工程技术最新文献

Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Aspergillus fumigatus is a ubiquitous pathogenic mold and causes several diseases, including mycotoxicosis, allergic reactions, and systemic diseases (invasive aspergillosis), with high mortality rates. In its ecological niche, the fungus has evolved and mastered many reply strategies to resist and survive against negative threats, including harsh environmental stress and deficiency of essential nutrients from natural environments, immunity responses and drug treatments in host, and competition from symbiotic microorganisms. Hence, treating A. fumigatus infection is a growing challenge. In this review, we summarized A. fumigatus reply strategies and escape mechanisms and clarified the main competitive or symbiotic relationships between A. fumigatus, viruses, bacteria, or fungi in host microecology. Additionally, we discussed the contemporary drug repertoire used to treat A. fumigatus and the latest evidence of potential resistance mechanisms. This review provides valuable knowledge which will stimulate further investigations and clinical applications for treating and preventing A. fumigatus infections. KEY POINTS: • Harsh living environment was a great challenge for A. fumigatus survival. • A. fumigatus has evolved multiple strategies to escape host immune responses. • A. fumigatus withstands antifungal drugs via intrinsic escape mechanisms.

Poly-β-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5 g L^-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

While glycoside hydrolase family 1 (GH1) enzymes mostly catalyze hydrolysis reactions, rice Os9BGlu31 preferentially catalyzes transglycosylation to transfer a glucosyl moiety to another aglycone moiety to form a new glycosylated compound through a retaining mechanism. In this study, Os9BGlu31 was used to synthesize eight phenolic acid glucosyl esters, which were evaluated for activities in cholangiocarcinoma cells. The transglycosylation products of Os9BGlu31 wild type and its mutant variants were detected, produced on a milligram scale, and purified, and their structures were characterized by NMR spectroscopy. The transglycosylation products were evaluated by antioxidant and anti-proliferative assays, followed by an anti-migration assay for the selected phenolic acid glucosyl ester. Os9BGlu31 mutants produced higher yield and activity than wild-type enzymes on phenolic acids to produce phenolic acid glucosyl esters. Among these, gallic acid glucosyl ester (β-glucogallin) had the highest antioxidant activity and anti-proliferative activity in cholangiocarcinoma cells. It also inhibited the migration of cholangiocarcinoma cells. Our study demonstrated that rice Os9BGlu31 transglucosidase is a promising enzyme for glycosylation of bioactive compounds in one-step reactions and provides evidence that β-glucogallin inhibits cell proliferation and migration of cholangiocarcinoma cells. KEY POINTS: • Os9BGlu31 transglucosidases produced phenolic acid glucosyl esters for bioactivity testing. • Phenolic acid glucosyl esters were tested for cytotoxicity in cholangiocarcinoma cells. • β-Glucogallin displayed the highest inhibition of cholangiocarcinoma cell growth.

The potential consequences for mankind could be disastrous due to global warming, which arises from an increase in the average temperature on Earth. The elevation in temperature primarily stems from the escalation in the concentration of greenhouse gases (GHG) such as CO₂, CH₄, and N₂O within the atmosphere. Among these gases, methane (CH₄) is particularly significant in driving alterations to the worldwide climate. Methanotrophic bacteria possess the distinctive ability to employ methane as both as source of carbon and energy. These bacteria show great potential as exceptional biocatalysts in advancing C1 bioconversion technology. The present review describes recent findings in methanotrophs including aerobic and anaerobic methanotroph bacteria, phenotypic characteristics, biotechnological potential, their physiology, ecology, and native multi-carbon utilizing pathways, and their molecular biology. The existing understanding of methanogenesis and methanotrophy in soil, as well as anaerobic methane oxidation and methanotrophy in temperate and extreme environments, is also covered in this discussion. New types of methanogens and communities of methanotrophic bacteria have been identified from various ecosystems and thoroughly examined for a range of biotechnological uses. Grasping the processes of methanogenesis and methanotrophy holds significant importance in the development of innovative agricultural techniques and industrial procedures that contribute to a more favorable equilibrium of GHG. This current review centers on the diversity of emerging methanogen and methanotroph species and their effects on the environment. By amalgamating advanced genetic analysis with ecological insights, this study pioneers a holistic approach to unraveling the biopotential of methanotrophs, offering unprecedented avenues for biotechnological applications. KEY POINTS: • The physiology of methanotrophic bacteria is fundamentally determined. • Native multi-carbon utilizing pathways in methanotrophic bacteria are summarized. • The genes responsible for encoding methane monooxygenase are discussed.

