Isolation, antibacterial characterization, and alternating tangential flow-based preparation of viable cells of Lacticaseibacillus paracasei XLK 401: Potential application in milk preservation.

It is desirable to obtain high levels of viable Lacticaseibacillus paracasei, a widely used food probiotic whose antibacterial activity and potential application in milk remain largely uninvestigated. Here, we isolated and purified the L. paracasei strain XLK 401 from food-grade blueberry ferments and found that it exhibited strong antibacterial activity against both gram-positive and gram-negative foodborne pathogens, including Staphylococcus aureus, Salmonella paratyphi B, Escherichia coli O157, and Shigella flexneri. Then, we applied alternating tangential flow (ATF) technology to produce viable L. paracasei XLK 401 cells and its cell-free supernatant (CFS). Compared with the conventional fed-batch method, 22 h of ATF-based processing markedly increased the number of viable cells of L. paracasei XLK 401 to 12.14 ± 0.13 log cfu/mL. Additionally, the CFS exhibited good thermal stability and pH tolerance, inhibiting biofilm formation in the abovementioned foodborne pathogens. According to liquid chromatography-mass spectrometry analysis, organic acids were the main antibacterial components of XLK 401 CFS, accounting for its inhibition activity. Moreover, the CFS of L. paracasei XLK 401 effectively inhibited the growth of multidrug-resistant gram-positive Staph. aureus and gram-negative E. coli O157 pathogens in milk, and caused a reduction in the pathogenic cell counts by 6 to 7 log cfu/mL compared with untreated control, thus considerably maintaining the safety of milk samples. For the first time to our knowledge, ATF-based technology was employed to obtain viable L. paracasei on a large scale, and its CFS could serve as a broad-spectrum biopreservative for potential application against foodborne pathogens in milk products.

A novel bacteriocin against Staphylococcus aureus from Lactobacillus paracasei isolated from Yunnan traditional fermented yogurt: Purification, antibacterial characterization, and antibiofilm activity.

Staphylococcus aureus and its biofilm have emerged as a significant threat to the safety of dairy products. In recent years, lactic acid bacteria (LAB) bacteriocins have been widely acknowledged as the potential natural antibacterial substance in food biopreservation due to their excellent antibacterial effects. However, few LAB bacteriocins with antibacterial and antibiofilm activity against S. aureus have been reported in dairy products. In the present study, a novel bacteriocin LSX01 of Lactobacillus paracasei LS-6 isolated from a traditional fermented yogurt produced in Yunnan, China, was purified and characterized extensively. The LSX01 possessed a molecular weight of 967.49 Da and an AA sequence of LDQAGISYT. The minimum inhibitory concentration of LSX01 against S. aureus_45 was 16.90 µg/mL, which was close to or lower than the previously reported bacteriocins. The LSX01 exhibited an extensive antimicrobial spectrum against both gram-positive and gram-negative bacteria. Moreover, LSX01 exhibited excellent tolerance to heat and acid-base treatments, and sensitivity to the proteolytic enzymes, such as pepsin and proteinase K. Furthermore, the treatment of S. aureus_45 planktonic cells with LSX01 significantly reduced their metabolic activity and disrupted the cell membrane integrity. Scan electron microscopy results demonstrated that LSX01 induced cytoplasmic content leakage and cell deformation. Additionally, biofilm formation of S. aureus_45 was also significantly inhibited by LSX01. Overall, the results suggested that the novel LAB bacteriocin LSX01 possessed antibacterial activity and antibiofilm activity against S. aureus and, hence, could have potential for improving safety of dairy products.

[Effects of Bupleurum chinense on the diversity of intestinal flora in depressed mice].

Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.

Comparative transcriptomic analysis of fireflies (Coleoptera: Lampyridae) to explore the molecular adaptations to fresh water.

