• Volume 51,Issue 7,2024 Table of Contents
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    • >REVIEWS
    • Advances in the relationship of IgA nephropathy with intestinal microecology and intestinal mucosal immunity

      2024, 51(7):2271-2279. DOI: 10.13344/j.microbiol.china.230993

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      Abstract:Immunoglobulin A nephropathy (IgAN) is a common primary glomerular disease characterized by multifaceted, immune-mediated mechanisms. Additionally, several comorbidities, including inflammatory bowel disease, hepatitis, HIV infection, and Sjögren’s syndrome, are intricately linked to the genesis and progression of IgAN and collectively denoted as secondary IgAN. The multiple-hit hypothesis is currently the widely embraced pathogenesis framework for IgAN, positing mucosal dysbiosis-induced disruption of the barrier function and immune aberrations as pivotal triggers for IgAN pathogenesis. The intestinal mucosa, constituting a pivotal element of the mucosal system, is increasingly acknowledged for its substantive involvement in IgAN development. Perturbations in intestinal microbiota, compromised mucosal barrier integrity, and immune dysregulation are recognized as pivotal players in IgAN pathophysiology. Investigating the therapeutic targets associated with these facets currently represents a focal point in research. We comprehensively review the research advances in the roles of intestinal microecology and mucosal immunity in the pathogenesis of IgAN. This review aims to lay a foundation for exploring the novel therapeutic targets for IgAN from intestinal microbiota and immune functionality.

    • Research progress in the mechanisms of interactions between Chlamydia and mitochondria

      2024, 51(7):2280-2288. DOI: 10.13344/j.microbiol.china.230775

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      Abstract:Chlamydia is a genus of prokaryotic microorganisms that lies between viruses and bacteria, strictly parasitizing eukaryotic cells and possessing a unique developmental cycle. Based on the characteristics of intracellular growth, Chlamydia can interact with multiple organelles in host cells to promote its own growth and replication. Mitochondria, multifunctional organelles in cells, not only serve as the metabolic center and energy factory but also are involved in various biological processes, playing an important role in the defense against pathogen invasion. Chlamydia infection is closely related to mitochondria. Studies have shown that Chlamydia relies on the mitochondrial metabolism of host cells to obtain energy and affects mitochondrial functions by changing mitochondrial protein composition and regulating mitochondrial dynamics, apoptosis, reactive oxygen species, etc. This article reviews the interaction mechanisms between Chlamydia and mitochondria in host cells.

    • Research advance of Clostridium perfringens epsilon toxin

      2024, 51(7):2289-2300. DOI: 10.13344/j.microbiol.china.230853

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      Abstract:Clostridium perfringens epsilon toxin (ETX) can cause necrotic enteritis and injuries in the lungs, kidneys, and brain to the host animals such as cattle and sheep, affecting the development of animal husbandry. Due to its potent toxicity, ETX has been categorized as a biological warfare and bioterrorism agent. ETX damages the target cells by forming pores in the cell membrane, resulting in abnormal release of cell contents, during which myelin and lymphocyte (MAL) is a specific receptor for ETX to exert toxic effects. There are only crude ETX vaccines for use in animals, while the vaccines for human application are still under development, and potential treatments are actively being explored. In recent years, researchers have achieved significant progress in understanding the pore-forming mechanism of ETX, identifying specific receptors, and developing vaccines and therapeutic drugs. This paper reviews the progress in the aforementioned aspects, aiming to offer valuable references for further ETX research.

    • Research progress in the application of gene knockout in multidrug-resistant bacteria

      2024, 51(7):2301-2311. DOI: 10.13344/j.microbiol.china.230840

      Abstract (202) HTML (327) PDF 619.96 K (459) Comment (0) Favorites

      Abstract:The widespread use of antibiotics has exacerbated the multidrug resistance of bacteria, posing a grave threat to public health. Gene knockout, as a precise method for intervening in bacterial genetics, exhibits immense potential in deciphering the mechanisms of multidrug resistance and exploring novel therapeutic approaches. This paper reviews the advancements in the application of gene knockout in the research on multidrug-resistant bacteria. We discovered that gene knockout not only facilitates the understanding of the drug resistance mechanisms in multidrug-resistant bacteria, but also provides crucial insights into identifying new drug targets. We further discuss the advantages and limitations of this technology and propose potential solutions. The further optimization of gene knockout and the integration of this technology with other biotechnologies will provide more effective strategies for addressing the multidrug-resistance in bacteria. We hope that gene knockout can be applied in a broader scope in clinical practice, offering new possibilities for the treatment of multidrug-resistant bacteria.

    • Research progress in straw-degrading psychrotrophic microorganisms

      2024, 51(7):2312-2325. DOI: 10.13344/j.microbiol.china.230819

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      Abstract:A large quantity of straw waste is produced in northern China, the main area of agricultural production. The complex three-dimensional structure formed by lignin, cellulose, and hemicellulose makes straw difficult to be degraded. Furthermore, the low temperature environment after harvest enhances the difficulty of straw degradation. Psychrotrophic microorganisms could promote straw degradation at low temperatures. Straw-degrading microorganisms include bacteria and fungi. Among them, fungi are powerful in lignin degradation. Bacteria have low lignin-degrading activities but high tolerance to low temperatures. Because the degradation of straw is coordinated by many enzymes, straw is difficult to be completely degraded by single strains. Psychrotrophic strain combinations with powerful straw decomposition performance are widely used in straw degradation. These combinations can be obtained by screening of strains from the nature, mixing of strains, and acclimation to temperature gradients. This review provides technical reference for efficiently screening straw-degrading microorganisms and promoting straw utilization under low temperature conditions.

    • Research progress in heteroresistance of Klebsiella pneumoniae to antibiotics

      2024, 51(7):2326-2336. DOI: 10.13344/j.microbiol.china.230482

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      Abstract:Klebsiella pneumoniae (KP), one of the most common pathogens causing clinical infections, often results in pneumonia, urinary tract infection, and bacteremia. In recent years, studies have reported the heteroresistance of K. pneumoniae. Heteroresistance refers to the phenomenon that there are both resistant and susceptible subpopulations of a strain to an antibiotic. Due to the phenotypic and genetic instability of heteroresistance, standard, low-cost, and efficient detection methods remain to be established. This article reviews the research progress in heteroresistance of K. pneumoniae, clarifies the definition and detection methods of heteroresistance, and probes into the heteroresistance mechanism of K. pneumoniae on the basis of our previous studies about the heteroresistance of Pseudomonas aeruginosa. This article will provide a reference for comprehensively understanding the heteroresistance mechanism of K. pneumoniae, optimizing the clinical use of antibiotics, and treating bacterial infections.

    • Gut microbiota-targeted therapeutic strategies for autism spectrum disorders

      2024, 51(7):2337-2352. DOI: 10.13344/j.microbiol.china.230781

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      Abstract:The role of gut microbiota in autism spectrum disorders (ASD) is receiving increasing attention and may become an important direction for ASD treatment in the future. Studies have shown that the gut microbiota in ASD patients is different from that in healthy people in terms of microbial relative abundance, diversity and so forth. These microorganisms can influence the development and function of the nervous system via multiple pathways along the microbiota-gut-brain axis, which affects the behaviors and cognitive abilities of ASD patients. Therefore, modulating the gut microbiota may be an effective therapy for ASD. Researchers have explored the efficacy of probiotics, fecal microbiota transplantation, and other therapies for ASD and have made preliminary progress. However, studies remain to be carried out to determine the optimal therapeutic strategies with long-term efficacy and high safety.

