Abstract:Staphylococcus aureus is considered as one of the most common foodborne pathogens causing infectious diseases in human and animals results in skin, soft tissue, and blood the infections, leading to sepsis and toxic shock syndrome. The overuse of antibiotics improves antibiotic resistance of S. aureus, leading to the emergence of methicillin-resistant S. aureus (MRSA), which has spread globally and seriously threatens the public health. Therefore, it is an urgent need to search for new therapies to effectively control S. aureus infection. This review focuses on the research progress in the prevention and treatment of S. aureus and makes an outlook on the controlling prospects, to provide theoretical guidance for the control of Staphylococcus aureus, especially MRSA.

Abstract:Biofilm (BF), a self-protective structure formed by bacteria and other microorganisms to resist external stress, is tolerant to antibacterial agents and could cause refractory chronic infections. BF dispersion refers to the process in which the cells in BF actively escape, resume the planktonic lifestyle, and find new colonization sites during the formation cycle of BF. Since bacteria in the planktonic state are more vulnerable to antimicrobial agents and immune responses, inducing BF dispersion has become a promising strategy for controlling biofilm-associated infections (BAI). We summarize the regulatory mechanisms and signaling molecules of BF dispersion, generalize a table of substances that could affect BF dispersion, and briefly expound potential hazards after BF dispersion and the directions of future research in this field. By this review, we sincerely hope to provide theoretical reference for the development of new dispersants and drug targets.

Abstract:The gastrointestinal tract is one of the most metabolically active organs and the largest reservoir of bacteria in the human body. The human gastrointestinal tract contains rich microbiota, which has a complex relationship with the host health. The gut microbiota is in a dynamic equilibrium, the disturbance of which can cause gastrointestinal diseases such as constipation, diarrhea, irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer. In recent years, there have been increasing studies on postbiotics. The protective effect of postbiotics on intestinal barrier is similar or even superior to that of probiotics. We reviewed the studies of postbiotics in animal experiments and clinical studies in alleviating gastrointestinal diseases and summarized the role and mechanisms of postbiotics in enhancing the epithelial barrier and regulating the immune system, gut microbiota, and nervous system.

Abstract:Lysophospholipids (LPLs), lipid metabolism intermediates in the cell membrane, are mainly generated by the hydrolysis of phospholipid molecules. LPLs differ significantly from their precursor phospholipids in the biological functions. In eukaryotic cells, LPLs are bioactive molecules involved in the regulation of multiple biological signals. However, the roles of LPLs in bacteria have not been fully revealed. LPLs are a secondary component in bacterial cell membrane and can be significantly increased under environmental stress conditions. In addition to participating in the phospholipid metabolism in the cell membrane, LPLs are considered to play a role in the environmental adaptability and pathogenicity of bacteria. LPLs accumulated in the cell membrane can improve the survival and proliferation efficiency of bacteria under environmental stress or act as signaling molecules in the pathogenic processes of bacteria. Recent studies suggest LPLs as a potential novel virulence factor of bacteria. We review the current knowledge about the biosynthetic pathways of LPLs and the roles of LPLs in bacterial adaptation and host-bacterium interaction, providing references for the further research on bacterial pathogenesis and prevention of bacterial infections.

Abstract:Agricultural soils generally have limited available phosphorus. Although the application of phosphorus fertilizers can alleviate phosphorus deficiency in crops, it brings environmental risks, thereby affecting agroecological stability. Microorganisms are integral components of soil phosphorus cycling and play a crucial role in mediating the availability of phosphorus to plants. Phosphate-solubilizing bacteria (PSB) can solubilize insoluble inorganic and organic phosphorus in the soil, facilitating phosphorus uptake by plant roots and enhancing crop resistance to biotic and abiotic stresses. Currently, the use of PSB as potential biofertilizers has attracted considerable attention and demonstrated broad prospects for sustainable agriculture. This article systematically elucidates the agroecological functions of PSB and expounds the physiological and molecular mechanisms of PSB-mediated phosphorus solubilization and plant growth-promoting effect with consideration to other factors such as organic acids, hydrolases, siderophores, and ACC deaminases. Moreover, we analyzed the impact of PSB on soil microbial communities and their interaction with root exudates, highlighted the key points and challenges in the application and promotion of PSB biofertilizers, and put forward the application of PSB biofertilizers as an effective measure to increase phosphorus fertilizer use efficiency and crop yield. Finally, we proposed suggestions for future research and application of PSB biofertilizers, aiming to promote the application of PSB biofertilizers in ecological agriculture. The efforts aim to mitigate the dual challenges posed by agricultural resources and the environment, meet future needs of global food security, and align with the development trend of green ecological agriculture.