Aquatic insects are well adapted to freshwater environments, but the molecular basis of these adaptations remains largely unknown. Most firefly species (Coleoptera: Lampyridae) are terrestrial, but the larvae of several species are aquatic. Here, larval and adult transcriptomes from Aquatica leii (freshwater) and Lychnuris praetexta (terrestrial) were generated to test whether the genes associated with metabolic efficiency and morphology have undergone adaptive evolution to fresh water. The aquatic fireflies had a significantly lower ratio of nonsynonymous to synonymous substitutions than the terrestrial insects, indicating a genomewide evolutionary constraint in the aquatic fireflies. We identified 341 fast-evolving genes and 116 positively selected genes in the aquatic fireflies. Of these, 76 genes exhibiting both fast evolution and positive selection were primarily involved in ATP production, energy metabolism and the hypoxia response. We identified 7,271 differentially expressed genes (DEGs) in A. leii (adults versus larvae) and 8,309 DEGs in L. praetexta (adults versus larvae). DEGs specific to the aquatic firefly (n = 1,445) were screened via interspecific comparisons (A. leii versus L. praetexta) and were significantly enriched for genes involved in metabolic efficiency (e.g., ATP production, hypoxia, and immune responses) and certain aspects of morphology (e.g., cuticle chitin, tracheal and compound eye morphology). These results indicate that sequence and expression-level changes in genes associated with both metabolic efficiency and morphological attributes related to the freshwater lifestyle contributed to freshwater adaptation in fireflies. This study provides new insights into the molecular mechanisms of aquatic adaptation in insects.

MicroRNA profile of immune response in gills of zebrafish (Danio rerio) upon Staphylococcus aureus infection.

The gills of fish are large mucosal surfaces that are very important portals for pathogen entry. Investigations have shown that microRNAs (miRNAs) are key regulators of immune response to bacterial infections in the gills of fish; however, how miRNA expression changes in response to infection by Gram-positive bacteria remains largely unknown. To further investigate the immunological role of miRNAs in fish gills under pathogen stress induced by Gram-positive bacterial infection, this study investigated Staphylococcus aureus (SA)-induced changes in the miRNAs levels in gills of adult zebrafish (Danio rerio). miRNA microarrays were used to analyze expression profiles of known miRNA in the gills of zebrafish in response to SA infection and compared these to uninfected control fish. A total of 30 differentially expressed miRNAs (DEMs) were identified. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses were performed. The results indicated that DEM targets were primarily involved in innate immune processes, apoptosis, defense responses, and antibacterial responses. Pathways involving bacterial infection, innate immunity, metabolic process, disease, and apoptosis were mediated by DEMs. Furthermore, real-time quantitative PCR experiments for nine key SA-responsive DEMs that regulated the "SA infection" pathway validated the accuracy of microarray results. Dynamic variations in gene expression were surveyed in detail for these key SA-responsive DEMs for PBS control and at 6, 12, 24, and 48 h after SA challenge in detail. This study provides novel insight into the mechanisms underlying the miRNA regulation during the SA-induced immune response in zebrafish gills, and provides basic knowledge on the innate immune response against Gram-positive bacterial infection in bony fish.

Identification of circular RNAs and their altered expression under poly(I:C) challenge in key antiviral immune pathways in amphioxus.

Amphioxus is a key model for studying comparative immunity of vertebrates. Circular RNA (circRNA), as RNAs with a circular structure, has received little attention until recently, where several studies have reported that circRNA expression changes are involved in the immune response in animals. However, circRNA and its immune role in amphioxus have not been previously studied. Here, circRNAs in Chinese amphioxus (Branchiostoma belcheri) were sequenced, and 1859 circRNAs were identified using two algorithms (find_circ and CIRI). The analysis of miRNA target sites on circRNAs showed that 332 circRNAs may function as miRNA sponges. Furthermore, we identified circRNAs that were conserved between B. belcheri and vertebrates, tracing the origin of these circRNAs within chordates. Additionally, in combination with several key antiviral immune (poly(I:C), pIC) pathways identified in our previous B. belcheri studies, nine circRNAs potentially involved in these pathways were identified using bioinformatic predictions. Among these nine circRNAs, eight were selected to examine their expression response in B. belcheri challenged by pIC in comparison to control using real-time quantitative PCR. The results showed that four circRNAs were induced as part of the antiviral response against pIC, while expression of two circRNAs was decreased, and the expression levels of the remaining two were not significantly altered after pIC challenge. This work is the first to identify circRNAs and reveal their antiviral role in amphioxus. Therefore, it opens a new window to explore the comparative immunology of circRNAs in chordates and the regulatory roles of circRNAs in antiviral immunity in amphioxus.