    • >Environmental Microbiology
    • Biodiversity and antimicrobial activities of culturable actinomycetes isolated from the soil of Dabancheng Salt Lake, Xinjiang, China

      2024, 51(7):2353-2367. DOI: 10.13344/j.microbiol.china.240010

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      Abstract:[Background] In recent years, new strains of actinomycetes have been discovered in the saline-alkali environments such as Ebinur Lake, Qijiaojing Salt Lake, and Lop Nur in Xinjiang of China. However, there are few reports about the actinomycetes strains isolated from the soil of Dabancheng Salt Lake. [Objective] We studied the diversity of culturable actinomycetes in the soil of Dabancheng Salt Lake in Xinjiang and screened out the strains with activities of functional enzymes, aiming to mine new medicinal microbial resources and lay a foundation for the discovery of new antibiotics. [Methods] The diversity of culturable actinomycetes in the soil of Dabancheng Salt Lake was investigated by the culture-dependent method and the phylogenetic analysis based on 16S rRNA gene sequences. PCR was employed to explore the distribution of the genes encoding types I and II polyketide synthase (pksI and pskII), non-ribosomal peptide synthase (nrps), and aminoglycoside phosphotransferase (aph) for antibiotic synthesis. The agar diffusion method was employed to screen the strains with antimicrobial activities and the strains capable of producing functional enzymes (with nine substrates as indicators). [Results] A total of 65 actinomycetes strains belonging to nine genera, five families of five orders were isolated from fourteen soil samples of Dabancheng Salt Lake. Streptomyces and Nocardiopsis were the dominant genera and two strains were potential new species. Abundant genes involved in antibiotic synthesis were identified. A strain (D21E05) with antagonistic effects on Gram-positive, Gram-negative, and fungi was screened out. The soil of Dabancheng Salt Lake harbored rich strains producing protease, lipase, catalase, cellulase, and amylase. [Conclusion] The soil of Dabancheng Salt Lake harbors rich medicinal actinomycetes resources, from which new actinomycetes species and new antibiotics can be discovered.

    • >Agricultural Microbiology
    • Screening of a selenium-enriching strain of Pleurotus ostreatus by ultraviolet mutagenesis and its application in nutrient enrichment

      2024, 51(7):2368-2380. DOI: 10.13344/j.microbiol.china.230811

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      Abstract:[Background] The cultivation of selenium-enriching edible fungi in their natural habitats often fails to meet the national standards for nutrient enrichment. [Objective] To screen out a selenium-enriching strain of Pleurotus ostreatus by ultraviolet mutagenesis, so as to provide a foundation for the product development with selenium-enriching edible fungi. [Methods] The strain to be mutated was determined by selenium resistance examination and then exposed to ultraviolet irradiation. The mycelial growth rate and the biological efficiency, total and organic selenium content of fruiting bodies were determined to evaluate the performance of the mutant strain. [Results] In the media with 100 mg/kg and 120 mg/kg selenium, respectively, the strain 120-5 showed the mycelial growth rates of 1.27 cm/d and 1.19 cm/d and the total selenium content of 199.77 mg/kg and 224.15 mg/kg in the fruiting bodies of 120-5. The organic selenium content in the fruiting bodies in the two treatments reached 198.83 mg/kg and 223.56 mg/kg, accounting for 99.53% and 99.74% of the total selenium, respectively. These values met the national standards for nutrient enrichment. However, in the media with 100 mg/kg and 120 mg/kg selenium, the original strain qiu-2020 showed the mycelial growth rates of 1.02 cm/d and 0.88 cm/d, respectively. It is noteworthy that in the medium with 120 mg/kg selenium, the original strain achieved the highest selenium content of 166.53 mg/kg, which fell short of the national standards. In the presence of 120 mg/kg selenium, the biological efficiency of the mutant strain and the original strain reached 92% and 77%, respectively. Remarkably, the agronomic traits of both the mutant and original strains remained unaltered. The whole-genome resequencing results unveiled single nucleotide polymorphism (SNP), insertion-deletion (Indel), and structure variation (SV) between the mutant and original strains. [Conclusion] The strain 120-5 with significant genomic mutations showcased a robust capacity of selenium enrichment, serving as a compelling candidate for nutrient enrichment.

    • Bacillus velezensis FH-1 promotes rice seedlings growth by regulating specific spatiotemporal soil microbiome

      2024, 51(7):2381-2410. DOI: 10.13344/j.microbiol.china.230799

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      Abstract:[Background] Microbial fertilizers prepared with plant growth-promoting rhizobacteria (PGPR) are an important way to achieve sustainable agricultural development. [Objective] To systematically study the distribution of Bacillus velezensis FH-1 capable of promoting rice growth in complex soil systems and the mechanism of the strain in promoting rice growth on a spatial-temporal scale. [Methods] The rice seedlings were inoculated with B. velezensis FH-1, and rice seedling and rhizosphere soil samples in different layers were collected at different time points. The plant height and dry weight of rice seedlings were measured by a ruler and a balance, respectively. After DNA was extracted from the rhizosphere soil, 16S rRNA gene amplicon sequencing was performed to analyze the microbial community composition. Spearman correlation analysis was performed to study the correlations between rice and microorganisms. [Results] On day 0 after inoculation, there was no significant difference between the B. velezensis FH-1 (F) group and the control group (CK). The rice growth-promoting effect of group F became increasingly obvious over time. On day 20 after inoculation, the plant height and dry weight of rice seedlings in group F were 6.02% and 12.52% higher than those in group CK, respectively (P<0.05). During days 5–10 after inoculation, the colonization abundance of B. velezensis FH-1 in the 0–3 cm underground soil layer was 1.19%–2.64% higher than that in the 3–6 cm underground soil layer. During this period, B. velezensis FH-1 promoted rice growth by enriching Kineosporiaceae and Chitinophagales and promoting fumarate respiration in the 0–3 cm underground soil layer and enriching Bacillaceae and Phycisphaerae and enhancing nitrate ammonification in the 3–6 cm underground soil layer. During days 15–20 after inoculation, the colonization abundance of B. velezensis FH-1 in the 0–3 cm underground soil layer was 134%–139% higher than that in the 3–6 cm underground soil layer. During this period, B. velezensis FH-1 promoted rice growth by enriching Bacillus and Ilumatobacteraceae and promoting xylanolysis in the 0–3 cm underground soil layer and enriching Acidimicrobiia and Mycobacterium and boosting respiration of sulfur compounds in the 3–6 cm underground soil layer. [Conclusion] In the soil ecosystem, B. velezensis FH-1 promotes the early growth of rice seedlings by regulating the structure and function of the microbial community in the soil on a spatial-temporal scale.

    • Effects of endophytic fungus Epichloë guerinii on disease resistance of the host Melica transsilvanica

      2024, 51(7):2411-2422. DOI: 10.13344/j.microbiol.china.230837

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      Abstract:[Background] Epichloë guerinii SC012 isolated from Melica transsilvanica, a plant only growing in the northern slope of the Tianshan Mountains in China, is a type of endophytic fungus of grasses. [Objective] To investigate the relationship between E. guerinii and the disease resistance of its host M. transsilvanica. [Methods] Dual-culture tests were conducted to assess the interactions between the endophytic fungus and four common plant pathogenic fungi. A pot experiment was performed to evaluate the resistance of M. transsilvanica with E. guerinii to pathogen invasion. [Results] In the dual-culture tests, E. guerinii isolated from M. transsilvanica exhibited inhibitory effects on Rhizoctonia solani, Alternaria tenuis, and Colletotrichum cereale, with the inhibition rates of 57.39%, 26.30%, and 20.92%, respectively. The fermentation broth of E. guerinii also inhibited the growth of the three pathogens, with the inhibition rates of 64.94%, 32.93%, and 15.61%, respectively. The endophytic fungus and its fermentation broth had no impact on the growth of Fusarium sp. The results of the pot experiment showed that E. guerinii reduced the disease indexes of M. transsilvanica exposed to the invasion of R. solani, A. tenuis, and C. cereale, with the relative protective effects of 36.61%, 16.01%, and 21.87%, respectively. Apart from reducing the incidence caused by R. solani, E. guerinii showed no significant effect on the incidence caused by other pathogens. M. transsilvanica plants with the endophytic fungus (E+) had higher polyphenol oxidase (PPO) and phenylalnine ammonialyase (PAL) activities than the plants without the endophytic fungus (E‒) (P<0.05), except that the PAL activity had no significant difference in the case of Fusarium sp. infection. [Conclusion] The endophytic fungus E. guerinii can enhance the resistance of M. transsilvanica to specific pathogens, while the resistance varies depending on the pathogens. The findings provide a basis for the use of endophytic fungi of grasses in the biocontrol and breeding of disease resistant crops.

