Xanthomonas maltophilia转化制备熊去氧胆酸及中间产物

以野生型嗜麦芽黄单胞菌(Xanthomonas maltophilia)为出发菌株,鹅去氧胆酸/β-环糊精包合物为反应底物,利用嗜麦芽黄单胞菌在液体发酵过程中产生的7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶,全细胞酶法催化制备熊去氧胆酸及中间产物。对嗜麦芽黄单胞菌形态鉴定,酶活测定并优化,制定产物检测方法。表明嗜麦芽黄单胞菌为杆状、单鞭毛、革兰氏阴性菌。菌株破碎后,SDS-PAGE分析表明在26-33 kDa之间存在蛋白条带,测定7α-羟基类固醇脱氢酶和7β-羟基类固醇脱氢酶酶活分别为79 U/mL、35 U/mL;优化显示,温度为35°C、pH值为9.0、添加30%甲醇时酶的活性提升;转化后UDCA得率为17.2 mg/L,7K-LCA得率为18.2 mg/L。作为一种野生型的底盘转化菌种,该研究为全细胞催化制备熊去氧胆酸及其中间产物提供了新的途径。

Enterogenic Stenotrophomonas maltophilia migrates to the mammary gland to induce mastitis by activating the calcium-ROS-AMPK-mTOR-autophagy pathway

Background Mastitis is an inflammatory disease of the mammary gland that has serious economic impacts on the dairy industry and endangers food safety.Our previous study found that the body has a gut/rumen-mammary gland axis and that disturbance of the gut/rumen microbiota could result in‘gastroenterogenic mastitis'.However,the mechanism has not been fully clarified.Recently,we found that long-term feeding of a high-concentrate diet induced mastitis in dairy cows,and the abundance of Stenotrophomonas maltophilia(S.maltophilia)was significantly increased in both the rumen and milk microbiota.Accordingly,we hypothesized that‘gastroenterogenic mastitis'can be induced by the migration of endogenous gut bacteria to the mammary gland.Therefore,this study investigated the mechanism by which enterogenic S.maltophilia induces mastitis.Results First,S.maltophilia was labelled with superfolder GFP and administered to mice via gavage.The results showed that treatment with S.maltophilia promoted the occurrence of mastitis and increased the permeability of the blood-milk barrier,leading to intestinal inflammation and intestinal leakage.Furthermore,tracking of ingested S.maltophilia revealed that S.maltophilia could migrate from the gut to the mammary gland and induce mastitis.Subsequently,mammary gland transcriptome analysis showed that the calcium and AMPK signalling pathways were significantly upregulated in mice treated with S.maltophilia.Then,using mouse mammary epithelial cells(MMECs),we verified that S.maltophilia induces mastitis through activation of the calcium-ROS-AMPK-mTOR-autophagy pathway.Conclusions In conclusion,the results showed that enterogenic S.maltophilia could migrate from the gut to the mammary gland via the gut-mammary axis and activate the calcium-ROS-AMPK-mTOR-autophagy pathway to induce mastitis.Targeting the gut-mammary gland axis may also be an effective method to treat mastitis.

纳米孔测序检测嗜麦芽窄食单胞菌重症肺炎1例报告

嗜麦芽窄食单胞菌肺炎是一种因嗜麦芽窄食单胞菌感染所致的肺炎。采用传统病原菌培养法检测,阳性率低,假阴性率高、周期长,病原学证据短期内无法正确获得,影响预后。本文探讨一种较为快速准确的检测方法。纳米孔测序(NTS)是一种第三代测序技术,相比传统方法优点是它的成本和时间效益,吞吐量高。本文检测了1例由嗜麦芽窄食单胞菌引起的重症肺炎。结果表明,该病是由嗜麦芽窄食单胞菌引起的感染。本文提供了NTS可以快速识别病原体的证据,这对临床诊断和治疗具有重要意义。

嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)DHHJ分解角蛋白的生化机制初探

对嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)DHHJ降解角蛋白的生化机制进行初步研究。结果发现:该细菌与羽毛共培养24 h后,电镜下观察到细菌紧密地生长在羽枝上,96 h后羽毛完全降解;该菌所产角蛋白酶属于胞外酶,胞内二硫键还原酶可提高该胞外酶活性3倍左右。单独的角蛋白酶和二硫键还原酶在没有活的细菌存在下,都不能完全降解羽毛,这说明细菌附着在降解过程中起了重要的作用,也可能是由于细菌持续提供了一种还原剂破坏二硫键。此外,羽毛降解过程中,在细菌与羽毛共培养液中检测到亚硫酸盐,说明亚硫酸盐解可能对羽毛降解也起了一定的作用。