Comparative transcriptomic analysis provides insights into antibacterial mechanisms of Branchiostoma belcheri under Vibrio parahaemolyticus infection.

Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus (Branchiostoma belcheri) infected with Vibrio parahaemolyticus (V. p.) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p.-infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p.-infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p. TREATMENT: Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri. This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus.

Lacticaseibacillus chiayiensis mediate intestinal microbiome and microbiota-derived metabolites regulating the growth and immunity of chicks.

Emerging evidence confirms beneficial properties of probiotics in promoting growth and immunity of farmed chicken. However, the molecular mechanisms underlying the host-microbiome interactions mediated by probiotics are not fully understood. In this study, the internal mechanisms of Lacticaseibacillus chiayiensis-mediated host-microbiome interactions and to elucidate how it promotes host growth were investigated by additional supplementation with L. chiayiensis. We conducted experiments, including intestinal cytokines, digestive enzymes test, intestinal microbiome, metabolome and transcriptome analysis. The results showed that chickens fed L. chiayiensis exhibited higher body weight gain and digestive enzyme activity, and lower pro-inflammatory cytokines, compared to controls. Microbiota sequencing analysis showed that the gut microbiota structure was reshaped with L. chiayiensis supplementation. Specifically, Lactobacillus and Escherichia increased in abundance and Enterococcus, Lactococcus, Corynebacterium, Weissella and Gallicola decreased. In addition, the bacterial community diversity was significantly increased compared to controls. Metabolomic and transcriptomic analyses revealed that higher bile acids and N-acyl amides concentrations and lower carbohydrates concentrations in L. chiayiensis-fed chickens. Meanwhile, the expression of genes related to nutrient transport and absorption in the intestine was upregulated, which reflected the enhanced digestion and absorption of nutrients in chickens supplemented with L. chiayiensis. Moreover, supplementation of L. chiayiensis down-regulated genes involved in inflammation-related, mainly involved in NF-κB signaling pathway and MHC-II mediated antigen presentation process. Cumulatively, these findings highlight that host-microbiota crosstalk enhances the host growth phenotype in two ways: by enhancing bile acid metabolism and digestive enzyme activity, and reducing the occurrence of intestinal inflammation to promote nutrient absorption and maintain intestinal health. This provides a basis for the application of LAB as an alternative to antibiotics in animal husbandry.

Genome-Wide and 16S rRNA Sequencing-Based Analysis on the Health Effects of Lacticaseibacillus paracasei XLK401 on Chicks.

Lacticaseibacillus paracasei, serves as a growth promoter used in the poultry industry, contributeing to broiler development. However, practical studies are needed to determine the probiotic potential and growth-promoting effects of specific L. paracasei strains. This study aims to determine whether L. paracasei XLK401 influences broiler chicken growth and the mechanisms involved. Notably, we identified several bile salt and acid tolerance-related genes (Asp23, atpD, atpA, atpH, and atpF) in L. paracasei XLK401. This bacterium demonstrates robust probiotic properties under acidic conditions (pH 2.0) and 0.3% bile salt conditions. It also contains a variety of antioxidant-related genes (trxA, trxB, and tpx), carbohydrate-related genes, gene-encoding glycosidases (e.g., GH and GT), and three clusters of genes associated with antimicrobial compounds. Supplementation with L. paracasei XLK401 significantly increased the body weight of the chicks. In addition, it significantly increased hepatic antioxidant enzyme activities (GSH-Px, SOD, and T-AOC) while significantly decreasing the levels of oxidative damage factors and inflammatory factors (MDA and IL-6), resulting in improved chick health. Improvements in body weight and health status were associated with significant increases in α-amylase activity and the remodeling of the host gut microbiota by L. paracasei XLK401. Among them, actinobacteria abundance in chicken intestines after feeding them L. paracasei XLK401 was significantly decreased, Bifidobacterium sp. abundance was also significantly decreased, and Subdoligranulum sp. abundance was significantly increased. This suggests that L. paracasei XLK401 can regulate the abundance of certain bacteria without changing the overall microbial structure. In addition, in the correlation analysis, Subdoligranulums sp. were positively correlated with SOD and negatively correlated with IL-1ß and MDA. Overall, our study demonstrates that L. paracasei XLK401 effectively promotes healthy chick growth. This is made possible by the modulation of gut microbe abundance and the underlying probiotic effect of L. paracasei XLK401. Based on these findings, we postulate L. paracasei XLK401 as a potential efficient growth promoter in broiler farming.

