Streptococcus pneumoniae, a common pathogen to cause pneumonia and otitis media, is also the most important causative agent of bacterial meningitis in humans. Previous studies have shown that S. pneumoniae strains can be pilus-negative, type I pilus- and/or type II pilus-positive, and pneumococcal pili promote adhesiveness and induce inflammation in hosts. However, the role of S. pneumoniae pili in pneumococcal migration through human blood-brain barrier (BBB) to cause meningitis remains unknown. Since no standard/model S. pneumoniae strains with determined whole genome sequences containing the genes responsible for encoding type I and II pili are available, a serotype-19 F S. pneumoniae strain named SP007 with both type I and II pili-encoding genes was isolated from a meningitis patient and its whole genome DNA sequence was determined (GenBank: CP096809). S. pneumoniae SP007 possessed a unique chimeric rrgA-B gene encoding a type I pilus and a common pitB gene encoding a type II pilus. Knockout of the rrgA-B or pitB gene significantly attenuated pneumococcal invasion into human BBB-derived brain microvascular endothelial cells (HBMECs), brain vascular pericytes (HBVPs), astrocytes (HAs), and pneumococcal transcytosis across the three cell monolayers. Complementation of the rrgA-B and pitB genes recovered the pneumococcal pathogenicity such as invasion into the BBB-derived cells and transcytosis across the three cell monolayers. Both the rrgA-B and pitB genes contributed to pneumococcal lethal virulence and caused typical histopathological changes in meningitis in mice. In particular, all the tested pneumococcal strains were found to proliferate in HBMECs, HBVPs, and HAs, in which S. pneumoniae SP007 displayed the strongest intracellular proliferation ability. Both type I and II pili mediate invasion of S. pneumoniae into human BBB-derived cells and promote pneumococcal transcytosis across the BBB, contributing to the development of pneumococcal meningitis in mice. These findings identify an important role of pili-mediated invasion and transcytosis in pneumococcal BBB penetration and provide new insights into the pathogenesis of pneumococcal meningitis.

BACKGROUD:Streptococcus pneumoniae is the most common pathogen of bacterial meningitis, but mechanisms of this pathogen invading blood-brain barrier (BBB) remain unknown. METHODS:Invasion of S. pneumoniae strains and microbeads coated with recombinant pilin, rRrgA-B or rPitB, into human BBB-derived brain microvascular endothelial cells (HBMECs), brain vascular pericytes (HBVPs), and astrocytes (HAs) by laser scanning confocal microscopy (LSCM). Fibronectin (FN), laminin (LN), and collagen-1/3/4 (COL-1/3/4) in the extracellular matrix (ECM) were detected by LSCM and flow cytometry. rRrgA-B and rPitB binding to the ECM proteins were identified using surface plasmon resonance. Pneumococcal endocytosis pathways, Ca2+ level, microfilament (MF) and microtubule (MT) polymerization in the cells were determined by LSCM-based inhibition assay. RESULTS:Type I/II pili-positive S. pneumoniae SP007 displayed natably stronger invasiveness into the cells than pilus-free S. pneumoniae ATCC49619. rRrgA-B-/rPitB-coated microbeads also exhibited invasion ability. HBMECs express FN, LN, and COL1, while HBVPs and HAs express all the ECM proteins. rRrgA-B and rPitB displayed a rapid binding to all the ECM proteins with KD values ranging from 3.25 × 10-7 to 9.58 × 10-8 M. Intracellular free Ca2+ increase and MF-dependent cytoskeleton rearrangement occurred during invasion of the pneumococcal strains and rRrgA-B-/rPitB-coated microbeads. Invasion of the strains and rRrgA-B-/rPitB-coated microbeads into these cells involved integrin (ITG), focal adhesion kinase (FAK), Rho-associated coiled-coil forming kinase (ROCK) or phosphatidylinositol-3-kinase (PI3K), clathrin (CLN)-, caveolae (CAV)- or macropinocytosis (MPC)-dependent endocytosis. CONCLUSION:Type I and II pili play important roles to mediate S. pneumoniae invasion into BBB-direived cells through FN/LN/COL1-ITG-FAK/ROCK-Ca2+-MF-CLN/CAV/MPC- or FN/LN/COL1/3/4-ITG-FAK/PI3K-Ca2+-MF-CLN/CAV-dependent endocytosis pathways to cause meningitis.