    • Overexpression of Vvmapk affects growth, stress responses, and virulence of Beauveria bassiana

      2024, 51(7):2423-2434. DOI: 10.13344/j.microbiol.china.230849

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      Abstract:[Background] Mitogen-activated protein kinase (MAPK) plays pivotal roles in cell proliferation, differentiation, apoptosis, and stress responses in eukaryotes. [Objective] To identify the roles of Vvmapk in Volvariella volvacea for applications. [Methods] Vvmapk was overexpressed under the control of Vvpgd promoter in Beauveria bassiana, and the roles of Vvmapk in the growth, development, stress responses, and virulence were examined. Compared with the wild type, the overexpression of Vvmapk in B. bassiana increased the pigment level and conidial yield but did not affect the growth. The strains overexpressing this gene displayed higher conidial germination rates under heat shock, salt, and UV-irradiation stress conditions than the control. Furthermore, Vvmapk overexpression endowed the fungi with stronger virulence against Galleria mellonella but weaker antagonistic effects against Fusarium solani and Fusarium oxysporum than the wild type. The relative transcription levels of BbBrlA, BbabaA, and BbwetA associated with the conidial development were significantly up-regulated in the recombinant strain. [Conclusion] VvMAPK positively regulated the conidial development, pigmentation level, and virulence of B. bassiana. The findings laid a theoretical foundation for the functional identification and applications of V. volvacea genes.

    • Metabolomics of Volvariella volvacea in response to infection by Pseudomonas sp. Z9

      2024, 51(7):2435-2449. DOI: 10.13344/j.microbiol.china.230852

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      Abstract:[Background] Pseudomonas sp. Z9 is the pathogen responsible for causing yellow blotch disease in Volvariella volvacea. However, the mechanism of this pathogen in inducing yellow blotch disease remains unclear. [Objective] To investigate the differential metabolites and metabolic pathways of V. volvacea infected by Pseudomonas sp. Z9, providing a basis for understanding the underlying mechanism. [Methods] V. volvacea fruiting bodies inoculated with the pathogen and sterile water (CK) were sampled every 12 h within 48 h. The content of malondialdehyde (MDA) and the relative conductivity of the fruiting bodies were measured. Meanwhile, the activities of superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO) associated with the metabolism of reactive oxygen species were determined. Non-targeted metabolomics was employed to explore the differential metabolites and pathways of Pseudomonas sp. Z9-infected V. volvacea 24 h post-infection. [Results] The MDA content of V. volvacea increased over time within 48 h after inoculation of the pathogen and was higher in the inoculation group than in the CK group (P<0.05). CAT and SOD activities gradually increased over time and reached the peak at the time point of 24 h, while the PPO activity first decreased and then increased. The relative conductivity of the two groups first increased and then reached stability after 12 h. A total of 131 differential metabolites between the two groups were screened out and identified. The KEGG pathway enrichment results revealed that the differential metabolites were mainly enriched in amino acid metabolism pathways, especially tryptophan metabolism and tyrosine metabolism. Syringic acid was the metabolite with the largest increase in content after infection by Pseudomonas sp. Z9 in V. volvacea, with a variable important for the projection (VIP) value of 1.355 6. [Conclusion] The cell membrane of V. volvacea infected by Pseudomonas sp. Z9 experiences persistent peroxidation. We hypothesize that V. volvacea utilizes syringic acid to eliminate reactive oxygen species produced upon infection of Pseudomonas sp. Z9, thus avoiding cellular oxidative damage. Tyrosine metabolism is an important metabolic pathway involved in the formation of yellow blotches caused by Pseudomonas sp. Z9 in V. volvacea.

    • Identification and biocontrol potential of a strain against two pepper diseases

      2024, 51(7):2450-2462. DOI: 10.13344/j.microbiol.china.230835

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      Abstract:[Background] Pepper southern blight caused by Sclerotium rolfsii and pepper blight caused by Phytophthora capsici have seriously affected the yield and quality of peppers (Capsicum annuum). [Objective] To obtain efficient antagonistic bacteria for the biocontrol of two pepper diseases and provide a theoretical basis for the development of biocontrol agents. [Methods] From the pre-preserved microbial resource library, the strain SEC-197 with strong inhibitory effects on both pathogens was screened out. It was identified by morphological, physiological, biochemical, and molecular analyses. The production of extracellular enzymes and plant growth-promoting effect of the strain were assessed. PCR amplification was employed to mine the genes associated with the inhibitory and promoting effects. Furthermore, the biocontrol and plant growth-promoting effects of the strain were evaluated by pot experiments. [Results] The strain SEC-197 showed inhibitory rates of 76.59%±0.59% and 74.85%±0.35% against S. rolfsii and P. capsici, respectively. Strain SEC-197 was identified as Bacillus velezensis, which can form biofilms and had the ability to secrete proteases and cellulases, dissolve organic and inorganic phosphorus, produce siderophores, and fix nitrogen. The strain carried biocontrol and growth promotion-related genes such as srfAB, fenB, and ynsE. The pot experiment results showed that the strain had the control effects of 72.08% and 70.42% against pepper southern blight and pepper blight, respectively. Moreover, the strain’s fermentation broth significantly increased growth indicators such as stem width, fresh weight, and dry weight of pepper plants. [Conclusion] The strain SEC-197 has significant antagonistic activity against both pepper southern blight and pepper blight and an obvious plant growth-promoting effect. The research finding provide a theoretical basis and experimental foundation for the practical application of pepper production.

    • Identification of strain KF-43-1 against cotton verticillium wilt and comparison of biocontrol effects among three application methods in the field

      2024, 51(7):2463-2474. DOI: 10.13344/j.microbiol.china.230780

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      Abstract:[Background] Cotton verticillium wilt, known as the “cancer” of cotton, causes serious harm to the yield and fiber quality of cotton. Biocontrol with eco-friendliness and high safety has become the key research content in the prevention and treatment of cotton verticillium wilt. Therefore, developing new biological agents to control cotton verticillium wilt is of great significance for cotton production. [Objective] To preliminarily identify and characterize the actinomycete strain KF-43-1 with antagonistic effect on cotton verticillium wilt and determine the best application method of this strain, so as to provide support for the development of new biocontrol agents against cotton verticillium wilt. [Methods] Strain KF-43-1 was identified based on molecular evidence, and its morphological and growth characteristics were observed. The inhibitory effects of KF-43-1 on pathogenic fungi were determined. The physiological and biochemical characteristics of the strain were determined, and verify the field control effect of different uses of actinomyces KF-43-1 on cotton verticillium wilt. [Results] Strain KF-43-1 was identified as Streptomyces albogriseolus. It showed inhibitory effect of 82.05% on the pathogen V991, which was higher than that of the actinomycete strain 5406. It demonstrated the inhibitory effect of 25.81% on Fusarium oxysporum ST89, which was lower than that of strain 5406. Strain KF-43-1 had no fluorescence reaction and can liquefy gelatin, hydrolyze starch, yield positive results in the methyl red test, and produce melanin. However, it did not have the ability to decompose cellulose. Strain KF-43-1 grew well at pH 7.0–8.0, and was inhibited in Actinomycetes culture medium with 2% salt concentration and showed some salt tolerance. The effect of antagonistic KF-43-1 on cotton verticillium wilt was higher than that of centrifuge coating and centrifuge spraying, indicating that antagonistic KF-43-1 had better inhibition effect on cotton verticillium wilt outbreak after colonizing in soil. [Conclusion] The actinomycete strain KF-43-1 serves as a candidate for the development and application of biological agents for the prevention and control of cotton verticillium wilt.