Antibacterial activity and mechanism of action of bacteriocin LFX01 against Staphylococcus aureus and Escherichia coli and its application on pork model.

Antibacterial activity and mechanism of action of bacteriocins against bacteria that cause pork contamination remain unclear. Here, antibacterial activity of bacteriocin LFX01 against two important indicator strains (i.e., Staphylococcus aureus and Escherichia coli) and its mechanism of action were investigated. The results showed antibacterial activity of LFX01 against growth and biofilm formation of S. aureus_26 (strain 2612:1606BL1486) and E. coli_02 (strain CMCC(B)44102). Additionally, the results demonstrated that LFX01 could decrease cell metabolic activity, disrupt cell membrane permeability and integrity, and trigger leakage of intracellular contents (e.g., K+, ATP, and lactic dehydrogenase). Furthermore, gel retardation showed that LFX01 could bind to the genomic DNA of indicator strains, disrupting DNA structure. These results uncovered mechanism of action of LFX01 against indicator strains from physiological and phenotypic levels. When applied to the surface of fresh pork models, the antibacterial activity of LFX01 against indicator strains was further confirmed. These findings suggested that LFX01 could be a potential pork preservative for controlling foodborne pathogens.

Assessment of the safety and probiotic characteristics of Lactobacillus salivarius CGMCC20700 based on whole-genome sequencing and phenotypic analysis.

Lactic acid bacteria are generally regarded as alternatives to antibiotics in livestock and poultry farming, especially Lactobacillus strains, which are safe and have probiotic potential. Although Lactobacillus salivarius has long been proposed to be a probiotic, the understanding of the roles of this species is still in its infancy. Here, a strain of L. salivarius CGMCC20700 isolated from the intestinal mucosa of Yunnan black-bone chicken broilers was investigated in the context of its safety and probiotic characteristics by whole-genome sequencing in parallel with phenotypic analysis. Whole-genome sequencing results showed that L. salivarius CGMCC20700 has a single scaffold of 1,737,577 bp with an average guanine-to-cytosine (GC) ratio of 33.51% and 1,757 protein-coding genes. The annotation of Clusters of Orthologous Groups (COG) classified the predicted proteins from the assembled genome as possessing cellular, metabolic, and information-related functions. Sequences related to risk assessment, such as antibiotic resistance and virulence genes, were identified, and the strain was further confirmed as safe according to the results of antibiotic resistance, hemolytic, and acute oral toxicology tests. Two gene clusters of antibacterial compounds and broad-spectrum antimicrobial activity were identified using genome mining tools and antibacterial spectrum tests. Stress resistance genes, active stressor removal genes, and adhesion related genes that were identified and examined with various phenotypic assays (such as stress tolerance tests in acids and bile salts and auto aggregation and hydrophobicity assays). The strain showed a high survival rate in the presence of bile salts and under acidic conditions and exhibited significant auto aggregation capacity and hydrophobicity. Overall, L. salivarius CGMCC20700 demonstrated excellent safety and probiotic potential at both the genomic and physiological levels and can be considered an appropriate candidate probiotic for livestock and poultry farming.

A novel bacteriocin against multiple foodborne pathogens from Lacticaseibacillus rhamnosus isolated from juice ferments: ATF perfusion-based preparation of viable cells, characterization, antibacterial and antibiofilm activity.