Abstract:Anaerobic ammonium-oxidizing bacteria (AnAOB) are a newly developed taxonomic group. Due to the slow growth and difficult cultivation, pure cultures of AnAOB have not yet been obtained. AnAOB exhibit unique cellular structures and functions compared with the well-known bacterial groups. Although being chemolithotrophic bacteria, AnAOB often contain cellular inclusions, e.g., glycogen particles. Studying the characteristics and dynamics of glycogen particles will deepen our understanding into AnAOB. According to relevant literature and our previous work, we systematically summarized the properties of the structure, metabolism, and function of the glycogen particles in AnAOB. The glycogen particles in AnAOB exist in the riboplasm and are in dynamic balance between synthesis via various pathways and degradation at multiple sites. Additionally, glycogen particles can serve as energy and carbon sources and provide stress protection, which are of great significance for the survival of AnAOB under adverse conditions. This review is expected to underpin the further investigation and application of AnAOB.

Abstract:Drug metabolism is a key step in the disposal of drugs, and the role of the gut as an important micro-ecosystem in the body is crucial in drug metabolism. The gut microbiota is capable of transforming and accumulating exogenous compounds (e.g., drugs) and modifying the activity and toxicity of these compounds, thereby influencing the host responses to the compounds. The interactions between gut microbiota and drugs are complex and need to be studied in detail. In recent years, the understanding of the metabolism of gut microbiota, gut microbiota-drug interactions, and the co-metabolism between gut microbiota and host are deepening, and increasing studies have shown that gut microorganisms play a crucial role in pharmacokinetics. By reviewing the relevant papers at home and abroad, we summarized the classification and functions of gut microorganisms, the effects of some commonly used drugs on gut microorganisms, and the metabolic effects and mechanisms of gut microbiota on drugs. In addition, we discussed the interactions between gut microbiota and drugs. The review helps to improve our understanding of the influences of gut microbiota on drug efficacy and the pathways and mechanisms of drug metabolism by gut microbiota, increase the possibility of modulating gut microbiota to improve treatment, and provide a reference for the rational and precise use of drugs, individualized treatment, and drug evaluation and development.

Abstract:[Objective] To optimize the soliD-state fermentation conditions of Cordyceps javanica Bd01, determine the virulence of the conidia to the 3rd-instar larvae of Spodoptera litura, and study the changes of protective enzyme activities in S. litura after infection by C. javanica. [Methods] The optimum soliD-state medium and culture conditions of C. javanica Bd01 were first determined by single factor experiments and then optimized by Box-Behnken response surface method. The virulence of the conidia to the 3rd-instar larvae of S. litura was determined by the immersion method, and the changes of enzyme activities in the larvae were determined by a spectrophotometer. [Results] With the spore yield as an indicator, the sporulation conditions of C. javanica were optimized as medium nutrient content of 30.24 g/L, pH 7.55, and light duration of 12.06 h. Under these conditions, the spore yield reached 2.78×10⁸ spores/mL. The conidial suspension of C. javanica at the concentration of 1×10⁸ spores/mL was virulent to the 3rd-instar larvae of S. litura. After seven days of treatment, the LT₅₀, LC₅₀, and corrected mortality rate were 3.11 d, 4.68×10⁵ spores/mL, and 88.68%, respectively. The activities of protective enzymes in the survived larvae of S. litura after treatment were significantly different from those in the control group. [Conclusion] The optimized culture medium can significantly increase the spore production of C. javanica. The lethal rate and lethal efficiency of C. javanica to S. litura larvae were dependent on the concentration. The protective enzymes in S. litura larvae may play a key role in the defense against the infection of C. javanica.

Abstract:[Objective] To optimize the embedding-based immobilization conditions of Aspergillus niger, preliminarily study the degradation mechanism of deltamethrin (DM) and its intermediate product 3-phenoxybenzoic acid (3-PBA) by immobilized A. niger, and further evaluate the performance of immobilized A. niger applied to agricultural cultivation. [Methods] With biochar-doped sodium alginate as an immobilized carrier, we employed single factor test and response surface methodology to optimize the immobilization conditions of A. niger. The content of DM and 3-PBA was determined by high performance liquid chromatography. [Results] Sodium alginate concentration, biochar concentration, and inoculum size were significant factors affecting DM removal rate, with the optimal values of 25.27, 1.28, and 125.28 g/L, respectively, for A. niger immobilization. After the application of immobilized A. niger, the half-life of DM in soil was shortened from 7.6 d to 5.2 d. Moreover, A. niger had a degradation effect on 3-PBA, which reached the lowest concentration of 1.45 mg/kg at the time point of 21 h. Impressively, the restoration with A. niger immobilization significantly improved the germination rate of tomato seeds, and recovered 6 growth indicators such as plant height and root length to varying degrees compared with the DM alone group. After 28 days of remediation by immobilized A. niger, both root enzyme activity and microbial number in the contaminated soil were improved to different degrees. [Conclusion] After optimization of the immobilization scheme of A. niger, the removal rate of DM in soil was significantly improved. The immobilization of A. niger can not only accelerate the degradation of DM and simultaneously degrade 3-PBA, but also enhance the tolerance of tomato to DM in the contaminated soil.