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different beta-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56-9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s(-1)) than the other rPBPs (Kcat = 3.20-7.49 x 10(-2) s(-1)). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s(-1)). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (K-D = 3.71-9.35 x 10(-4) M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

The functional amyloid of Pseudomonas (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of Pseudomonas fluorescens PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of fap genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our in vivo data showed that the REC domain deletion of BrfA promoted fap gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of fapA in a BrfA-dependent manner. In in vitro experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of fapA, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the fapA promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. In vivo experiments using a lacZ fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of fapA transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in P. fluorescens. Fap functional amyloids and BrfA-type transcription factors are widespread in Pseudomonas species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of fap provided by this work will contribute to the development of antibiofilm strategies.

Mycobacterium marinum ( M. marinum ) is a non-tuberculous mycobacterium (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing NTM infection. However, this method is time-consuming and has low positivity rates for fastidious organisms. Oxford Nanopore MinION sequencing is an emerging third-generation sequencing technology that can sequence DNA or RNA directly in a culture-independent manner and offers rapid microbial identification. Further benefits include low cost, short turnaround time, long read lengths, and small equipment size. Nanopore sequencing plays a crucial role in assessing drug resistance, clinical identification of microbes, and monitoring infectious diseases. Some reports on Mycobacterium tuberculosis (MTB) using nanopore sequencing have been published, however, there are few reports on NTM, such as M. marinum . Here, we report the use of nanopore sequencing for the diagnosis of M. marinum .

目的 分析全身麻醉患者术后医院感染的病原学特点,并构建风险预测模型.方法 回顾性收集2021年9月-2022年11月义乌市中心医院收治的798例全身麻醉患者临床资料,根据患者术后是否发生医院感染分为感染组65例和未感染组733例,统计全身麻醉患者术后医院感染部位分布、感染病原学特点,单因素及多因素Logistic回归分析术后医院感染的危险因素,并构建风险预测模型,受试者工作特征(ROC)曲线分析模型的预测价值.结果 65例全身麻醉患者术后医院感染主要类型为呼吸道和手术部位感染,占比分别为58.46％和26.15％;共分离出79株病原菌,主要感染病原菌种类为鲍氏不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、表皮葡萄球菌;合并糖尿病、合并低蛋白血症、美国麻醉医师协会(ASA)分级Ⅲ级、麻醉通气时间≥2 h、麻醉机过滤器重复性使用、住院时间≥14 d均为全身麻醉患者术后医院感染的独立危险因素(P＜0.05);根据上述因素构建的模型具有较好的预测价值,曲线下面积(AUC)为0.831,敏感度为78.46％,特异度为72.71％.结论 全身麻醉患者术后医院感染主要病原菌种类为鲍氏不动杆菌、铜绿假单胞菌、金黄色葡萄球菌、表皮葡萄球菌,危险因素包括合并糖尿病、合并低蛋白血症、ASA分级Ⅲ级、麻醉通气时间≥2 h、麻醉机过滤器重复性使用、住院时间≥14 d,据此构建的预测模型具有较好的预测价值.