Foodborne pathogens and their biofilms pose a risk to human health through food chain. However, the bacteriocin resources combating this threat are still limited. Here, Lacticaseibacillus rhamnosus, one of the most used probiotics in food industry, was prepared on a large scale using alternating tangential flow (ATF) perfusion-based technology. Compared to the conventional fed-batch approach, ATF perfusion remarkably increased the viable cells of L. rhamnosus CLK 101 to 11.93 ± 0.14 log CFU/mL. Based on obtained viable cells, we purified and characterized a novel bacteriocin CLK_01 with a broad spectrum of activity against both Gram-positive and Gram-negative foodborne pathogens. LC-MS/MS analysis revealed that CLK_01 has a molecular mass of 701.49 Da and a hydrophobic amino acid composition of I-K-K-V-T-I. As a novel bacteriocin, CLK_01 showed high thermal stability and acid-base tolerance over 25-121 °C and pH 2-10. It significantly reduced cell viability of bacterial pathogens (p < 0.001), and strongly inhibited their biofilm formation. Scanning electron microscopy demonstrated deformation of pathogenic cells caused by CLK_01, leading to cytoplasmic content leakage and bacterial death. Summarily, we employed ATF perfusion to obtain viable L. rhamnosus, and presented that bacteriocin CLK_01 could serve as a promising biopreservative for controlling foodborne pathogenic bacteria and their biofilms.

Effects of Dietary Supplementation of Lacticaseibacillus Chiayiensis AACE3 on Hepatic Antioxidant Capacity, Immune Factors and Gut Microbiology in Nandan Yao Chicks.

The growing issue of antibiotic resistance has restrained the utilization of antibiotics as growth enhancers in the poultry industry. Probiotics are candidates for replacing antibiotics in the poultry industry. However, probiotics are strain-specific and their efficacy needs to be investigated before applying them. The aim of this study was to assess the positive effects of Lacticaseibacillus chiayiensis AACE3 on the health and gut microbiota of Nandan Yao chicks. The results showed that compared with the blank control (NC) and aureomycin (PC) groups, L. chiayiensis AACE3 increased final body weight (BW), villus height and improved the ratio of villus height to crypt depth in chicken jejunal tissues. L. chiayiensis AACE3 also increased the activity of hepatic antioxidant enzymes (SOD, CAT and T-AOC) and reduced hepatic oxidative damage (MDA). Furthermore, compared to NC, L. chiayiensis AACE3, the activity of intestinal digestive enzymes (i.e., α-amylase, lipase and trypsin) was increased. L. chiayiensis AACE3 upregulated the production of IgA and IgG and downregulated the production of IL-6, IL-1ß and TNF-α in chicken serum. Moreover, supplementation of L. chiayiensis AACE3 enhances the diversity of gut microbes. At the phylum level, the abundance of Actinobacteriota and Proteobacteria decreased with L. chiayiensis AACE3 supplementation, while the abundance of Verrucomicrobiota and Bacteroidetes increased. At the genus level, there was an increase in the abundance of potential probiotics Akkermansia, Romboutsia, Subdoligranulum, and Lactobacillus. This study confirms that L. chiayiensis AACE3 is an excellent feed additive as an alternative to aureomycin and offers various advantages for the healthy growth of chickens during the brooding period by positively affecting their gut microbiome.

Genome-wide transcriptomics and microRNAomics analyses uncover multi-faceted mechanisms to cope with copper stress in ancient macrobenthos amphioxus.

The mechanisms underlying the toxicity of environmental stress are unclear for marine macrobenthos. Copper/Cu has posed the most serious threats to amphioxus, an ancient and model benthic cephalochordate. Herein, a dynamic change in the physiological parameters (GR, SOD, ATP, and MDA) was detected with ROS accumulation in Branchiostoma belcheri exposed to 0.3 mg·L-1 Cu. Transcriptomes and microRNAomes of B. belcheri were generated to investigate the molecular mechanisms by which this amphioxus copes with Cu exposure. Time-specific genes identified at different time points after exposure were involved in the stimulus and immune response, detoxification and ionic homeostasis, aging and the nervous system, sequentially, with prolongation of exposure time, forming a dynamic process of molecular response to Cu stress. In total, 57 differentially expressed miRNAs were identified under Cu stress. Transcriptomics-miRNAomics analyses indicate that these miRNAs targeted genes associated with many key biological processes such as xenobiotics degradation, oxidative stress, and energy metabolism. The constructed miRNA-mRNA-pathway network uncovered a broad post-transcriptional regulatory mechanism in B. belcheri to cope with Cu stress. Overall, this integrated analyses show that enhanced defense response, accelerated ROS elimination, and repressed ATP production constitute a comprehensive strategy to cope with Cu toxicity in the ancient macrobenthos.