    • Fungal diversity in the plant and rhizosphere soil of Valeriana jatamansi Jones

      2024, 51(7):2475-2485. DOI: 10.13344/j.microbiol.china.230857

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      Abstract:[Background] Valeriana jatamansi Jones has a long history of medical use and is widely used. However, the fungal diversity in the plant and rhizosphere soil of V. jatamansi remains to be reported. [Objective] To explore the diversity and structures of endophytic fungal communities in the rhizosphere soil and different tissues of V. jatamansi and lay a foundation for the development and utilization of endophytic fungal resources in this plant. [Methods] The ITS1 region of fungal rRNA gene was sequenced by the Illumina Miseq PE250 platform to analyze the endophytic fungi in the root, stem, leaf, and rhizosphere soil samples of V. jatamansi. The bioinformatics tools were used to analyze the sequencing results and reveal the fungal community structures in the plant and rhizosphere soil of V. jatamansi. [Results] A total of 1 776 731 valid sequences and 7 399 OTUs were obtained from the root, stem, leaf, and rhizosphere soil samples of V. jatamansi, belonging to 1 269 species, 767 genera, 331 families, 146 orders, 65 classes of 17 phyla. The richness of endophytic fungi followed the trend of rhizosphere soil>stem>root>leaf, and the diversity was in the order of rhizosphere soil>stem>leaf>root. There were 192 common OTUs shared by the four samples, and the number of unique OTUs showed the trend of rhizosphere soil>stem>root>leaf. At the phylum level, the dominant fungi in the root, stem, leaf, and rhizosphere soil samples were Ascomycota and Basidiomycota. At the genus level, Hannaella was the dominant genus in the leaf and stem samples, Plectosphaerella was the dominant genus in the root samples, and the unclassified_fungi accounted for a relatively high proportion in the rhizosphere soil samples. The fungal network showed that each genus was associated with one or more genera in the average abundance, most of the genera were positively correlated with each other. [Conclusion] There were abundant endophytic fungal resources in V. jatamansi, and the endophytic fungal community composition showed significant differences in different tissues and rhizosphere soil of the plant. This study explored the biological information of endophytic fungal resources in V. jatamansi, providing a theoretical basis for the protection of V. jatamansi resources and the development and utilization of endophytic fungal resources.

    • >Food Microbiology
    • Contamination status, multilocus sequence typing, and drug resistance of Salmonella in poultry meat in Liaoning Province

      2024, 51(7):2486-2493. DOI: 10.13344/j.microbiol.china.230957

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      Abstract:[Background] Salmonella is a major group of zoonosis pathogens that can be transmitted to humans through poultry and its products, posing a threat to human health. It is important for public health to understand the pathogenic characteristics and drug resistance of Salmonella in poultry meat. [Objective] To investigate the contamination status, serotype distribution, drug sensitivity, and molecular epidemic characteristics of Salmonella in poultry meat sold in Liaoning Province in 2022, and to explore the relationship between different serotypes, multilocus sequence typing (MLST) results, and drug resistance. [Methods] The collected samples were tested according to the method in the National Standard for Food Safety: Microbiology Inspection of Salmonella (GB 4789.4—2016), and the strains were identified by biochemical tests. The slide agglutination method was used for the serological test, and the micro-broth method was employed to examine the drug sensitivity of the strains. BioNumerics 7.6 was used to analyze the results of drug sensitivity tests and MLST. [Results] The 53 Salmonella strains belonged to 11 serotypes, mainly S. enteritidis. The resistance rate of Salmonella to nalidixic acid (NAL) was the highest, 92.5% (49/53). A total of 25 multiple drug resistance spectra were identified, and the total resistance rate was 66.0% (35/53). Among the drug resistance genes, the tetracycline resistance gene tetC showed the highest carrying rate of 45.3% (24/53). The strains of Salmonella were classified into 10 sequence types (STs) and 1 unclassified type according to the sequences of 7 housekeeping genes. [Conclusion] The Salmonella in poultry meat sold in Liaoning Province demonstrates serious contamination, with diverse serotypes, high overall drug resistance, and serious multidrug resistance. Efforts should be doubled for the standardization of poultry breeding.

    • Silymarin inhibits the food-borne pathogen Staphylococcus aureus ATCC 25923 in vitro

      2024, 51(7):2494-2507. DOI: 10.13344/j.microbiol.china.230809

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      Abstract:[Background] As a common food-borne pathogen, Staphylococcus aureus can do harm to food safety and human health. Although chemical preservatives can inhibit the growth of food-borne pathogens, they pose a threat to human health and the environment. [Objective] To decipher the mechanism of silymarin in inhibiting S. aureus ATCC 25923 for the development of plant-derived bacteriostatic agents. [Methods] We investigated the effects of silymarin on the membrane structure of bacteria by measuring alkaline phosphatase, β-galactosidase activities, and PI staining. The effects of silymarin on the macromolecules of bacteria were explored by DNA electrophoresis and determination of DNA and protein concentrations in bacteria cells. Furthermore, the effects of silymarin on the synthesis of polysaccharide intercellular adhesion (PIA), bacteria in biofilms, and the proteins and polysaccharides in mature biofilms were explored. Fourier transform infrared spectrometry was employed to reveal the changes of bacterial composition. On the basis of the results, the diversity of the mechanisms of silymarin in inhibiting S. aureus ATCC 25923 was expounded. [Results] The minimum inhibitory concentration (MIC) of silymarin against S. aureus ATCC 25923 was 0.5 mg/mL. Silymarin can inhibit the growth of bacteria by destroying the membrane structure of bacteria cells, reducing the concentrations of DNA and protein, Inhibit the synthesis of PIA in biofilm, and degrading the mature biofilm components. [Conclusion] Silymarin can inhibit the growth of S. aureus in multiple ways, which is expected to provide a theoretical basis for the further application of natural products from plants.

    • Composite organic acids for decontamination of Listeria monocytogenes in chicken

      2024, 51(7):2508-2520. DOI: 10.13344/j.microbiol.china.230838

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      Abstract:[Background] Listeria monocytogenes (Lm) is a major zoonotic pathogen. Lm carried by domesticated animals can spread along slaughter and sale chains, posing a threat to consumers’ health. [Objective] To establish and apply a green efficient technology for the control of Lm contamination in the broiler industrial chain. [Methods] We prepared a composite organic acid disinfectant composed of lactic acid and citric acid, optimized the sterilization conditions by response surface methodology, conducted preliminary applications of the disinfectant in slaughterhouses, and explored the sterilization mechanism. [Results] The optimal sterilization conditions were as follows: lactic acid content of 0.98%, citric acid content of 0.72%, and treatment for 3.6 min. The treatment under these conditions reduced the Lm on the chicken surface by more than 99%, extended the shelf life of chicken, and decreased the isolation rate of Lm on the surface of knives and broiler carcasses in the pre-cooling and segmentation stages in broiler slaughterhouses. Moreover, the composite organic acid disinfect significantly down-regulated the expression of genes associated with Lm virulence, inhibited the biofilm formation, and destroyed the cell structure of Lm. [Conclusion] The composite organic acid disinfect prepared in this study can effectively reduce Lm on the broiler surface in a short time, demonstrating good application prospects in preventing and controlling Lm contamination in the slaughter process of broilers.