嗜麦芽窄食单胞菌耐药率变化与抗菌药物使用强度的相关性分析

目的分析嗜麦芽窄食单胞菌的耐药率变化与抗菌药物使用强度之间的相关性。方法统计某三级医院2018~2022年住院患者的嗜麦芽窄食单胞菌的临床分布、耐药率和常用抗菌药物使用强度,采用Spearman进行相关性分析。结果药敏检出438株嗜麦芽窄食单胞菌,其对米诺环素、复方新诺明、左氧氟沙星和氯霉素的耐药率较低,分别为2.19%、7.84%、11.00%和17.59%,对头孢他啶的耐药率相对较高,为36.30%。嗜麦芽窄食单胞菌对头孢他啶的耐药率与碳氢霉希类抗菌药物使用强度呈正相关(r=0.880,P<0.05)。结论抗菌药物使用强度可影响嗜麦芽窄食单胞菌对部分抗菌药物的耐药率,合理控制抗菌药物使用、降低使用强度可减少嗜麦芽窄食单胞菌的诱导耐药。

重组酶聚合酶扩增结合侧向流动试纸条技术快速可视化检测嗜麦芽窄食单胞菌方法的建立与应用

目的:建立一种基于重组酶聚合酶扩增(recombinant polymerase amplification,RPA)和侧向流动试纸条(lateral flow strips,LFS)的嗜麦芽窄食单胞菌快速可视化检测方法。方法:以嗜麦芽窄食单胞菌特异性序列(NC_010943.1)为模板,设计RPA引物。通过基础型RPA反应和琼脂糖凝胶电泳,根据候选引物对的扩增性能及引物二聚体的形成情况筛选最佳引物对。根据最佳引物对,设计探针和修饰引物。在引物和探针中引入碱基错配以消除假阳性信号,建立RPA-LFS反应体系。根据检测线的显色情况,优化RPA-LFS的最佳反应条件。使用临床常见的12种致病菌和12个临床来源的嗜麦芽窄食单胞菌检测该方法的特异性。以10倍梯度稀释的嗜麦芽窄食单胞菌基因组为模板,检测该方法的灵敏度。收集108例临床样本,将该方法与qPCR检测法和生化培养法对比,对RPA-LFS检测法进行kappa一致性检验及临床应用评价。结果:RPA-LFS检测法在37℃恒温条件下8 min即可完成扩增反应,1 min内可在LFS上观察到结果。该方法灵敏度高,最低检出限为1.107 CFU,并且与其他病原菌无交叉反应,特异性强。应用于临床样本检测时,该方法与qPCR相比,检测结果准确性一致。与生化培养方法的符合率为99.07%,kappa指数值为0.972,具有良好的一致性。结论:本研究建立了一种不依赖于精密仪器和专业技术人员的RPA-LFS检测方法,能够短时间内精准鉴定嗜麦芽窄食单胞菌。该方法的建立可为及时制定合理的抗菌治疗方案提供信息,具有较大的临床应用潜力。

嗜麦芽窄食单胞菌S．maltophilia YHJ-1角蛋白酶基因的克隆表达及活性鉴定

从长期堆积废弃羽毛的土壤中分离得到一株高效降解羽毛的菌株YHJ—1，经16S rDNA序列分析鉴定为嗜麦芽窄食单胞菌，命名为嗜麦芽窄食单胞菌S．mahophilia YHJ-1。以菌株YHJ-1基因组为模板，通过PCR方法克隆了该菌的角蛋白酶基因（GenBank登录号：FJ765514），命名为kerD。将其编码区插入到表达载体pET28a（＋）中，转化大肠杆菌（Escherichia coli）BL21（DE3），筛选获得重组菌。通过对重组菌破碎上清液酶活测定和SDS—PAG分析，证实该酶在大肠杆菌中获得了高效表达，但酶活较低，最高酶活为10．5U／mL。酶学性质研究结果表明，该酶的最适反应温度为50℃，最适反应pH值为7．8，为中性酶。实验结果为嗜麦芽窄食单胞菌角蛋白酶的基础理论和应用研究奠定了基础。