The role of symbiotic fungi in the life cycle of Gastrodia elata Blume (Orchidaceae): a comprehensive review.

Gastrodia elata Blume, a fully mycoheterotrophic perennial plant of the family Orchidaceae, is a traditional Chinese herb with medicinal and edible value. Interestingly, G. elata requires symbiotic relationships with Mycena and Armillaria strains for seed germination and plant growth, respectively. However, there is no comprehensive summary of the symbiotic mechanism between fungi and G. elata. Here, the colonization and digestion of hyphae, the bidirectional exchange of nutrients, the adaptation of fungi and G. elata to symbiosis, and the role of microorganisms and secondary metabolites in the symbiotic relationship between fungi and G. elata are summarized. We comprehensively and deeply analyzed the mechanism of symbiosis between G. elata and fungi from three perspectives: morphology, nutrition, and molecules. The aim of this review was to enrich the understanding of the mutualistic symbiosis mechanisms between plants and fungi and lay a theoretical foundation for the ecological cultivation of G. elata.

Genomic and in-vitro characteristics of a novel strain Lacticaseibacillus chiayiensis AACE3 isolated from fermented blueberry.

Numerous different species of LAB are used in different fields due to their unique characteristics. However, Lacticaseibacillus chiayiensis, a newly established species in 2018, has limited microorganism resources, and lacks comprehensive evaluations of its properties. In this study, L. chiayiensis AACE3, isolated from fermented blueberry, was evaluated by genomic analysis and in vitro assays of the properties. The genome identified genes associated with biofilm formation (luxS, ccpA, brpA), resistance to oxidative stress (tpx, trxA, trxB, hslO), tolerance to acidic conditions (dltA, dltC), resistance to unfavorable osmotic pressure (opuBB, gbuA, gbuB, gbuC), and adhesion (luxS, dltA, dltC). The AACE3 showed 112 unique genes, relative to the other three L. chiayiensis strains. Among them, the presence of genes such as clpP, pepO, and feoA suggests a possible advantage of AACE3 over other L. chiayiensis in terms of environmental adaptation. In vitro evaluation of the properties revealed that AACE3 had robust antibacterial activity against eight common pathogens: Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Salmonella choleraesuis, Shigella flexneri, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In addition, AACE3 showed more than 80% survival rate in all tests simulating gastrointestinal fluid, and it exhibited high antioxidant capacity. Interestingly, the cell culture supernatant was superior to intact organisms and ultrasonically crushed bacterial extracts in all tests of antioxidant capacity. These results suggested that the antioxidant capacity may originate from certain metabolites and extracellular enzymes produced by AACE3. Moreover, AACE3 was a moderate biofilm producer due to the self-agglomeration effect. Taken together, L. chiayiensis AACE3 appears to be a candidate strain for combating the growing incidence of pathogen infections and antioxidant production.

Laceyella sediminis sp. nov., a thermophilic bacterium isolated from a hot spring.

A thermophilic bacterium, designated strain RHA1(T), was isolated from a sediment sample collected from a hot spring in Tengchong county, Yunnan province, south-west China, and was characterized by using a polyphasic approach. Based on its phenotypic and phylogenetic characteristics, strain RHA1(T) was affiliated to the genus Laceyella. The strain formed white aerial and yellow-white substrate mycelia, bearing single endospores on short sporophores. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Whole-cell hydrolysates contained ribose and glucose. The major fatty acids were iso-C(15:0) (62.39%) and anteiso-C(15:0) (17.55%)(.) The predominant menaquinone was MK-9. The G+C content of the genomic DNA of strain RHA1(T) was 47.9 mol%. Based on DNA-DNA hybridization data, chemotaxonomic characteristics and differential physiological properties, strain RHA1(T) is considered to represent a novel species of the genus Laceyella, for which the name Laceyella sediminis sp. nov. is proposed; the type strain is RHA1(T) (=DSM 45263(T)=CCTCC AA 208058(T)).