Abstract:[Objective] To establish a fluorescent probe-based loop-mediated isothermal amplification method for the specific detection of Burkholderia gladioli pv. cocovenenans. [Methods] According to the results of the multiple sequence alignment analysis, a conserved sequence, bonA, was selected for the specific detection of B. gladioli pv. cocovenenans. The primers and fluorescent probes were designed according to the conserved sequence for the establishment of a fluorescent probe-based loop-mediated isothermal amplification method. The established method was employed to detect 19 strains of B. gladioli and 3 non-target strains, and thus the specificity of this method was examined. Furthermore, the specificity of this method was compared with that of the real-time fluorescent PCR. Additionally, we investigated the limit of detection of this method for genomic DNA. [Results] The established method exhibited specificity towards B. gladioli pv. cocovenenans and could obtain the result within 30 min. Moreover, this method could visualize the detection results. The limits of detection for the genomic DNA and bacterial suspension were 1.98 pg/μL and 2.7×10² CFU/mL, respectively. In addition, this method can be applied to the detection of B. gladioli pv. cocovenenans in food samples. [Conclusion] A rapid, visualizable, and specific method was established for detecting B. gladioli pv. cocovenenans and can serve as a rapid and accurate molecular detection method for ensuring food safety.

Abstract:[Objective] Alcanivorax is a genus of dominant hydrocarbon-degrading bacteria in the marine environment, and the knowledge about the regulation mechanism of its alkane metabolism is limited. This study aims to decipher the regulation mechanism of alkane degradation by Alcanivorax at both transcriptional and translational levels. [Methods] The transcriptome and translatome data of A. dieselolei B5 grown in the medium with n-hexadecane as the sole carbon and energy source were obtained. The changes in the gene translation efficiency were calculated with sodium acetate as the control. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment was performed for the differentially translated genes (DTGs) and differential translation efficiency genes (DTEGs). [Results] Both the transcriptional and translational levels of the key genes involved in alkane metabolism were significantly up-regulated when A. dieselolei was grown on n-hexadecane. These key genes mainly encoded alkane monooxygenase, cytochrome P450 oxidase, alcohol dehydrogenase, and aldehyde dehydrogenase. KEGG enrichment analysis revealed that the up-regulated DTGs were involved in peptidoglycan biosynthesis, fatty acid degradation, chloroalkane degradation, oxidative phosphorylation, biofilm formation, etc. DTEGs were mainly involved in the biosynthesis of siderophore non-ribosomal peptides, oxidative phosphorylation, biosynthesis of unsaturated fatty acids, etc. The combined analysis of transcriptome and proteome data showed that A. dieselolei efficiently coordinated the transcription and translation processes to adapt to alkane oxidation. The gene expression level and translational efficiency showed a negative correlation under both culture conditions. The global protein regulators CsrA and sRNAs may be involved in post-transcriptional regulation of genes involved in alkane metabolism, leading to differences in the translational efficiency. [Conclusion] The combined analysis of transcriptome and translatome data suggested that post-transcriptional regulation was involved in the alkane metabolism of A. dieselolei. This study underpins further exploration of the post-transcriptional regulatory mechanisms controlling alkane metabolism.

Abstract:[Objective] D-pantothenic acid (DPA) is a functional compound widely used in medical care, cosmetics, animal food and feed and other fields, with good market prospects. This study aims to use CRISPRi to mine the endogenous gene targets affecting the biosynthesis of DPA by the lab-stored Escherichia coli strain DPAP10. [Methods] We constructed a dual-plasmid CRISPRi system with pTarget and pdCas9 to achieve the inhibition of gene expression individually or in combination. Shaking flask fermentation was carried out to validate the effect of gene blocking on DPA production. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was employed to determine the transcription level after gene blocking. The changes of metabolic pathways were analyzed by high performance liquid chromatography (HPLC). [Results] We screened out 5 key genes (pgk, gltA, ptsH, ptsI, and crp) and 7 gene combinations (pgk-gltA, pgk-ptsH, gltA-ptsH, pgk-ptsI, gltA-ptsI, pgk-crp, and gltA-crp) from 126 target genes. The engineered strain DPAP10/pdCas9+pT-gltA-ptsH showed the DPA yield of 5.3 g/L in a shaking flask, which increased by 49.5% compared with that by the start strain DPAP10. Furthermore, we down-regulated the expression of gltA and ptsH to construct an engineered strain DPAP10-gltA^TTG-ptsH^TTG. This strain showed the DPA yield of 75.4g/L in 5 L fermentor which compared with the control strain DPAP10 under the same culture conditions increased by 19.5%. [Conclusion] We confirmed that CRISPRi could mine the genes involved in DPA synthesis. Changing the pyruvate flux and reducing the tricarboxylic acid (TCA) cycling rate would promote the carbon flow into the DPA synthesis, thus increasing DPA production. This finding provides a new idea for constructing higher yielding strains.