    • Screening, identification, and mechanism elucidation of zearalenone-reducing lactic acid bacteria from Chinese sourdough

      2024, 51(7):2521-2533. DOI: 10.13344/j.microbiol.china.230789

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      Abstract:[Background] Zearalenone (ZEN), a mycotoxin with estrogenic effects produced by Fusarium, is one of the pollutants that seriously jeopardize human health and agricultural safety worldwide. As a class of food-grade non-pathogenic microorganisms generally recognized as safe (GRAS), lactic acid bacteria have been proved to have good mycotoxin-reducing ability in recent years, demonstrating the application potential in safeguarding food safety. [Objective] To screen the lactic acid bacteria capable of reducing ZEN from seven Chinese sourdough samples collected from Shandong, Henan, and Gansu, and investigate the reducing mechanisms of ZEN by the bacteria. [Methods] The strains were isolated by the dilution-plate coating method. Ultra-high performance liquid chromatography was employed to screen the strains with ZEN-reducing activity, mass spectrometry (Q exactive) to identify the metabolites, and transmission electron microscopy (SEM) to observe the morphology of the strains. The adsorption effects were examined at different initial toxin concentrations and bacterial concentrations, and a kinetic model was fitted. Fourier transform infrared spectroscopy (FTIR) was employed to identify the functional groups involved in the adsorption of ZEN and elucidate the adsorption mechanism of ZEN. [Results] A total of 63 strains of Lactobacillus were isolated from the preliminary screening, and then three strains with ZEN-reducing activity were obtained. Strains 6-8 and 18-2 were identified as Levilactobacillus brevis, and strain 12-6 as Acetobacter tropicalis. The two strains of L. brevis had ZEN-degrading effects, with the degradation rates of 85.5% and 87.3%, respectively, within 48 h at ZEN concentration of 10 mg/L. The mass spectrometry results showed that the degradation product was α-ZEL. The other strain had a ZEN-adsorbing effect, with the adsorption rate reaching 62.9% within 20 min at a bacterial concentration of 4.26×1010 CFU/mL and a ZEN concentration of 10 mg/L. Moreover, the adsorption rate increased by 20% after strain inactivation. The adsorption process was fitted with both pseudo-first-order and pseudo-second-order models. Fourier transform infrared spectroscopy (FTIR) showed that the main adsorption sites of strain 12-6 were peptidoglycan and teichoic acid in the cell wall, involving hydroxyl, methenyl, carboxyl, and amide groups. [Conclusion] L. brevis 6-8 and 18-2 showed strong degradation capacity against ZEN, and A. tropicalis12-6 had a strong adsorption capacity for ZEN. The adsorption kinetics conformed to pseudo-first-order and pseudo-second-order models, with the adsorption sites at teichoic acid and peptidoglycan in the cell wall. This study provides a theoretical basis for the application of lactic acid bacteria in the removal of harmful substances from food and feed.

    • >Veterinary Microbiology
    • Rescue and identification of a recombinant porcine epidemic diarrhea virus carrying a tagged peptide at the N terminus of S protein

      2024, 51(7):2534-2545. DOI: 10.13344/j.microbiol.china.240078

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      Abstract:[Background] Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) is a severe porcine infectious disease causing serious losses to the pig industry. The spike (S) protein, one of the structural proteins of PEDV, is responsible for viral infection and entry in host cells. The structure and function of S protein are a research hotspot in the molecular biology of PEDV. The S protein of PEDV has two functional domains: the N-terminal S1 domain and the C-terminal S2 domain. Deletion of the S1N domain (position: 19−233 aa) did not impair the survival or propagation of the PEDV strain DR13att in vitro. [Objective] To know whether a tagged protein or peptide can substitute the S1N domain, we introduced the HA tag and soybean ascorbate peroxidase (APEX2) gene respectively into the S1Ndomain of DR13att to establish recombinant viruses by targeted RNA recombination. [Methods] The S1N domain (position: 1−234 aa) of the transfer vector p-PEDV-DR13att was respectively substituted with HA gene and APEX2 gene and the transfer vectors were linearized. The RNA of linearized transfer vectors were electrotransferred to the LR7 cells infected with mPEDV, and the recombinant PEDV was rescued on Vero cells. The recombinant virus was validated by observation of cytopathic effect (CPE), RT-PCR, sequencing, indirect immunofluorescence assay (IFA), and Western blotting. Finally, the titer of each recombinant virus was measured and the growth curve of each recombinant virus was plotted to reveal the growth characteristics of the recombinant viruses and their parent virus. [Results] The constructed recombinant viruses were rescued and passaged. The recombinant virus rPEDV-DR13-S1N-HA (rPEDV-DSH) carrying the HA tag instead of the S1N domain showed CPE in P1. The recombinant virus rPEDV-DR13-S1N-APEX2 (rPEDV-DSA) carrying APEX2 had not presented CPE until blind passage to P4. The RT-PCR, sequencing, IFA, and Western blotting of rPEDV-DSH and rPEDV-DSA in P4 confirmed that rPEDV-DSH with the HA tag was successfully rescued, while rPEDV-DSA was not rescued. The growth curves indicated that rPEDV-DSH had a similar growth trend but decreased proliferation level compared with the parent strain DR13att. [Conclusion] The recombinant virus rPEDV-DSH carrying a HA tag in the S1N domain (position: 1−234 aa) was successfully rescued, which laid a foundation for further research on the interaction mechanism between the S protein and host cells.

    • Isolation and identification of Enterococcus mundtii and Enterococcus faecium with probiotic properties from the intestine of sheep

      2024, 51(7):2546-2562. DOI: 10.13344/j.microbiol.china.231049

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      Abstract:[Background] As the resistance of pathogens to antibiotics is enhancing, it is increasingly important to develop safe and reliable antibiotic alternatives in the animal husbandry. Probiotics with no toxicity, no residues, and low costs have emerged as potential solutions, offering effective supplements of beneficial microorganisms in the intestinal tract. [Objective] To isolate probiotics from the intestine of sheep. [Methods] The intestinal contents and feces of healthy sheep were cultured and purified with the MRS medium, and the isolates were then identified by morphological analysis and 16S rRNA gene sequencing. The probiotic properties of the isolates were assessed, including the antimicrobial activity, acid and bile salt tolerance, self-aggregation capacity, antibiotic sensitivity, and safety in mice. [Results] Several strains were isolated from the intestine of healthy sheep, among which A12 and C1 were identified as Enterococcus mundtii and E. faecium, respectively. The cell-free supernatants of isolates A12 and C1 had strong inhibitory activities against Staphylococcus aureus and Salmonella choleraesuis. The isolates A12 and C1 had good acid and bile salt tolerance and strong self-aggregation ability and were susceptible to a variety of antibiotics. In addition, the two isolates did not induce obvious pathological lesions in mice, demonstrating good safety. [Conclusion] E. mundtii strain A12 and E. faecium strain C1 isolated from the sheep intestine have excellent probiotic properties and good safety, showcasing the potential to be developed as probiotics.

    • Isolation, identification and antibiotic resistance analysis of pathogenic Escherichia coli and Salmonella from white feather broiler

      2024, 51(7):2563-2575. DOI: 10.13344/j.microbiol.china.230913

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      Abstract:[Background] Escherichia coli and Salmonella are the main pathogens in broiler breeding, which can cause huge economic losses, and their antibiotic resistance shouldn’t be ignored. [Objective] To isolate and identify pathogens from dead chickens, and analyze their antibiotic resistance characteristics. [Methods] The target strains were isolated and purified by conventional method. Biochemical tests, 16S rRNA gene sequence analysis and growth curve verification were used for identification. The drug susceptibility test was carried out by disk diffusion method, and the drug resistance rate and multiple drug resistance were analyzed. [Results] A total of 10 strains of E. coli and 10 strains of Salmonella were isolated, including 4 enterica subspecies, 1 arizonae subspecies and 5 Salmonella bongori. E. coli isolates were only sensitive to carbapenems and resistant to other antibiotics. While Salmonella isolates were sensitive to carbapenems and showed different degree of resistance to other antibiotics. All the isolates were multi-drug resistant. [Conclusion] The pathogens were isolated and their antibiotic resistance were determined, which provided reference for the prevention and cure of E. coli and Salmonella in white feather broiler breeding.