Cloning, expression, and characterization of an alkaline thermostable GH11 xylanase from Thermobifida halotolerans YIM 90462T.

A xylanase gene (thxyn11A) from the Thermobifida halotolerans strain YIM 90462(T) was cloned and expressed in Escherichia coli. The open reading frame (ORF) of thxyn11A has 1,008 bp encoding a mature xylanase with a high degree of similarity (80 %) to the xylanase from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111. This enzyme (Thxyn11A) also possesses a glycosyl hydrolases family 11 (GH11) domain and a high isoelectric point (pI = 9.1). However, Thxyn11A varies from most GH11 xylanases, due to its large molecular mass (34 kDa). Recombinant Thxyn11A demonstrated a strong pH and temperature tolerance with a maximum activity at pH 9.0 and 70 °C. Xylotriose, the end-product of xylan hydrolysis by Thxyn11A, serves as a catalyst for hemicellulose pretreatment in industrial applications and can also function as a food source or supplement for enterobacteria. Due to its attractive biochemical properties, Thxyn11A may have potential value in many commercial applications.

Bacteriophage Lytic Enzyme P9ly as an Alternative Antibacterial Agent Against Antibiotic-Resistant Shigella dysenteriae and Staphylococcus aureus.

Developing new strategies to replace or supplement antibiotics to combat bacterial infection is a pressing task in the field of microbiological research. In this study, we report a lytic enzyme named P9ly deriving from the bacteriophage PSD9 that could infect multidrug-resistant Shigella. This enzyme was identified through whole-genome sequencing of PSD9. The results show that P9ly contains a conserved T4-like_lys domain and belongs to the phage lysozyme family. Recombinant P9ly obtained from protein purification presented biological activity and could digest bacterial cell walls (CW), resulting in the destruction of cell structure and leakage of intracellular components. Furthermore, P9ly exhibited bacteriolytic and bactericidal activity on different strains, especially multidrug-resistant Gram-negative Shigella dysenteriae and Gram-positive Staphylococcus aureus. Additionally, combined use of P9ly with ceftriaxone sodium (CRO) could decrease necessary dose of the antibiotic used and improve the antibacterial effect. In summary, under the current backdrop of extensive antibiotic usage and the continuous emergence of bacterial resistance, this study provides an insight into developing bacteriophage-based antibacterial agents against both Gram-negative and Gram-positive pathogens.

Antibacterial effect of bacteriocin XJS01 and its application as antibiofilm agents to treat multidrug-resistant Staphylococcus aureus infection.

Multidrug-resistant (MDR) Staphylococcus aureus biofilms have emerged as a serious threat to human health. Recently, the development of antibiotic replacement therapy has gained much attention due to the potential application of bacteriocin. The present study sought to evaluate the antibacterial effect of bacteriocin XJS01 against MDR S. aureus, a previously reported bacteriocin against S. aureus strain 2612:1606BL1486 (S. aureus_26, an MDR strain demonstrated here), and its potential application as an antibiofilm agent. The minimum bactericide concentration of XJS01 against MDR S. aureus_26 was 33.18 µg/mL. XJS01 exhibited excellent storage stability and resistance against acid and reduced the density of established MDR S. aureus_26 biofilm. The hemolytic and HEK293T cytotoxicity activities of XJS01 and the histological analyses in mice confirmed its safety. Moreover, XJS01 effectively disrupted the MDR S. aureus_26 biofilm established on the skin wound surface and reduced the biofilm-isolated bacteria, thereby decreasing the release of pro-inflammatory cytokines and the proliferation of alternatively activated macrophages. Compared to mupirocin, XJS01 exhibited an excellent therapeutic effect on mice skin wounds, confirming it to be a potential alternative to antibiotics.