羟基化酶产生菌Stenorophomonas maltophilia CGMCC 1.1788对农药氯噻啉生物转化的条件研究

[目的]探究羟基化酶产生菌Stenorophomonas maltophilia CGMCC 1.1788菌株中羟基化酶的转化活性。[方法]采用摇瓶发酵转化方法对羟基化酶产生菌S.maltophilia CGMCC 1.1788对农药氯噻啉的生物转化条件(不同生长时期的细胞、温度、pH等影响因子)进行了优化。[结果]确立了转化条件:100 ml锥形瓶最佳装液量10 ml,最佳反应温度25℃,最适pH 6.5,最佳反应底物起始浓度0.2 g/L,最佳反应时间48 h。[结论]为较好地发挥该菌的微生物转化功能及了解羟基化酶的生物学特性提供了理论依据。

Stenotropho monasmaltophilia DHHJ角蛋白酶特性及酶促动力学研究

通过对嗜麦芽窄食单胞菌DHHJ发酵液中的角蛋白酶进行盐析、层析纯化后,进一步研究角蛋白酶纯酶的理化性质。角蛋白酶对温度敏感,易于保存在低温环境(4℃)下,4 d后,纯酶液的酶活损失率为22.2%,在室温环境(25℃)下,纯酶液几乎失活。10 mmol/L Ca^(2+),Ba^(2+),有机溶剂甘油和乙醇(浓度范围在0%-20%之间)对角蛋白酶活有显著的促进作用,10 mmol/L Na^+对酶活有一定促进作用,而10 mmol/L Zn^(2+),Cd^(2+),表面活性剂Tween 80和SDS则可显著的抑制酶活,其中,ZnCl_2抑制角蛋白酶的类型主要是与底物竞争性地抑制角蛋白酶活性。比起羽毛底物,角蛋白酶与酪蛋白的亲和能力更好。本研究为工业化应用提供一定的理论基础。

角蛋白单体获得及诱导S.maltophilia DHHJ产角蛋白酶研究

可降解羽毛微生物能以羽毛粉为唯一营养源生长,而羽毛粉是一种大颗粒的不溶性底物,不能直接进入细胞内作为营养源同时作为一级信使来诱导酶基因表达。本文通过化学还原法水解羽毛得到可溶性羽毛角蛋白溶液,利用电泳和质谱验证其分子量约10 k D,为角蛋白单体。分别以该可溶性羽毛角蛋白及角蛋白单体酶解片段为诱导源,在无诱导源、羽毛粉为对照的情况下,测定72 h内嗜麦芽窄食单胞菌(S.maltophilia)DHHJ产生的角蛋白酶的酶活。在无诱导源时,角蛋白酶基因表现本底表达(0.5 U/m L);培养基中添加羽毛粉及角蛋白单体时,角蛋白酶表达量高,分别可达15 U/m L和20 U/m L。并且角蛋白单体酶解片段诱导酶表达较低(2.3 U/m L)。该结果初步表明,水溶性角蛋白单体作为信号源与细菌细胞接触或进入细胞内,控制角蛋白酶基因表达。该结果为细菌降解角蛋白分子机制研究奠定基础。

嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)R551．3四环素抗性消除的研究

嗜麦芽寡养单胞菌可共代谢降解烟碱类农药吡虫啉,并且对多种抗生素具有抗性.本文通过电穿孔法消除了该菌的四环素抗性,获得了四环素敏感型菌株R551-3Tcr-,可以用作遗传转化系统的受体菌,能被转化吸收遗传载体质粒pJB866H::ndhSL,构成一对很好的嗜麦芽寡养单胞菌遗传转化系统.同时HPLC结果表明所获得的四环素敏感型菌株羟基化吡虫啉的活性并未发生改变,为进一步开展敲除和回补共代谢途径关键酶的研究工作奠定了基础.

Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment

Objectives: To investigate possible sources of Stenotrophomonas maltophilia(S. maltophilia) in the clinical environment.Methods: Different samples were collected from Amol City of Iran. Steps for the identification of S. maltophilia included culturing, biochemical tests, polymerase chain reaction(PCR) of 16 S r RNA gene and 23 S r RNA gene. In addition, production of melanin pigment and patterns of motility of the bacteria, were also investigated.Results: In our study, 20 S. maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/110(6.36%), blood was 1/777(0.13%),sputum was 4/40(4%), urine was 1/2 947(0.03%), tap water was 1/240(0.42%) and dental suction was 6/120(5%). The isolated bacteria showed production of melanin pigment with rates of strong, moderate, weak, and lack of pigment. Types of motilities were seen in isolates.Conclusions: The highest percentage of bacteria is isolated of oxygen manometer system and dental suction, yet has not been reported from oxygen manometer system. These bacteria have also been associated with patients who have respiratory problems, so it is essential for staffs of hospitals to draw attention to this source of bacteria.