Mildew poses a significant threat to tobacco production; however, there is limited information on the structure of the abundant and rare microbial subcommunities in moldy tobacco leaves. In this study, we employed high-throughput sequencing technology to discern the disparities in the composition, diversity, and co-occurrence patterns of abundant and rare fungal and bacterial subcommunities between moldy and normal tobacco leaves collected from Guizhou, Shanghai, and Jilin provinces, China. Furthermore, we explored the correlation between microorganisms and metabolites by integrating the metabolic profiles of moldy and normal tobacco leaves. The results showed that the fungi are more sensitive to mildew than bacteria, and that the fungal abundant taxa exhibit greater resistance and environmental adaptability than the rare taxa. The loss of rare taxa results in irreversible changes in the diversity, richness, and composition of the fungal community. Moreover, rare fungal taxa and abundant bacterial taxa played crucial roles in maintaining the stability and functionality of the tobacco microecosystem. In moldy tobacco, however, the disappearance of rare taxa as key nodes resulted in reduced connectivity and stability within the fungal network. In addition, metabolomic analysis showed that the contents of indoles, pyridines, polyketones, phenols, and peptides were significantly enriched in the moldy tobacco leaves, while the contents of amino acids, carbohydrates, lipids, and other compounds were significantly reduced in these leaves. Most metabolites showed negative correlations with Dothideomycetes, Alphaproteobacteria, and Gammaproteobacteria, but showed positive correlations with Eurotiales and Bacilli. This study has demonstrated that abundant fungal taxa are the predominant biological agents responsible for tobacco mildew, while bacteria may indirectly contribute to this process through the production and degradation of metabolites. KEY POINTS: • Fungi exhibited greater sensitivity to mildew of tobacco leaf compared to bacteria • Rare fungal taxa underwent significant damage during the mildew process • Mildew may damage the defense system of the tobacco leaf microecosystem.

Donor human milk (DHM) provides myriad nutritional and immunological benefits for preterm and low birthweight infants. However, pasteurization leaves DHM devoid of potentially beneficial milk microbiota. In the present study, we performed milk microbiome transplantation from freshly collected mother's own milk (MOM) into pasteurized DHM. Small volumes of MOM (5%, 10%, or 30% v/v) were inoculated into pasteurized DHM and incubated at 37 °C for up to 8 h. Further, we compared microbiome recolonization in UV-C-treated and Holder-pasteurized DHM, as UV-C treatment has been shown to conserve important biochemical components of DHM that are lost during Holder pasteurization. Bacterial culture and viability-coupled metataxonomic sequencing were employed to assess the effectiveness of milk microbiome transplantation. Growth of transplanted MOM bacteria occurred rapidly in recolonized DHM samples; however, a greater level of growth was observed in Holder-pasteurized DHM compared to UV-C-treated DHM, potentially due to the conserved antimicrobial properties in UV-C-treated DHM. Viability-coupled metataxonomic analysis demonstrated similarity between recolonized DHM samples and fresh MOM samples, suggesting that the milk microbiome can be successfully transplanted into pasteurized DHM. These results highlight the potential of MOM microbiota transplantation to restore the microbial composition of UV-C-treated and Holder-pasteurized DHM and enhance the nutritional and immunological benefits of DHM for preterm and vulnerable infants. KEY POINTS: • Mother's own milk microbiome can be successfully transplanted into donor human milk. • Recolonization is equally successful in UV-C-treated and Holder-pasteurized milk. • Recolonization time should be restricted due to rapid bacterial growth.