Abstract:[Objective] To reveal the function of PE_PGRS15 from Mycobacterium tuberculosis. [Methods] A recombinant Mycolicibacterium smegmatis strain heterologously expressing PE_PGRS15 (MS-PE_PGRS15) was generated. The colony morphology, cell surface morphology, and envelope structure were observed by a plating method, a scanning electron microscope, and a transmission electron microscope, respectively. The localization of PE_PGRS15 was detected by the cell fractionation assay. The resistance of the recombinant strain to environmental stresses and antibiotics was measured by the killing curve method and micro-broth dilution method. The permeability and fatty acid profile of the cell wall of the recombinant strain were determined by dye uptake assay and gas chromatography-mass spectrometry, respectively. The functions of different domains of PE_PGRS15 were analyzed by protein truncation and fusion experiments. [Results] PE_PGRS15 was located on the cell envelope of MS-PE_PGRS15. MS-PE_PGRS15 showed altered colony morphology, envelope structure, and cell wall fatty acid profile, with noticeable increase in resistance to multiple environmental stresses and antibiotics. The dye uptake experiments with ethidium bromide and Nile red suggested that the cell wall of MS-PE_PGRS15 was more impermeable than that of the control strain. The PGRS domain of PE_PGRS15 affected mycobacterial cell wall permeability and stress resistance, while the PE domain was involved in the transport of the protein to the cell surface. [Conclusion] PE_PGRS15 was present in the cell wall fraction of MS-PE_PGRS15 and influenced cell wall permeability and colony morphology, ultimately enhancing the resistance of recombinant M. smegmatis to stresses.

Abstract:[Objective] To explore the effects of feeding methods on the digestive enzyme activity, microflora structure, and short chain fatty acids in the colon of Tibetan pigs. [Methods] Five free-ranging Tibetan pigs and 5 captive Tibetan pigs with the same age were randomly selected. The pigs were slaughtered for the collection of colonic feces samples. The digestive enzyme activity, microflora structure, and content of short chain fatty acids in the samples were determined by enzyme linked immunosorbent assay (ELISA), high-throughput sequencing, and gas chromatography, respectively. [Results] The free-ranging Tibetan pigs had lower average daily gain and higher activities of cellulase and hemicellulase in the colon than the captive Tibetan pigs (P<0.05). The alpha diversity indexes of colon microflora had no significant difference (P>0.05) and the principal component analysis showed similarity in the microflora structure between the two groups of pigs. At the phylum and family levels, the colon of the free-ranging Tibetan pigs had higher relative abundance of Verrucomicrobia, Flavobacteriaceae, Selenomonadaceae, Planctomycetaceae, and Eggerthellaceae and lower relative abundance of Streptococcaceae, Veillonellaceae, Pseudomonadaceae, Rhodocyclaceae, Rhodospirillaceae, Lactobacillaceae, Rikenellaceae, and Pasteurellaceae than that of the captive Tibetan pigs (P<0.05). At the genus and species levels, 7 genera and 4 species showed significant differences in the relative abundance between the pigs fed with the two methods, including Treponema, Ruminococcus, Eggerthella, Megasphaera, Alistipes, Pseudomonas, Streptococcus, Prevotella copri, Lactobacillus reuteri, Megasphaera elsdenii, and Faecalibacterium prausnitzii. The content of acetic acid in the colon of free-ranging Tibetan pigs was higher in that in the colon of captive Tibetan pigs (P<0.05). [Conclusion] Feeding methods affect the activities of cellulase and hemicellulase, microflora structure, and acetic acid content in the colon of Tibetan pigs. The free-ranging grazing Tibetan pigs showed poorer growth performance and stronger cellulose-degrading ability than the captive Tibetan pigs.

