellular levels of granzyme B and perforin, cytokine production, including IFN-γ, and cytotoxicity against tumor cells. Our results revealed that JW15 induced the activation of NK cells, as shown by increased NK cell cytotoxicity activity, as well as increased activation markers and IFN-γ production. In addition, EPS activated NK cells and splenocytes, leading to the secretion of various cytokines. Furthermore, oral administration of live JW15 increased NK cell cytotoxicity. Taken together, our findings suggest JW15 as a promising probiotic candidate strain with the immune-enhancing potential to prevent or treat viral infection and cancer.

Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains.

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples.

The rescue and selection of thermally stable type O vaccine candidate strains of foot-and-mouth disease virus.

Inactivated foot-and-mouth disease virus (FMDV) vaccines have been used widely to control foot-and-mouth disease (FMD). However, the virions (146S) of this virus are easily dissociated into pentamer subunits (12S), which limits the immune protective efficacy of inactivated vaccines when the temperature is higher than 30 °C. A cold-chain system can maintain the quality of the vaccines, but such systems are usually not reliable in limited-resource settings. Thus, it is imperative to improve the thermostability of vaccine strains to guarantee the quality of the vaccines. In this study, four recombinant FMDV strains containing single or multiple amino acid substitutions in the structural proteins were rescued using a previously constructed FMDV type O full-length infectious clone (pO/DY-VP1). We found that single or multiple amino acid substitutions in the structural proteins affected viral replication to different degrees. Furthermore, the heat and acid stability of the recombinant viruses was significantly increased when compared with the parental virus. Three thermally stable recombinant viruses (rHN/DY-VP1Y2098F, rHN/DY-VP1V2090A-S2093H, and rHN/DY-VP1V2090A-S2093H-Y2098F) were prepared as inactivated vaccines to immunize pigs. Blood samples were collected every week to prepare sera, and a virus neutralization test showed that the substitutions S2093H and Y2098F, separately or in combination, did not affect the immunogenicity of the virus, but the Y2098F mutation increased the thermostability significantly (p < 0.05). Therefore, the rHN/DY-VP1Y2098F mutant should be considered for use in future vaccines.

Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

Antioxidant and Anti-Inflammatory Properties of Probiotic Candidate Strains Isolated during Fermentation of Agave (Agave angustifolia Haw).

Agave species are a source of diverse products for human use, such as food, fiber, and beverages, which include mezcal, a distilled beverage produced by spontaneous fermentation. Agave is an excellent source of high amounts of sugars, minerals, and phenolic compounds, which favor the growth of lactic acid bacteria (LAB) and yeast communities. In this work, 20 promising LAB strains with probiotic characteristics were isolated from the agave fermentation stage in mezcal production. The strains belonged to Lactobacillus plantarum (15), Lactobacillus rhamnosus (2), Enterococcus faecium (2), and Lactococcus lactis (1). These isolates were characterized for their resistance under gastrointestinal conditions, such as lysozyme, acid pH, and bile salts. In addition, the adherence of these LABs to human intestinal epithelial cells (Caco-2 and HT-29 cells) was tested in vitro and their antioxidant and immunomodulatory profile was determined using cellular models. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains were selected for their antioxidant properties, and their capacities in an oxidative stress model in intestinal epithelial cells IECs (Caco-2 and HT-29 cells) in the presence of hydrogen peroxide were evaluated. Interestingly, Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 and LM19 strains showed anti-inflammatory properties in TNF-α-stimulated HT-29 cells. Subsequently, bacterial strains exhibiting antioxidant and anti-inflammatory properties were tested in vivo in a mouse model with dinitrobenzene sulfonic acid (DNBS)-induced chronic colitis. Weight loss, intestinal permeability, and cytokine profiles were measured in mice as indicators of inflammation. One of the selected strains, Lactobacillus plantarum LM17, improved the health of the mice, as observed by reduced weight loss, and significantly decreased intestinal permeability. Altogether, our results demonstrate the potential of LAB (and lactobacilli in particular) isolated from the agave fermentation stage in mezcal production. Lactobacillus rhamnosus LM07 and Lactobacillus plantarum LM17 strains represent potential candidates for developing new probiotic supplements to treat inflammatory bowel disease (IBD).

Pathogenicity and immunogenicity of a gI/gE/TK/UL13-gene-deleted variant pseudorabies virus strain in swine

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease.

Insertion of cellulase gene in yeast expression vector (pYY1 and pWYH257 Plasmid) as a candidate for cellulosic ethanol-producing strain

Abstract. Muammar A, Oktaviana I, Chohansandhika J, Handayani NSN. 2021. Insertion of cellulase gene in yeast expression vector (pYY1 and pWYH257 Plasmid) as a candidate for cellulosic ethanol-producing strain. Biodiversitas 22: 2715-2721. Production of ethanol from cellulose-based materials often faces obstacles because it requires extensive pretreatment and large amounts of cellulase enzymes. Another method that is more efficient is by inserting cellulase genes from cellulolytic microorganisms such as Aspergillus niger which possess cellulase enzymes encoded by cbhA gene. This gene is inserted into the host which will then be used to produce candidate strains able to produce cellulosic ethanol. This study aimed to insert cbhA gene into vector with a strong promoter to form the candidate. Total RNA was isolated from Aspergillus niger and converted into cDNA. The cbhA gene is amplified from cDNA, then used as insert and inserted into yeast expression vector pYY1 and pWYH257. Vector and inserts were cut using restriction enzymes. Ligation products were transformed into competent cells of Escherichia coli DH10B, the transformation results were confirmed by Colony PCR. Recombinant plasmids were transformed into Saccharomyces cerevisiae INVSc1. The results obtained from the study are that cbhA gene amplification has been successfully carried out by producing DNA bands with the size of 1514 bp as expected for the target gene. The gene was successfully inserted into pYY1 and pWYH257 vector and the recombinant pYY1 plasmid was successfully transformed into Saccharomyces cerevisiae INVSc1