Disseminated ankle mycosis is a life-threatening systemic infection caused by the emerging opportunistic and lethal fungal pathogen Talaromyces marneffei which is more common in HIV-positive patients. However, an increasing number of infections are occurring in HIV-negative patients. Here, we report a case of Talaromyces marneffei infection in HIV-negative patient. A 50s HIV-negative male patient with fever, cough, bloody sputum expectoration, pulmonary sarcoidosis and body rashes was hospitalized at Zhejiang Provincial People's Hospital. CT scanning showed pulmonary multiple nodules with apical bronchial occlusion, patchy infiltration and pathological biopsy demonstrated bronchiolitis obliterans with organized pneumonia and chronic active inflammation of lung tissue with infiltration of numerous lymphocytes, plasma cells, phagocytes and neutrophils. Laboratory tests revealed significantly increased white blood cells count 18.3 ×109/L, neutrophil count 15.34 ×109/L, monocyte count 0.66 ×109/L, platelet count 517 ×109/L, C-reactive protein 116 mg/L, erythrocyte sedimentation rate 112mm/h. The β-D-glucan test was negative (33.06 pg/mL) while fungal culture of broncho alveolar lavage fluid revealed colonies with temperature-dependent dimorphic growth character and Talaromyces marneffei was confirmed by ITS sequencing of the colonies. The patient exhibited radiological improvement and clinical recuperation after intravenously guttae of voriconazole. Talaromycosis in immunocompetent and HIV-negative individuals is relatively rare and is characterized by an insidious onset, various clinical manifestations, and is clinically challenging. Fungal culture and ITS sequencing are warranted for diagnosis Talaromyces marneffei infection. This is the first report on identification of Talaromyces marneffei infection in an HIV-negative patient with skin involvement by ITS sequencing in Zhejiang.

Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest owing to their excellent ability to modulate the composition of the gut microbiota. The acid hydrolysis-based processing of xylan-containing materials has been proposed to represent a cost-effective approach to XOS preparation, with organic acids being preferable in this context. As such, in the present study, maleic acid was selected as a mild, edible organic acid for use in the hydrolysis of xylan to produce XOS. A response surface methodology (RSM) approach with a central composite design was employed to optimize maleic acid-mediated XOS production, resulting in a yield of 50.3% following a 15 min treatment with 0.08% maleic acid at 168°C. Under these conditions, the desired XOS degree of polymerization (2-3) was successfully achieved, demonstrating the viability of this using a low acid dose and a high reaction temperature to expedite the production of desired functional products. Moreover, as maleic acid is a relatively stable carboxylic acid, it has the potential to be recycled. These results suggest that dilute maleic acid-based thermal treatment of corncob-derived xylan can achieve satisfactory XOS yields, highlighting a promising and cost-effective approach to XOS production.

目的 分析绍兴地区淋病奈瑟菌对临床常用抗生素的耐药性,分析阿奇霉素耐药菌株耐药相关基因的突变情况.方法 琼脂稀释法检测6种抗生素的最小抑菌浓度(MIC),采用头孢硝噻吩纸片法测定PPNG.PCR扩增淋病奈瑟菌阿奇霉素(AZM)耐药相关基因23S rRNA、rplD、rplV和mtrR(启动区和编码区)并测序.结果 PPNG和TRNG的阳性率分别为38.72％和45.79％,未出现头孢曲松和大观霉素耐药株,54株AZM耐药株中呈高度、中度和低度耐药的分别有15株、18株和21株.23S rRNA基因4个等位基因a2143g突变和c2599t突变分别与AZM高度和中低度耐药相关,mtrR基因启动区a缺失与AZM耐药相关但与耐药程度无关,mtrR基因编码蛋白G45D突变与AZM高度耐药相关,而H105Y突变与AZM中度、低度耐药相关.结论 头孢曲松和大观霉素可以作为本地区淋病奈瑟菌感染治疗的推荐用药,23SrRNA4个等位基因、mtrR(启动区和编码区)突变与AZM耐药相关,本地区淋病奈瑟菌阿奇霉素耐药率已达18.18％,应根据药敏结果确定是否用阿奇霉素治疗淋病奈瑟菌感染.

目的 分析解脲脲原体（Uu）和人型支原体（Mh）感染对精液质量的影响，探讨支原体感染与男性不育的相关性。方法 回顾性分析323例男性不育患者（不育组）精液常规检测、支原体培养结果，并与89例有正常生育能力男性（对照组）比较。结果 不育组中精液质量异常者占55.7%。精子的存活率、活力、正常形态和液化时间是影响精液质量的主要因素。不育组患者的精子密度、总数、存活率、向前运动占比（PR）、正常形态百分比均明显低于对照组，而液化时间则高于对照组（P ＜0.05）。不育组支原体总感染率、Uu感染率和Uu+Mh混合感染率均明显高于对照组（P ＜0.05）。支原体阳性组患者精子存活率、PR、正常形态比例明显小于阴性组（P ＜0.05）。结论 支原体感染可影响精子存活率、PR、正常形态比例等精液质量的主要参数，精液质量下降是引起男性不育的重要因素。