Abstract:[Objective] To identify the biosynthetic gene cluster (BGC) of gombapyrones (GOMs), the trialkyl-substituted aromatic polyketides derived from Streptomyces rubellomurinus ATCC 31215, and deduce the biosynthetic pathway. [Methods] GOM-B and GOM-D were extracted for the large-scale fermentation broth of S. rubellomurinus ATCC 31215. The P450 monooxygenase catalyzing the polyene chain aromatization is conserved in the biosynthetic pathways of trialkyl-substituted aromatic polyketides in bacteria. Thus, BLAST searching was carried out with the P450 monooxygenase as a probe to identify the candidate BGC for GOMs (gom BGC) from the genome of S. rubellomurinus. Through deletion of the polyketide synthase (PKS) gene in gom BGC and high performance liquid chromatography-mass spectrometry (HPLC-MS) identification of the fermentation products of the mutant strain, the gom BGC can be confirmed to direct the production of GOMs. Furthermore, bioinformatics tools were used to deduce the biosynthetic pathway of GOM-B. [Results] GOM-B and GOM-D were extracted from the fermentation broth of S. rubellomurinus and their structures were identified by NMR. The gom BGC identified in the work was submitted to the PubMed, with the GenBank accession number: OQ831859. The deletion of a PKS gene gomB resulted in the disappearance of GOM-B and GOM-D in the fermentation broth. The biosynthetic pathway of GOM-B was deduced as a type I PKS based on bioinformatics analysis. [Conclusion] This work identified a new BGC which directs the biosynthesis of trialkyl-substituted aromatic polyketides, including GOM-B and GOM-D. The type I PKS involves a P450 monooxygenase GomJ putatively catalyzing the unique polyene chain aromatization. Compared with the gbn BGC recently reported to direct the GOM-G biosynthesis, the PKS assembly line encoded by gom BGC lacks one elongation module, which is consistent with the carbon skeleton of GOM-B. The gom BGC could serve as an example that the gene evolution of bacterial type I PKS leads to the structural diversity of polyketides. Moreover, GomJ shows 78.3% sequence identity to the P450 monooxygenase GbnP functionally identified from gbn BGC, whereas the putative substrate of GomJ has two carbons less than the substrate of GbnP. Thus, the discovery of this work helps to decipher the substrate-specificity mechanism of the unusual P450 monooxygenase which catalyzes polyene chain aromatization in the biosynthetic pathways of trialkyl-substituted aromatic polyketides in bacteria.

Abstract:[Objective] To establish a zebrafish model of Edwardsiella tarda infection for the pathological studies and the development of drugs. [Methods] Zebrafish were artificially infected through different routes to simulate natural infection states, and the pathogenic mechanism of E. tarda in zebrafish was studied based on mortality, behavioral changes, biochemical indicators, and changes in the antioxidant capacity. [Results] Among the three infection routes, intraperitoneal injection showed the strongest pathogenicity. The zebrafish infected with E. tarda presented symptoms such as exophthalmos, anal bleeding, surface ulcers, and ascites. Pathological examination showed that the infected zebrafish developed acute inflammation, with extensive necrosis, degeneration, and shedding of hepatocytes and phagocytes gathering around. The TX strain was isolated from the diseased zebrafish and identified as E. tarda by polymerase chain reaction (PCR) with specific primers. The median lethal dose (LD₅₀) of the strain was determined to be 3.65×10² colony forming units (CFU)/individual. Compared with the control group, the infection model established by injection showed a decrease of 22.26% in superoxide dismutase activity and increases of 16 folds in malondialdehyde content, 38.99% in acid phosphatase activity, and 24.36% in alkaline phosphatase activity. [Conclusion] An E. tarda infection model was established by intraperitoneal injection in zebrafish, which exhibited typical disease symptoms and physiological and biochemical characteristics, and the LD₅₀ was determined. The findings provide a theoretical reference for the pathological studies and the drug development for E. tarda infections in aquatic animals.

Abstract:[Objective] Biogenic amines (BAs) widely exist in fermented food as a group of potentially harmful substances which can be degraded by amine oxidases. This study aims to explore, the enzymatic properties and amine-degrading abilities of amine oxidases from lactic acid bacteria. [Methods] The recombinant protein of the multicopper oxidase gene SufI in Lactobacillus plantarum was obtained through heterologous expression. After expression condition optimization and purification, the optimal reaction conditions, enzyme stability, amine-reducing ability, spectrum, and structural characteristics of the recombinant protein were investigated. [Results] The recombinant enzymes had the optimal performance at pH 3.5 and 20 ℃. The relative activity of the recombinant enzyme was above 70% within pH 4.0–10.0 and 15–65 ℃. This enzyme showed good stability, and was not affected by inhibitors such as ethanol. In a reaction system containing eight BAs, the recombinant enzyme could degrade 403.23 μg/mL and showed the highest degradation rate of 34.99% (70 μg/mL) for tyramine. In a reaction system containing single BAs, the recombinant enzyme had highest substrate specificity affinity toward tyramine, demonstrating the activity of 18.33 U/mL. The UV-visible scanning spectrum showed that the enzyme had a characteristic absorption peak at 600 nm. According to Fourier transform infrared spectrum, the relative content of alpha helixes, beta sheets, beta turns, and random coils in the secondary structure of amide was 21.52%, 20.72%, 33.80%, and 23.97%, respectively. Homologous modeling predicted that the enzyme possessed three copper-binding domains, which contained histidine, cysteine, methionine, glutamate, and other residues. The structure indicated that the recombinant enzyme belonged to the multicopper oxidase family. [Conclusion] The multicopper oxidase from L. plantarum JB1 showed good aciD-base and thermal stability, and was capable of degrading BAs, especially tyramine. It is expected to be further developed for fermented food and beverages.