Vulvovaginal candidiasis (VVC) affects approximately 30-50% of women at least once during their lifetime, causing uncomfortable symptoms and limitations in their daily quality of life. Antifungal therapy is not very effective, does not prevent recurrencies and usually causes side effects. Therefore, alternative therapies are urgently needed. The goal of this work was to investigate the potential benefits of using mannan oligosaccharides (MOS) extracts together with a Lactobacillus sp. pool, composed by the most significant species present in the vaginal environment, to prevent infections by Candida albicans. Microbial growth of isolated strains of the main vaginal lactobacilli and Candida strains was assessed in the presence of MOS, to screen their impact upon growth. A pool of the lactobacilli was then tested against C. albicans in competition and prophylaxis studies; bacterial and yeast cell numbers were quantified in specific time points, and the above-mentioned studies were assessed in simulated vaginal fluid (SVF). Finally, adhesion to vaginal epithelial cells (HeLa) was also evaluated, once again resorting to simultaneous exposure (competition) or prophylaxis assays, aiming to measure the effect of MOS presence in pathogen adherence. Results demonstrated that MOS extracts have potential to prevent vaginal candidiasis in synergy with vaginal lactobacilli, with improved results than those obtained when using lactobacilli alone. KEY POINTS: Potential benefits of MOS extracts with vaginal lactobacilli to prevent C. albicans infections. MOS impacts on growth of vaginal lactobacilli pool and C. albicans in SVF. MOS extracts in synergy with L. crispatus inhibit C. albicans adhesion in HeLa cells.

Menaquinone-7 (MK-7), a subtype of vitamin K₂ (VK₂), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K₃ (VK₃) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK₃ concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76% and 22% under static and shaking cultivation conditions, respectively. The molecular basis underlying the superior performance of BS012 was elucidated through genome and transcriptome analyses, encompassing observations of alterations in cell morphology, variations in central carbon and nitrogen metabolism, spore formation, and antioxidant systems. In summation, ALE technology can notably enhance the tolerance of B. subtilis to VK and increase MK-7 production, thus offering a theoretical framework for the microbial fermentation production of other VK₂ subtypes. Additionally, the evolved strain BS012 can be developed for integration into probiotic formulations within the food industry to maintain intestinal flora homeostasis, mitigate osteoporosis risk, and reduce the incidence of cardiovascular disease. KEY POINTS: • Bacillus subtilis was evolved for improved vitamin K tolerance and menaquinone-7 (MK-7) production • Evolved strains formed wrinkled biofilms and elongated almost twofold in length • Evolved strains induced sporulation to improve tolerance when carbon was limited.

Infection and invasion are the prerequisites for developing the disease symptoms in a host. While the probable mechanism of host invasion and pathogenesis is known in many pathogens, very little information is available on Leptospira invasion/pathogenesis. For causing systemic infection Leptospira must transmigrate across epithelial barriers, which is the most critical and challenging step. Extracellular and membrane-bound proteases play a crucial role in the invasion process. An extensive search for the proteins experimentally proven to be involved in the invasion process through cell junction cleavage in other pathogens has resulted in identifying 26 proteins. The similarity searches on the Leptospira genome for counterparts of these 26 pathogenesis-related proteins identified at least 12 probable coding sequences. The proteins were either extracellular or membrane-bound with a proteolytic domain to cleave the cell junction proteins. This review will emphasize our current understanding of the pathogenic aspects of host cell junction-pathogenic protein interactions involved in the invasion process. Further, potential candidate proteins with cell junction cleavage properties that may be exploited in the diagnostic/therapeutic aspects of leptospirosis will also be discussed. KEY POINTS: • The review focussed on the cell junction cleavage proteins in bacterial pathogenesis • Cell junction disruptors from Leptospira genome are identified using bioinformatics • The review provides insights into the therapeutic/diagnostic interventions possible.