    • Isolation, identification, genetic evolution analysis, and pathogenicity test of Riemerella anatipestifer PT-RA1

      2024, 51(7):2576-2585. DOI: 10.13344/j.microbiol.china.230885

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      Abstract:[Background] Riemerella anatipestifer is a pathogen causing fibrinous pericarditis, perihepatitis, and airsacculitis in ducks. With a variety of serotypes, this pathogen is difficult to be prevented and controlled. [Objective] To understand the genetic characteristics, drug resistance and pathogenicity of Riemerella anatipestifer in Putian city, and provide scientific basis for its treatment and prevention. [Methods] Isolation and purification, 16S rRNA gene identification, and serotype identification were performed on the brain tissue of 15-day-old ducklings suspected to be infected with orrhomeningitis from a breeding farm in Putian. The 16S rRNA gene of this isolate was sequenced, based on which the genetic evolution analysis was performed. Furthermore, the antibiotic resistance of the strain and the pathogenicity to ducklings were determined. [Results] A strain of Riemerella anatipestifer serotype 11 was isolated, named PT-RA1. The 16S rRNA gene of PT-RA1 was in the same branch on the genetic evolution tree as those of the reference strains (GZ-RA12, CZ-RA03, and SD-RA2018) of R. anatipestifer, with 100% similarity, which suggested that R. anatipestifer may have spread across different regions. The isolate was resistant to 15 antibiotics including streptomycin, gentamicin, and kanamycin, while it was sensitive to ampicillin, amoxicillin, cefazolin, ceftriaxone, cefixime, cefepime, tetracycline, doxycycline, oxfloxacin, and florfenicol. The isolate caused a mortality rate of 80% in ducklings, and the dead ducks showed typical symptoms such as fibrinous pericarditis, perihepatitis, airsacculitis, and spleen enlargement of R. anatipestifer infection. [Conclusion] We isolated and characterized a R. anatipestifer strain PT-RA1, which provided a basis for the prevention and control of R. anatipestifer infection and subsequent research.

    • Isolation, identification and biological characteristics analysis of a strain of Klebsiella pneumoniae

      2024, 51(7):2586-2598. DOI: 10.13344/j.microbiol.china.230804

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      Abstract:[Background] Klebsiella pneumoniae is a zoonotic opportunistic pathogen, which can cause infections in many animals and is difficult to be controlled. [Objective] To investigate the existence of pathogenic bacteria in a rhinoceros domestication farm. [Methods] Bacteria isolation and identification, drug resistance and drug resistance genes, virulence gene screening, and pathogenicity test in mice were performed on six samples of rhinoceros feces randomly collected. [Results] One Klebsiella pneumoniae strain was isolated, with the isolation rate of 16.67%. The isolate was resistant to six antibiotics, including doxycycline, polymyxin B and bacitracin, and carried six resistance genes and six virulence genes (kfuBC, fimH, ureA, uge, wabG, and wcaG). In the pathogenicity test, the strain suspension of 1×1010 CFU/mL led to a mortality rate as high as 80% in mice and lesions appeared in the liver, lung, and small intestine tissues of dead mice. [Conclusion] The K. pneumoniae strain isolated from the rhinoceros domestication farm had strong drug resistance, a low combination rate of drug resistance gene and drug resistance phenotype, and a complex drug resistance mechanism. The results of virulence gene prediction were in good agreement with the results of pathogenicity test. The data could provide reference for the prevention and control of K. pneumoniae infection in domestication farms.

    • Bacteria from porcine colon regulate cytokine and transcription factor expression in the gut of the mouse model of colitis

      2024, 51(7):2599-2613. DOI: 10.13344/j.microbiol.china.230731

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      Abstract:[Background] Microorganisms in the mammalian digestive tract are important for maintaining gut homeostasis, while the regulatory role of gut bacteria in cytokine expression remains unclarified. [Objective] To compare the regulatory effects of different isolated from porcine intestinal tract on the expression of cytokines and transcription factors in the model of colitis. [Methods] Bacteria were isolated from the porcine colon, and the culture supernatant of each isolate was used to treat the Caco-2 cell model of lipopolysaccharide-induced inflammation. Furthermore, a mouse model of colitis was induced by dextran sodium sulfate (DSS) and then administrated with bacterial suspension by gavage. The gene expression of cytokines and transcription factors in Caco-2 cells and mouse colon tissue, the cell growth rate, and the mouse weight after bacterial treatment were determined. [Results] Six strains of bacteria were isolated from porcine colon, belonging to Enterococcus, Lactobacillus, Sharpea, and Mitsuokella. In the Caco-2 cell model of inflammation, Lactobacillus amylovorus LGM down-regulated the mRNA levels of interleukin (IL)-4 and IL-17 (P<0.05); Enterococcus cecorum LGM down-regulated the mRNA levels of T-box expressed in T cells (T-bet), forkhead box P3 (Foxp3), IL-17, and transforming growth factor-β (TGF-β) (P<0.05). In the mouse model of colitis, only L. amylovorus LGM alleviated the colon inflammation, restored the colon length, and up-regulated the mRNA levels of Foxp3, GATA Binding Protein 3 (GATA-3), and interferon-γ (IFN-γ) in the colon tissue (P<0.05). However, Mitsuokella jalaludinii LGM aggravated colon injury and up-regulated the mRNA level of interferon-γ in the tissue (P<0.05). [Conclusion] The bacterial isolates have different regulatory effects on porcine colitis, which is related to the regulation of cytokine gene expression. L. amylovorus LGM exerts an anti-inflammatory effect by up-regulating the expression of Foxp3 and GATA-3 in the colon.

    • >Microbial Functional Genomics
    • Secondary metabolism potential of Saccharothrix and establishment of gene editing systems in representative strains

      2024, 51(7):2614-2629. DOI: 10.13344/j.microbiol.china.240067

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      Abstract:[Background] Rare actinomycetes represent a “new treasure trove” for discovering natural products. Studies remain to be carried out to explore the natural product-producing potential of Saccharothrix as typical rare actinomycetes. Furthermore, few studies reported gene editing systems specifically tailored for Saccharothrix. [Objective] This study aims to elucidate the potential of Saccharothrix in synthesizing natural products with diverse structures. Additionally, we seek to establish gene editing systems for representative strains, thereby fostering the discovery of novel natural compounds and advancing the research on related biosynthetic pathways. [Methods] Multi-locus sequence analysis (MLSA) was employed to assess the similarity among 34 publicly available Saccharothrix genomes. The tool antiSMASH was utilized to analyze the gene clusters and the structural information of associated products. Additionally, BiG-SCAPE was applied for clustering analysis of these gene clusters. The representative strains, Saccharothrix australiensis DSM 43800 and S. syringae NRRL B-16468, were selected, for which the conjugation transfer and gene editing systems were established via integrative vectors and gene knockout vectors. [Results] The analysis of the 34 Saccharothrix genomes revealed a total of 1 348 natural product biosynthetic gene clusters, with an average of approximately 40 clusters per genome. The gene clusters were abundant in the biosynthesis of polyketides, non-ribosomal peptides, hybrid products of polyketides and non-ribosomal peptides, as well as ribosomally synthesized and post-translational modified peptides. The 1 348 gene clusters were grouped into 852 gene cluster families (GCFs), which were further grouped into 130 gene cluster clans (GCCs). This study established and optimized a conjugation transfer system applicable to S. australiensis DSM 43800 and S. syringae NRRL B-16468. Additionally, gene editing systems were successfully established for the two representative strains, with the establishment of corresponding mutant strains. [Conclusion] As rare actinomycetes, Saccharothrix exhibit a rich array of natural product biosynthetic gene clusters, highlighting robust potential for synthesizing diverse natural compounds, particularly polyketides and polypeptides. Moreover, this study achieves precise editing of the Saccharothrix genome, laying a solid foundation for probing into the gene clusters and associated products.