Streptococcus pneumoniae is a common diplococcus pathogen found worldwide. The characterization of predominant serotypes, drug resistance, and virulence genes of S. pneumoniae isolates prevailing in different areas and countries is clinically important for choice of antibiotics and improvement of vaccines. In this study, pneumonia (78.7%) and meningitis (37.0%) were the predominant diseases observed in the 282 (children) and 27 (adults) S. pneumoniae-infected patients (p < 0.05) from seven hospitals in different areas of East China. Of the 309 pneumococcal isolates, 90.3% were classified by PCR into 15 serotypes, with serotypes 19F (27.2%) and the 6A/B (19.1%) being most predominant (p < 0.05). Importantly, serotypes 15A and 15B/C combined for a total of 10.4% of the isolates, but these serotypes are not included in the 13-valent pneumococcal capsule conjugate vaccine used in China. Antimicrobial susceptibility analysis by the E-test showed that >95% of the 309 pneumococcal isolates were susceptible to moxifloxacin and levofloxacin, as well as 18.4, 85.8, and 81.6% of the isolates displayed susceptibility to penicillin, cefotaxime, and imipenem, respectively. A significant correlation between the prevalence of predominant serotypes and their penicillin resistance was observed (p < 0.05). In particular, >95% of all the pneumococcal isolates showed resistance to erythromycin and azithromycin. Of the nine detected virulence genes, the lytA, ply, hysA, and nanA were the most common with 95–100% positive rates in the 309 pneumococcal isolates, while the pavA and psaA genes displayed a significant correlation with pneumococcal bacteremia and meningitis (p < 0.05). Overall, our data suggested that the predominant serotypes, drug resistance, and virulence genes of the S. pneumoniae isolates prevailing in East China are distinct from those observed in other areas of China and adjacent countries.

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter’s syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC , rpoN , and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in Δ brfA , as in Δ rpoN . These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments.

目的 分析解脲脲原体(Uu)和人型支原体(Mh)感染对精液质量的影响,探讨支原体感染与男性不育的相关性.方法 回顾性分析323例男性不育患者(不育组)精液常规检测、支原体培养结果,并与89例有正常生育能力男性(对照组)比较.结果 不育组中精液质量异常者占55.7％.精子的存活率、活力、正常形态和液化时间是影响精液质量的主要因素.不育组患者的精子密度、总数、存活率、向前运动占比(PR)、正常形态百分比均明显低于对照组,而液化时间则高于对照组(P＜0.05).不育组支原体总感染率、Uu感染率和Uu+Mh混合感染率均明显高于对照组(P＜0.05).支原体阳性组患者精子存活率、PR、正常形态比例明显小于阴性组(P＜0.05).结论 支原体感染可影响精子存活率、PR、正常形态比例等精液质量的主要参数,精液质量下降是引起男性不育的重要因素.

Staphylococcus epidermidis (S. epidermidis) is a clinically important conditioned pathogen that can cause a troublesome chronic implant-related infection once a biofilm is formed. The nitric oxide synthase (NOS) gene, which is responsible for endogenous nitric oxide synthesis, has already been found in the genome of S. epidermidis; however, the specific mechanisms associated with the effects of NOS on S. epidermidis pathogenicity are still unknown. The purpose of the current study was to investigate whether the NOS gene has an impact on biofilm formation in S. epidermidis. Bioinformatics analysis of the NOS gene was performed, and homologous recombination was subsequently employed to delete this gene. The effects of the NOS gene on biofilm formation of S. epidermidis and its underlying mechanisms were analyzed by bacterial growth assays, biofilm semiquantitative determination, Triton X-100-induced autolysis assays, and bacterial biofilm dispersal assays. Additionally, the transcription levels of fbe, aap, icaA, icaR and sigB, which are related to biofilm formation, were further investigated by qRT-PCR following NOS deletion. Phylogenetic analysis revealed that the NOS gene was conserved between bacterial species originating from different genera. The NOS deletion strain of S. epidermidis 1457 and its counterpart were successfully constructed. Disruption of the NOS gene resulted in significantly enhanced biofilm formation, slightly retarded bacterial growth, a markedly decreased autolysis rate, and drastically weakened bacterial biofilm dispersal. Our data showed that the fbe, aap and icaA genes were significantly upregulated, while the icaR and sigB genes were significantly downregulated, compared with the wild strain. Therefore, these data strongly suggested that the NOS gene can negatively regulate biofilm formation in S. epidermidis by affecting biofilm aggregation and dispersal.