Abstract:[Objective] Streptococcus suis is a zoonotic pathogen that can cause diseases in pigs and humans. Healthy pigs in slaughterhouses often carry diverse serotypes of S. suis, which can be a major source of infections for susceptible animals and humans. The outbreaks of S. suis occurred in Sichuan and Guangxi, resulting in severe illness and deaths of pigs and humans. Therefore, it is crucial to investigate the infection rate of S. suis in healthy pigs in slaughterhouses and unveil the pathogenic characteristics of S. suis in these two areas. [Methods] We isolated S. suis from the tonsils of healthy pigs collected from slaughterhouses in Guangxi and Sichuan during 2021–2022 and performed serotype-specific PCR for serotyping the isolates. Furthermore, we examined the virulence of the isolates in zebrafish and mice and performed genome sequencing and multilocus-sequence typing (MLST) on the virulent strains to explore their pathogenic characteristics. [Results] The positive rate of S. suis was 84.21% (48/57) in the 57 tonsil samples collected from Guangxi. Among the 60 strains isolated from Guangxi, serotype 31 (16.67%, 10/60) had the highest isolation rate, followed by serotype 9 (11.67%, 7/60), serotype 4 (10.00%, 6/60), and serotype 12 (8.33%, 5/60). The tonsil samples carrying at least two serotypes of strains accounted for 33.33% (16/48) in Guangxi. The positive rate of S. suis in 250 tonsil samples collected from Sichuan was 10.8% (27/250). Among the 41 strains isolated from Sichuan, serotype 31 (34.15%, 14/41) had the highest isolation rate, followed by serotype 16 (17.07%, 7/41), serotype 9 (12.20%, 5/41), and serotype 28 (9.76%, 4/41). The tonsil samples carrying at least 2 serotypes of strains accounted for 33.33% (9/27) in Sichuan. Zebrafish infection experiments with 28 strains from Guangxi and 16 strains from Sichuan showed that five strains were highly virulent, causing the mortality rates ranging from 70% to 100% in the zebrafish challenged with the dose of 3×10⁶ CFU/fish. The five strains included three strains from Guangxi (serotypes 2, 9 and NCL18) and two strains from Sichuan (serotypes 2 and 9). Furthermore, the 5 strains were used to infect mice, with four strains causing the mortality rates ranging from 80% to 100% and 1 strain causing the mortality rate of 60%, which were consistent with the results of the zebrafish infection experiments. MLST and virulence gene analysis revealed that the two strains of serotype 2 belonged to ST1 and exhibited the virulence genotypes mrp+, epf+, and sly+, showing the potential as zoonotic pathogens. The two strains of serotype 9 had the virulence genotypes mrp-, epf-, and sly- and belonged to ST1198 and ST2104, respectively. The NCL18 strain was typed as ST2102 and had the virulence genotypes mrp+, epf-, and sly-. [Conclusion] The S. suis infection rates differed between slaughterhouses in Guangxi and Sichuan, and the isolation rate was higher in Guangxi. The slaughterhouses in both Guangxi and Sichuan harbor serotype 2 ST1 strains with zoonotic potential that are widespread worldwide, warranting attention.

Abstract:[Objective] Listeria monocytogenes is a fooD-borne bacterial pathogen that is widely distributed in the environment. This study aims to investigate the regulatory role of the orphan response regulator DegU in the infection and high-temperature adaptation of L. monocytogenes. [Methods] The regulatory roles of DegU in three strains including wilD-type EGD-e, ΔdegU, and CΔdegU of L. monocytogenes were studied. The infection models established with human epithelial cell line Caco-2, mouse RAW264.7 macrophages, and murine fibroblast L929, real time quantitative polymerase chain reaction (RT-qPCR), and electrophoretic mobility shift assay (EMSA) were employed to investigate the roles of DegU in the infection and high-temperature adaptation. [Results] Compared with wilD-type EGD-e, ΔdegU showed weakened abilities to adhere, invade, proliferate, and migrate between host cells. The RT-qPCR results revealed that the transcription levels of the multiple virulence factors were down-regulated while that of clpE was up-regulated in ΔdegU. However, the transcription level of clpE was down-regulated at 43 ℃. Moreover, EMSA demonstrated that DegU bound directly with the promoter of clpE. [Conclusion] DegU plays a vital role in adhesion, invasion, proliferation, and migration of L. monocytogenes. Additionally, DegU directly binds with the promoter and regulates the transcriptional level of clpE to adapt to the high-temperature environment. The findings provide a basis for probing into the infection and environmental adaptation mechanisms of L. monocytogenes.