Characterization of tricalcium phosphate solubilization by Stenotrophomonas maltophilia YC isolated from phosphate mines

The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tricalcium phosphate (TCP) as sole phosphorus (P) source. The strain YC is identified as Stenotrophomonas maltophilia (S. maltophilia) based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis. The results show that the strain S. maltophilia YC can solubilize TCP and release soluble P in NBRIP growth medium. A positive correlation between concentration of soluble P and population of the isolate and a negative correlation between concentration of soluble P and pH in the culture medium are observed from statistical analysis results. Moreover, gluconic acid is detected in the culture medium by HPLC analysis. It indicates that the isolate can release gluconic acid during the solubilizing experiment, which causes acidification of the culture medium and then TCP solubilization. S. maltophilia YC has a maximal TCP solubilizing capability when using maltose as carbon source and ammonium nitrate as nitrogen source, respectively, in NBRIP growth medium.

Characteristics of copper removal and ion release during copper biosorption by Stenotrophomonas maltophilia in presence of benzo[a]pyrene

The ability of Stenotrophomonas maltophilia was demonstrated to selectively remove Cu2+from Cu(NO3)2 solution under the circumstance that 1 mg/L benzo[a]pyrene(BaP) was either present or not. The removal ratios of 2 and 10 mg/L Cu2+by 0.25 g/L biosorbent are up to 80% and 49% at 10 min, respectively. The biosorption includes ion exchange, NO3 reduction, ion release, and cell oxidation by Cu2+. BaP does not significantly affect Cu2+removal and ion release. Although 2 mg/L Cu2+increases the release of PO4 3, K+, NH4 +and Ca2+, 10 mg/L Cu2+has strong oxidation on cell, and then decreases NO3 reduction and hinders the release of K+, NH4 +and Ca2+. Exogenous cations inhibit the Cu2+biosorption, while additional anions increase the removal ratios of 10 mg/L Cu2+from 52% to 88%.

Physiological Study of Stenotrophomonas maltophilia DHHJ in Feather Biodegradation:Structural Remodeling of Cell Surface and Dynamic Change in Kerastinase Activity

Poultry industry produces a vast amount of feather waste annually, which forms a burden for environment protection.However, feathers are valuable bio-resources with high keratinaceous protein content and can be converted into more valuable materials through some approaches such as biodegradation by microorganism-derived keratinases. The characters of keratinases in microorganisms remain largely undetermined. In this study,it is reported that the morphological change of cell surface and the activities of intracellular and extracellular keratinases in Stenotrophomonas maltophilia( S. maltophilia) DHHJ. S. maltophilia DHHJ was cultured on lysogeny broth( LB) and feather broth(FB) through fermenter technology,and ultrastructure of cell and keratinase activity including extracellular and intracellular enzyme were observed respectively. Ultrastructural change on the cell surface was only observed for the bacteria cultured on FB medium,but not on LB,suggesting that the change could be induced by feather keratin. Therefore, the results showed that extracellular keratinase is a kind of induction enzyme while intracellular keratinase is a kind of constitute enzyme in S. maltophilia DHHJ.

<i>Stenotrophomonas maltophilia</i>Keratitis Related to Therapeutic Contact Lens Misidentified with an Automated Identification System

We present a case of keratitis caused by Stenotrophomonas maltophilia in a therapeutic contact lens user with trichiasis and symblepharon. This keratitis was initially diagnosed as caused by Achromobacter xylosoxidans, but the strain was sent for species confirmation and the isolate was finally identified as S. maltophilia by means of 16S rDNA sequencing. The patient rapidly improved on administration of fortified ceftazidime. Physicians should be aware that the definitive identification of the pathogenic agent and prolonged antimicrobial treatment according to culture sensitivities in keratitis are mandatory as treatment success depends greatly on them.

Comparative Study of the Mutant Prevention Concentrations of Sulfamethoxazole-Trimethoprim Alone and in Combination with Levofloxacin against <i>Stenotrophomonas maltophilia</i>

Objectives: To determine the mutant prevention concentration (MPC) of sulfamethoxazole-trimethoprim (SXT) alone and in combination with levofloxacin (LVX) against Stenotrophomonas maltophilia (S. maltophilia) and to determine if the combination may decrease the emergence of resistant mutants. Methods: The MPC with 20 S. maltophilia strains which were both susceptible to SXT and LVX were determined by inhibiting visible growth among 1010 CFU on four agar plates after 72 hours incubation at 37°C. Results: All except two strains (18/20) showed a mutant prevention concentration ≥ 152/8 μg/mL for SXT and the range of the mutant prevention concentration for the SXT in combination with LVX is 9.5/0.5~608/32 μg/mL, which demonstrates at least 2 fold reduction except one strain. There was a significant difference (P < 0.01) between SXT alone and in combination with LVX on the mutant prevention concentration and mutant prevention concentration/minimum inhibitory concentration values. Conclusions: The MPC/MIC values were narrowed for SXT by combining with LVX against the S maltophilia. The combination may decrease the enrichment of mutant bacterial populations. Much study is needed to verify whether the using of drug combinations may restrict or even block the selection of S. maltophilia mutants.