Objective:To analyze the binding ability of motifs in the serine/threonine kinase StkP extracellular region (EC-StkP) of Streptococcus pneumoniae to β-lactam antibiotics. Methods:Three motifs (SXXK) in the EC-StkP were mutated into AXXA, respectively or simultaneously. Four mutant plasmids (EC- stkp-AXXA1, EC- stkp-AXXA2, EC- stkp-AXXA3 and EC- stkp-AXXA4) were transfected into recipient cells for cloning and expression. SDS-PAGE combined with gel image analysis was used to detect the expression of the recombinant mutant proteins (EC-rStkP-AXXA1, EC-rStkP-AXXA2, EC-rStkP-AXXA3 and EC-rStkP-AXXA4). The expressed mutated proteins were extracted and purified by Ni-NTA affinity chromatography. The binding abilities of the mutant proteins to penicillin (PCN) and cefotaxime (CTX) were detected by isothermal titration calorimetry (ITC 200) and surface plasmon resonance (Biacore t200). Results:PCN and CTX could not bind to the expressed proteins with mutations in the first or the third motif (EC-rStkP-AXXA1, EC-rStkP-AXXA3, EC-rStkP-AXXA4). EC-rStkP-AXXA2 could weakly bind to CTX, but not to PCN.Conclusions:All three motifs in the EC-StkP of Streptococcus pneumoniae could bind to β-lactam antibiotics with the first and the third motifs being more important.

Pseudomonas fluorescens is a typical spoiler of proteinaceous foods, and it is characterized by high spoilage activity. The sigma factor RpoN is a well-known regulator controlling nitrogen assimilation and virulence in many pathogens. However, its exact role in regulating the spoilage caused by P. fluorescens is unknown. Here, an in-frame deletion mutation of rpoN was constructed to investigate its global regulatory function through phenotypic and RNA-seq analysis. The results of phenotypic assays showed that the rpoN mutant was deficient in swimming motility, biofilm formation, and resistance to heat and nine antibiotics, while the mutant increased the resistance to H2O2. Moreover, the rpoN mutant markedly reduced extracellular protease and total volatile basic nitrogen (TVB-N) production in sterilized fish juice at 4°C; meanwhile, the juice with the rpoN mutant showed significantly higher sensory scores than that with the wild-type strain. To identify RpoN-controlled genes, RNA-seq-dependent transcriptomics analysis of the wild-type strain and the rpoN mutant was performed. A total of 1224 genes were significantly downregulated, and 474 genes were significantly upregulated by at least two folds at the RNA level in the rpoN mutant compared with the wild-type strain, revealing the involvement of RpoN in several cellular processes, mainly flagellar mobility, adhesion, polysaccharide metabolism, resistance, and amino acid transport and metabolism; this may contribute to the swimming motility, biofilm formation, stress and antibiotic resistance, and spoilage activities of P. fluorescens. Our results provide insights into the regulatory role of RpoN of P. fluorescens in food spoilage, which can be valuable to ensure food quality and safety.