Abstract:Apple Valsa canker caused by Valsa mali is a destructive disease of apple trees. MicroRNA-like RNAs (milRNAs) play an important role in fungal growth and development, pathogenicity, and stress responses. Our previous study had demonstrated that the expression of Vm-milR21 was significantly down-regulated in the infection, suggesting Vm-milR21 might be involved in the pathogenicity of V. mali. [Objective] To reveal the role of Vm-milR21. [Methods] The strains with overexpression and silencing of Vm-milR21 precursor were constructed, and the phenotypic characteristics were compared between different transformants and the wild type. Furthermore, the targeting relationship between Vm-milR21 and Vm-03494 was verified by Quantitative real time polymerase chain reaction (qRT-PCR) and co-transformation. On this basis, the mutant with the knockout of Vm-03494 was constructed and the phenotype of the mutant was characterized. [Results] Compared with the wild type, the transformant overexpressing Vm-milR21 precursor presented slow mycelial growth and weakened pathogenicity, while the silencing of Vm-milR21 did not cause significant changes. Vm-milR21 inhibited the expression of Vm-03494 in a sequence-specific manner. Compare with the wild type, the mutant without Vm-03494 presented slow mycelial growth and diminished pathogenicity. [Conclusion] Vm-milR21 plays a role in the infection of V. mali by regulating the expression of Vm-03494. The results enrich the understanding of the functions of fungal milRNAs and provide a new theoretical basis for comprehensively deciphering the pathogenesis of V. mali.

Abstract:[Objective] The emergence of multidrug-resistant bacteria represents a serious challenge to public health security. In this study, we isolated the phages against multidrug-resistant Escherichia coli and studied their biological and genomic features, aiming to provide a theoretical foundation for the development of phage therapies against drug-resistant bacteria. [Methods] We used the double plate method to isolate E. coli phages from sewage. We employed phosphotungstic acid staining and transmission electron microscopy to observe the phage morphology, and subsequently assessed the biological properties, including host range, thermal and pH stability, one-step growth curve, and in vitro antibacterial effect of the phages. Furthermore, we conducted an in vivo bacteriostasis test to evaluate the protective effects of the phages on Galleria mellonella larvae infected with multidrug-resistant E. coli N1203-1Af. Finally, we carried out whole genome sequencing to analyze the genomic characteristics of the phages. [Results] Five E. coli phages (pEC-S163-2.1, pEC-S163-2.2, pEC-M1167-5Ar.1, pEC-m1291-2DR.1, and pEC-N1203-2Af.1) were isolated. The phage pEC-N1203-2Af.1 presented a C3 morphotype rare among short-tailed phages and was characterized by a long head with the length 2–3 times that of the width. Furthermore, pEC-N1203-2Af.1 demonstrated lytic activity against three out of the 15 tested E. coli strains. Ten minutes after infection, the phage entered an exponential growth phase and exhibited stable activity within the temperature range of –20 to 50 ℃ and pH 4.0–10.0. After treatment with pEC-N1203-2Af.1 at the multiplicity of infection (MOI) levels of 100, 1, and 0.01 for 48 h, the survival of G. mellonella larvae infected with E. coli N1203-2Af reached over 70% (7/10). The genome of phage pEC-N1203-2Af.1 had a total length of 77 334 bp and the G+C content of 42.18%. Notably, the phage did not carry any drug resistance gene or virulence factor. Functional gene prediction revealed a total of 121 CDS in the phage genome, with CDS53–CDS64 encoding the phage structure and lysis module. [Conclusion] The phage pEC-N1203-2Af.1 against multidrug-resistant E. coli exhibits potent antibacterial activity and stable biological characteristics. Our findings suggest that pEC-N1203-2Af.1 may represent a new member of the Kuravirus genus, characterized by a rare C3 morphotype that may be attributed to the main head protein encoded by CDS63. Notably, the distal trimeric protein of bacteriophages with a C3 morphotype exhibits less than 50% consistency across different regions, which indicates that the bacteriophages with this morphotype may have evolved in diverse directions to adapt to varying environmental conditions.