嗜麦芽窄食单胞菌的临床分布及耐药性分析

目的分析嗜麦芽窄食单胞菌临床分布情况及对常用抗菌药物耐药性分析。方法收集2019年1月至2021年12月河北医科大学第二医院住院患者分离的嗜麦芽窄食单胞菌,采用WHONET 5.6软件对嗜麦芽窄食单胞菌的临床科室分布、标本来源及药物敏感性试验开展回顾性研究。结果2019年1月至2021年12月共分离得到嗜麦芽窄食单胞菌共966株,在革兰氏阴性杆菌中的检出率为2.54%,检出率最高科室为小儿内科;临床科室分布中呼吸内科构成比最高(30.85%);感染患者以60岁以上老年患者为主,占57.04%;标本来源以痰标本为主,共分离771株,占79.81%。药敏结果分析显示,嗜麦芽窄食单胞菌对米诺环素、复方新诺明和左氧氟沙星的耐药率较低,耐药率分别为2.60%、10.40%、13.50%;对头孢他啶耐药率较高,耐药率为70.9%。不同标本来源嗜麦芽窄食单胞菌药敏结果显示,灌洗液、尿、血标本中嗜麦芽窄食单胞菌对米诺环素、复方新诺明、左氧氟沙星、头孢他啶的耐药率均高于痰标本。结论嗜麦芽窄食单胞菌主要来自呼吸内科,以痰为主。易感人群为老年人,耐药性高,临床医师应根据药敏结果合理选择抗菌药物,减少耐药菌株的产生及流行。

Stenotrophomonas maltophilia,an emerging pathogen in newborns:Three case reports and a review of the literature

BACKGROUND Stenotrophomonas maltophilia(S.maltophilia)is a rare cause of neonatal sepsis with significant morbidity and mortality and has extensive resistance to several antibiotics leaving few options for antimicrobial therapy.Only a few cases have been reported in neonates from developing countries.We report three cases of critically ill,extramural babies with neonatal S.maltophilia sepsis.All three babies recovered and were discharged.CASE SUMMARY All three cases were term extramural babies,who were critically ill at the time of presentation at our neonatal intensive care unit.They had features of multiorgan dysfunction at admission.Blood culture was positive for S.maltophilia in two babies and one had a positive tracheal aspirate culture.The babies were treated according to the antibiogram available.They recovered and were subsequently discharged.CONCLUSION Although various authors have reported S.maltophilia in pediatric and adult populations,only a few cases have been reported in the newborn period and this infection is even rarer in developing countries.Although S.maltophilia infection has a grave outcome,our three babies were successfully treated and subsequently discharged.

菌株C8和B4的分离鉴定及其耐盐促生效果和机制

土壤盐渍化是影响农业生产的主要环境因素,合理使用根际促生菌是改良修复盐渍化土壤的有效途径。本研究从东营地区盐渍化土壤中分离筛选到两株耐盐促生菌株C8和B4,经形态学特征、生理生化特性、16S rDNA和gyrB基因序列分析,分别鉴定为氧化微杆菌(Microbacterium oxydans)和嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)。含盐LB培养基上检测结果显示,菌株C8耐6%NaCl,具有解钾、溶有机磷、溶无机磷和分泌生长素的功能;菌株B4耐8%NaCl,具有溶有机磷和分泌生长素的功能。C8和B4单独施用及配施对盐胁迫下番茄的促生作用及机制的试验结果表明,C8和B4单独施用及配施均显著促进盐胁迫下番茄种子萌发和幼苗生长,提高过氧化氢酶(CAT)和过氧化物酶(POD)活性,上调过氧化氢酶基因CAT1和CAT2表达量,增加植株K+含量,降低Na+含量和Na+/K+,上调液泡膜Na+/H+逆向转运蛋白基因NHX1和NHX3的表达量。C8和B4配施具有增效作用。以上结果表明,C8和B4通过调节抗氧化酶及Na+转运蛋白基因表达,提高植株抗氧化能力,维持体内离子稳态,提高植株耐盐性。