目的 观察亚抑制浓度鱼腥草素钠和红霉素体外诱导对肺炎链球菌(Streptococcus pneumoniae,Sp)耐药性影响,探讨Sp红霉素耐药的分子机制.方法 琼脂稀释法测定ATCC6306和10株红霉素敏感临床分离株对鱼腥草素钠和红霉素MICs;1/2MIC的鱼腥草素钠和红霉素为起始浓度进行连续诱导,检测诱导前后菌株对鱼腥草素钠和红霉素的MICs;PCR扩增药物诱导前后菌株L4和L22编码基因rplD和rplV并测序,比较诱导前后氨基酸序列的变化.结果 Sp经鱼腥草素钠诱导传代的各临床菌株MICs改变不超过2倍,无耐药株产生;诱导前所有菌株对红霉素的MICs为0.125 μg/ml～0.25 μg,/ml,诱导后ATCC6306和5株临床菌株成为红霉素耐药株,MICs为4μg/ml～ 32μg/ml,其中ATCC6306和3株临床L4和L22氨基酸序列存在1处～3处替换突变.结论 红霉素体外诱导肺炎链球菌获得稳定性耐药,耐药机制可能与核糖体蛋白L4和L22氨基酸突变相关.相对于化学合成药物肺炎链球菌对鱼腥草素钠不易产生耐药.

目的 探讨血清抗磷脂酶A2 受体(PLA2R)抗体在特发性膜性肾病(IMN)发生和发展中的作用.方法 选取2018 年1月-2019 年8 月在河南大学第一附属医院肾脏内科住院行肾穿刺活检112 例膜性肾病患者为研究对象,根据肾组织活检病理结果分为IMN 组(62 例)和非特发性膜性肾病(N-IMN)组(50 例),另选取同期本院行健康体检者为对照组(50 例).酶联免疫吸附法检测3 组研究对象血清抗PLA2R 抗体,并收集相关临床资料进行统计分析.结果 IMN组和N-IMN组比较,血清CRP、抗PLA2R抗体、C3和24 h尿蛋白定量均明显高于对照组(P＜0.05),TP、Alb、IgG 和eGFR水平均明显低于对照组(P＜0.05).IMN 组血清CRP、抗PLA2R抗体、C3 和IgG 与N-IMN 组差异有统计学意义(P＜0.05).IMN组患者抗体阳性者血清TP、Alb、C3、IgG和eGFR明显低于抗体阴性者,24 h蛋白尿定量明显高于抗体阴性组,差异均有统计学意义(P＜0.05).IMN患者抗PLA2R抗体与TP、Alb、IgG、C3呈负相关(r值分别为-0.55、-0.41、-0.41、-0.37,P＜0.01).Ⅲ期IMN患者抗PLA2R抗体阳性明显高于Ⅰ期～Ⅱ期IMN患者.结论 血清抗PLA2R 抗体在特发性膜性肾病中诊断及在疾病发展监测中有一定价值.

Objective:To investigate the possible mechanism of high mobility group box-1 (HMGB1) in amplifing inflammatory responses in Leptospira interrogans hemolysin Sph2-treated J774A.1 macrophages. Methods:Recombinant Sph2 was incubated with J774A.1 macrophages. The damage of cell membrane was detected by lactate dehydrogenase(LDH) determination; the changes of cell structure were observed by cryo-electron microscope; ELISA was used to determine the expression of HMGB1. After the commercial recombinant HMGB1 was incubated with mouse J774A.1 macrophages, the phosphorylation of NF-κB, p38-MAPK and JNK signaling pathway wsa detected by Western blot, and the expression of IL-1β, IL-6, and KC (IL-8) was detected by ELISA.Results:Recombinant hemolysin rSph2 induced significant changes in the structures of J774A.1 cells, including nucleus disappearance, cell membrane structure damage, cell lysis and membrane swelling. The yields of LDH and HMGB1 also increased significantly. Phosphorylated-NF-κB, -p38-MAPK and -JNK were increased by HMGB1. The expression of IL-1β, IL-6 and KC in J774A.1 cells was up-regulated by HMGB1 and inhibited via inhibitors of NF-κB, p38-MAPK and JNK signal pathways.Conclusions:Hemolysin rSph2 damaged the membrane of J774A.1 cells, and induced the secretion of HMGB1. Secreted-HMGB1 might induce the expression of IL-1β, IL-6 and KC in J774A.1 cells via NF-κB, p38-MAPK and JNK signal pathways, thus amplifying the inflammatory responses caused by Sph2.