Abstract:[Objective] This study aims to isolate and characterize the bacteria with tolerance to cadmium ions and the abilities to remove cadmium and promote plant growth from the rice rhizosphere soil in Hubei province, China. [Methods] Using the dilution-plate spreading and gradient domestication methods with cadmium concentration gradients, we isolated a bacterial strain named Y01Z, which demonstrated stable growth and could tolerate 700 mg/L CdCl₂. The strain was then identified based on morphological, physiological, biochemical, and molecular biological characteristics. [Results] Y01Z was identified as Brevundimonas diminuta, with the optimal growth conditions of pH 7.0, 30 °C, and 0.5% NaCl. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that Y01Z elongated its cells under high cadmium concentrations to ensure survival and reproduction. Moreover, it accumulated cadmium ions and transported them intracellularly. Fourier transform infrared spectroscopy indicated that Cd²⁺ bound to carboxyl, hydroxyl, carbonyl, and amide functional groups on the cell surface. After 104 hours of cultivation, B. diminuta Y01Z removed 75% of the total added cadmium, reducing the cadmium concentration from 300 mg/L to 74.73 mg/L. Additionally, the bacterium exhibited plant growth-promoting properties, including solubilization of phosphorus, production of amino nitrogen and indole-3-acetic acid, and possession of iron-chelating agents. [Conclusion] The findings suggest that B. diminuta Y01Z has significant potential for rhizobacteria-mediated remediation of cadmium-contaminated soils and the promotion of sustainable agriculture practices.

Abstract:[Objective] O-SuccinyL-L-homoserine (OSH), an important precursor for the synthesis of L-methionine, L-glufosinate-ammonium and other chemicals, has wide applications in the pharmaceutical, pesticide, and food industries. The green and efficient production of OSH has attracted a great deal of attention. This study investigated the metabolic pathway and metabolite changes during the microbial fermentation for production of OSH, aiming to establish an efficient regulation strategy to increase both the yield and sugar-acid conversion of OSH. [Methods] Metabolomics was employed to systematically investigate the metabolic changes during the fermentation, and the key metabolic pathways associated with OSH biosynthesis were determined. The key metabolites were added at different fermentation stages to balance the metabolic flux and reduce the competitive utilization of precursors. [Results] The efficient bio-production of OSH was realized in a 5 L fermentation tank. The titer reached 70.1 g/L with the conversion of 0.52 g/g (glucose). [Conclusion] The optimization of OSH fermentation system and regulation of fermentation process based on metabolomics have significantly improved the production of OSH, laying a foundation for the industrialization of OSH.

Abstract:[Objective] Saccharothrix is a genus of rare filamentous actinomycetes showing great application values in the biopharmaceutical field, industrial enzyme preparations, and environmental remediation. This study aims to establish a method to identify Saccharothrix spp. based on ribosomal protein biomarkers and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). [Methods] We searched two public genome databases to obtain the sequences of 15 ribosomal proteins from the genome-sequenced strains of Saccharothrix and calculated the theoretical molecular weights. We then compared the molecular weights to investigate the matching of 15 ribosomal proteins between different species of Saccharothrix and that between the type strains of Saccharothrix and its closely related three genera (Umezawaea, Actinosynnema, and Lentzea). The criteria for identifying Saccharothrix at the species and genus levels were proposed based on the number of ribosomal protein matches. Subsequently, the identification criteria were tested by MALDI-TOF MS with target and non-target organisms and improved. [Results] MALDI-TOF MS peaks from an unknown organism were compared with the weight list of 15 ribosomal protein biomarkers from each Saccharothrix type strain and the number of peaks that matched each strain’s biomarkers was obtained. Based on the maximum matches, peak intensity mode, and characteristic peaks, the organism can be identified at the genus or species level. [Conclusion] This study established a method for identifying Saccharothrix spp. based on 15 ribosomal protein biomarkers and MALDI-TOF MS, which can be used in directed screening and rapid identification of Saccharothrix spp.

Abstract:Bacteria modulate intracellular concentrations of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP), which facilitates the adaptation to environments, survival, and infection. [Objective] To establish an effective method for measuring the c-di-GMP levels in Escherichia coli. [Methods] We designed the primers for the dual fluorescence reporter plasmid based on the regulation pattern of the riboswitch receptor by c-di-GMP and the fluorescence reporter genes. The dual fluorescence reporter plasmid pAmCherry-Vc2EGFP (pACVcE) was constructed by overlap polymerase chain reaction (overlap PCR) and homologous recombination. Then, we constructed the mutants overexpressing and lacking the genes involved in c-di-GMP metabolism, and used pACVcE to measure the c-di-GMP levels in Escherichia coli. [Results] The targeted genes were successfully amplified with correct sequences. The intracellular c-di-GMP levels in Escherichia coli expressing the c-di-GMP synthase DgcZ were significantly increased, while the intracellular c-di-GMP levels expressing the c-di-GMP phosphodiesterase PdeK were significantly decreased. The deletion of the pdeK gene encoding c-di-GMP phosphodiesterase elevated the level of c-di-GMP in avian pathogenic Escherichia coli. [Conclusion] We constructed a dual fluorescence reporter plasmid based on riboswitch, which can facilitate the rapid determination of c-di-GMP levels in Escherichia coli.