    • Responses of ruminal carbohydrate-degrading enzymes and microbial metagenome in yaks to dietary supplementation with mixed isoacids

      2024, 51(7):2630-2646. DOI: 10.13344/j.microbiol.china.230714

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      Abstract:[Background] Isoacids are branched-chain volatile fatty acids, providing nutrients to the microorganisms and promoting the growth of fiber-degrading bacteria in the rumen. [Objective] This study aimed to investigate the effects of dietary supplementation with mixed isoacids (MI) on the activities of carbohydrate-degrading enzymes, microbiota composition, and functional genes in the yak rumen. In addition, the correlations of the enzyme activities with the relative abundance of rumen microorganisms and functional genes were analyzed. The results are expected to provide a reference for the rational use of MI to improve the fermentation performance in the yak rumen. [Methods] A single factorial experimental design was used and 16 male yaks weighing approximately 200 kg were randomized into four groups with four replicates per group and one yak per replicate. Yaks in the control group were fed with a basal diet, while those in the other groups were fed a basal diet supplemented with 0.1% (T1 group), 0.2% (T2 group), and 0.3% (T3 group) of MI, respectively. The pre-experiment lasted for 10 days, and the formal feeding lasted for 85 days. At the end of the experiment, rumen fluid samples were collected and the rumen microbiome was analyzed by metagenomic sequencing. Activities of carbohydrate-degrading enzymes were measured by and UV spectrophotometry. [Results] Compared with the control group, dietary MI supplementation significantly increased the activities of carboxymethyl cellulase and amylase in the yak rumen. Supplementation with 0.3% MI reduced the microbial richness and increased the similarity of microbiota composition and functional gene composition in the yak rumen. The differential bacteria such as Firmicutes, Erysipelotrichia, Saccharibacteria, and Aeromonadaceae had high relative abundance in the T2 group. The KEGG pathway enrichment analysis showed that the differentially expressed genes encoding phosphotransferase (2.7.1.68), ubiquitin hydrolase (3.4.19.12), aminoacyltransferase (2.3.2.27), and protein serine/threonine kinases (2.7.11.11) had high relative abundance in the T1 group. Rumen amylase activity was positively correlated with Profundibacterium, Aeromonas, Saccharibacteria, and Aeromonadaceae, as well as members of the GH13_20, GH77, and CBM72 gene families. The carboxymethyl cellulase activity in the rumen was positively correlated with members of the GT90 gene family. [Conclusion] Supplementation of MI in yak diets increased the activities of amylase and carboxymethyl cellulase, improved the similarity of rumen microbiota composition and functional gene composition, and increased the relative abundance of bacteria and functional genes in the yak rumen. In addition, rumen microbiome and their functional genes were correlated with the activities of rumen enzymes. The results suggest that MI can improve the rumen fermentation in yaks to a certain extent.

    • >Pharmaceutical Microbiology
    • Endophytic bacteria and fungi for high production of taxanes: screening and application in the fermentation of Taxus media

      2024, 51(7):2647-2662. DOI: 10.13344/j.microbiol.china.230822

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      Abstract:[Background] Taxus media are abundant in endophytic bacteria and fungi, which can produce a range of anti-tumor active compounds. Fermentation and processing can increase the concentrations of active components in Chinese herbal medicines. [Objective] To select the optimal strain from the taxane-producing endophytic strains of T. media for solid fermentation and optimize the fermentation duration, so as to innovate the solid fermentation method of T. media. [Methods] The plate streaking method was used to isolate the endophytic strains of T. media. HPLC and UPLC-MS were employed to measure taxanes, and the strains with high yields of taxanes were selected. The selected strains were used for solid fermentation of T. media, and the concentrations of seven components were determined by HPLC. Weighted scores were computed to determine the optimal strain for fermentation, and the optimal duration of the solid fermentation process was determined. The optimal strain was identified by 16S rRNA gene and whole-genome sequencing. [Results] A total of 30 fungal strains and 35 bacterial strains were isolated from T. media, and 7 strains had the ability to produce taxanes. Among them, 6, 4, and 2 strains produced baccatin III, cephalomannine, and taxol, respectively. The 6 strains producing baccatin III were LY2 (2.99 mg/L), LY9 (4.90 mg/L), LP22 (0.34 mg/L), PJ20 (1.43 mg/L), PJ25 (0.18 mg/L), and PJ30 (0.36 mg/L). The 4 strains yielding cephalomannine were LY2 (4.92 mg/L), LY9 (1.31 mg/L), PP4 (0.66 mg/L), and PJ25 (1.31 mg/L). The 2 strains capable of producing taxol were PP4 (0.70 mg/L) and PJ25 (0.34 mg/L). The HPLC results and weighted scores confirmed that LP22 was the predominant strain for solid fermentation and 48 h was the optimum fermentation period. The strain LP22 was identified as Bacillus amyloliquefaciens by 16S rRNA gene and whole genome sequencing. The full-length genome sequencing data was submitted to NCBI and received the accession number PRJNA1039587. [Conclusion] Certain endophytic strains of T. media have the ability to produce taxanes. The solid fermentation with strain LP22 for 48 h increased the content of taxanes and flavonoids in T. media. The findings provide a basis for further research on the fermentation and processing of T. media.

    • >Medical Microbiology
    • Virulence characteristics of Klebsiella pneumoniae isolates from the feces of children in neonatal intensive care unit

      2024, 51(7):2663-2675. DOI: 10.13344/j.microbiol.china.230982

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      Abstract:[Background] Klebsiella pneumoniae, a conditional pathogen, often colonizes the upper respiratory tract and intestinal tract of the human body. Due to the emergence of clinically multi-drug resistant strains and the continuous enhancement of strain virulence, this pathogen has received extensive attention. Children in the neonatal intensive care unit (NICU) have an imperfect immune system and are exposed to the risk of nosocomial transmission of highly virulent strains of K. pneumoniae. [Objective] To investigate the presence and virulence characteristics of K. pneumoniae isolates from feces of children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital in 2021. [Methods] The strain isolation, whole genome sequencing, and virulence gene analysis of K. pneumoniae were carried out on 421 fecal samples collected from children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital. The virulence factor database (VFDB) and Kleborate were used for annotation of the virulence genes and the multilocus sequence typing (MLST) of K. pneumoniae, and the virulence of each strain was examined by the string test, the Galleria mellonella survival test, and the cytotoxicity test. [Results] A total of 13 strains of K. pneumoniae were detected in the 421 fecal samples, with a detection rate of 3.1%. K. pneumoniae was mainly carried by the patients with ABO hemolytic, low birth weight, neonatal pneumonia, and neonatal hyperbilirubinemia. The genome of K. pneumoniae isolates was 5 Mb, with median sequence length after genome assembly N50>20 kb and G+C content of 57%. The 13 strains presented 13 ST types, 13 K serotypes, and 7 O serotypes. Among the 846 virulence genes annotated by the VFDB, strain Kp50 had the largest number of virulence proteins and virulence genes, and this strain had the highest virulence score determined by Kleborate. Phenotypic tests revealed that among the 13 K. pneumoniae strains, only strain Kp50 had a high mucoid phenotype and stronger cytotoxicity. Strains Kp50, Kp260, Kp185, and Kp273, which had siderophore expression ability, presented stronger virulence in the larvae of Galleria mellonella. [Conclusion] K. pneumoniae isolates from the fecal samples of children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital showed a low detection rate and high diversity. There are highly virulent strains carrying multiple virulence genes and phenotypes. High attention should be paid to the prevention and control of the spread of highly virulent strains in hospitals.

    • Composition and gene functions of intestinal IgA-coated microbiota in the mouse model of chronic obstructive pulmonary disease

      2024, 51(7):2676-2689. DOI: 10.13344/j.microbiol.china.230793

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      Abstract:[Background] Immunoglobulin A (IgA) secreted by small airways plays a key role in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the role of IgA derived from intestinal mucosa in the pathogenesis of COPD and the IgA-coated microbiota remain unclear. [Objective] To investigate the composition, abundance, and differential gene functions of intestinal-derived IgA-coated gut microbiota in the mouse model of COPD. [Methods] A mouse model of COPD was established by intranasal instillation of lipopolysaccharide combined with cigarette smoking. Twelve fecal samples from COPD mice and 12 fecal samples from wild-type mice were collected. IgA magnetic beads were used to separate IgA-coated gut microbiota, followed by 16S rRNA gene high-throughput sequencing. [Results] We examined the modeling of COPD in mice by comparing the staining of lung tissue sections, mean linear intercept (mLI), and inflammatory cytokine concentrations in the alveolar lavage fluid between the two groups. Both OTU and PCA showed that the intestinal IgA-coated microbiota in fecal samples were different and comparable between the two groups. The alpha diversity analysis showed no statistically significant difference in the species diversity between the two groups (P>0.05). The structure of intestinal-derived IgA-coated microbiota had differences between the two groups (P<0.05). In the COPD group, the bacterial orders that were significantly enriched were Bdellovibrionales, Clostridiales, and Bifidobacteriales; the mainly enriched bacterial families were Prevotellaceae, Clostridiaceae, Paenibacillaceae, Bdellovibrionaceae, and Bifidobacteriaceae; and the mainly enriched bacterial genera were Alloprevotella, Brevibacillus, Clostridium-sensu-stricto, Turicibacter, Faecalibacterium, Vampirovibrio, and Bifidobacterium. The results of KEGG pathway enrichment analysis of differentially expressed genes showed that the cell growth and death, nucleotide metabolism, and digestive system-related pathways in the COPD group were significantly up-regulated, while the membrane transport pathway was significantly down-regulated. [Conclusion] The CODP mice present altered intestinal-derived IgA-coated gut microbiota and gene dysfunction.

    • Screening of antioxidant strains and its protective effect on oxidative stress damage of HepG2 cells

      2024, 51(7):2690-2701. DOI: 10.13344/j.microbiol.china.230842

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      Abstract:[Background] The oxidative damage of the body can cause a variety of diseases and body aging. It had been found that probiotics can improve the metabolic level and the antioxidant capacity of the host. This provided a new direction for the development of antioxidant products. [Objective] To screen the strains with good antioxidant effects and provide strain materials for the development and research of antioxidant drugs and nutraceuticals. [Methods] The strains isolated from the feces of healthy people in Changshou Village, Wentang Town, Yichun City, Jiangxi Province were identified by 16S rRNA gene sequencing. We selected six strains of probiotics and then determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity of the culture supernatants of the strains. One strain with the best antioxidant capacity was selected for subsequent cellular experiments. A model of oxidative stress damage in HepG2 cells was established, and the protective effect of the culture supernatant of the selected strain on the cell model was evaluated by the CCK-8 method. The protective effect of the culture supernatant on SOD in the cells was determined by the enzyme assay kit, and the antioxidant effect of the culture supernatant was further verified. [Results] Among the six strains of probiotics, strain H0661 had the best antioxidant capacity with GSH-Px activity of 586.11 U/mL (P<0.01), SOD activity of 1 278.00 U/mL (P<0.01), and DPPH radical scavenging capacity of 40.5%. The strain was identified based on the 16S rRNA gene sequencing results and named Staphylococcus xylosus TG022. The culture supernatant of this strain at the concentration of 15% (volume fraction) showed the best protective effect against oxidative stress in HepG2 cells (P<0.01). In addition, the culture supernatant of S. xylosus TG022 was effective in protecting the SOD activity of the HepG2 cells exposed to oxidative stress (P<0.01). Moreover, the antioxidant activity of HepG2 cells was increased after treatment with the culture supernatant of S. xylosus TG022 (P<0.05). [Conclusion] S. xylosus TG022 was screened out, and its culture supernatant had strong antioxidant effects. This study provided a candidate strain for the development of antioxidant drugs and nutraceuticals.

    • >BIOLOGICAL LAB
    • Spectrophotometry for rapid determination of the concentration of Avibacterium paragallinarum

      2024, 51(7):2702-2710. DOI: 10.13344/j.microbiol.china.230850

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      Abstract:[Background] The infection with Avibacterium paragallinarum (Apg) results in an acute upper respiratory disease known as infectious coryza in chickens. In general, the disease exhibits a short incubation period, an acute onset, and a high degree of infectivity, thereby causing substantial economic losses of breeding farms. [Objective] To assess the feasibility of spectrophotometry (SP) as a potential substitution of plate colony count (PCC) in determining the concentration of Apg. [Methods] The Apg precipitate was suspended in tryptone soy broth (TSB) or sterile physiological saline solution (SPSS), as two sets of diluents respectively. The absorbance (OD) values of the bacterial suspensions at different dilutions were determined by SP and the viable cell count was determined by PCC. With OD values measured at different wavelengths (450, 540, 600 and 650 nm) as the independent variables and the viable cell counts as the dependent variables, the standard curves and linear equations were established. Set up a tryptone soy broth (TSB) group and a sterile physiological saline solution (SPSS) group, using TSB and SPSS as suspensions and dilutions. Use SP and PCC methods to measure the suspension of different dilutions of Streptococcus parahaemolyticus, and compare and analyze the effect of the two suspensions on the correlation between OD value and bacterial concentration through standard curves and linear equations. The OD values of the bacterial suspension samples collected at different culture stages (10, 12, 14, 16 and 18 h) were measured and substituted into the linear equation to obtain the concentration of the bacterial suspension. PCC was carried out at the same time. SPSS 17.0 was used for statistical analysis to verify the applicability of SP. [Results] The OD value measured at 600 nm (OD600) showed the best linear relationship with the PCC results (R2>0.995). At this wavelength, the OD values measured in the TSB and SPSS groups were not different from the PCC results (R2difference=0.000 5), and the TSB group was superior to the SPSS group (R2TSB=0.998 9> R2SPSS=0.998 4). The results of SP and PCC showed no differences at the incubation time points of 10, 12, and 14 h (P>0.05) but had differences at the time points of 16 and 18 h (P<0.05). [Conclusion] During the logarithmic and stationary phases, SP could rapidly measure OD600 to determine the concentration of Apg.

    • >PERSPECTIVES AND COMMENTS
    • Research progress and hotspots of antibiotic resistance genes in livestock and poultry manure based on CiteSpace

      2024, 51(7):2711-2726. DOI: 10.13344/j.microbiol.china.230999

      Abstract (98) HTML (274) PDF 1.38 M (313) Comment (0) Favorites

      Abstract:Antibiotics have been widely used in animal husbandry since they were discovered in the 1940s, promoting the growth and health of domesticated animals. As most antibiotics are water-soluble, 30% to 90% of antibiotics are released into the environment in the form of parent compounds through urine or feces. Improper use of antibiotics will pose selective pressure on the environments and enhance the transmission of antibiotic resistance genes (ARGs) in the environment, thereby posing a potential risk to human health and ecosystems. Revealing the research hotspots, frontiers, and trends of ARGs in livestock and poultry manure in the last 20 years can provide important information for controlling the contamination of ARGs derived from livestock and poultry manure and shed new lights for the future research. We retrieved the articles published in the Web of Science (WoS) Core Collection and China National Knowledge Infrastructure (CNKI) from 2002 to 2022 to analyze the research status and trends of ARGs in animal husbandry systems. CiteSpace was employed to visualize the keyword co-occurrence, keyword clustering, keyword bursts, number of publications, and collaboration of research institutions. A total of 896 valid publications were included in this study. The number of publications about ARGs in livestock and poultry manure in both China and the globe has been growing since 2011 and has accelerated significantly since 2014. The papers published in English were much more than those in Chinese. Additionally, China was the country with the largest number of publications and the Chinese Academy of Sciences was the institution with the largest number of publications. The research hotspots included the source and origin of ARGs, transmission of ARGs in the environment, the potential human exposure to ARGs, and the mechanisms for the mitigation of ARGs. The future research work can identify the underlying mechanisms by using advanced molecular methods including metagenomics, high-throughput qPCR array, and single-cell Raman spectroscopy. In addition, efforts should be made to promote the development of approaches for mitigating ARGs. This study visualized the main research progress in ARGs in livestock and poultry manure and put forward the future key research directions, providing new insights into the mitigation of ARGs.

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