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。

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.

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.

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%.

<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.

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.

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。

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。

嗜麦芽寡养单胞菌株对焦化废水中多环芳烃萘蒽的降解特性

生物降解是去除多环芳烃的有效方法之一,但仍存在降解不彻底、降解性能不稳定等问题,需采用细菌对其去除效果进行进一步研究。通过柠檬酸钠的共代谢作用强化嗜麦芽寡养单胞菌株(Stenotrophomonas maltophilia)JWJ-Y6对萘和蒽的降解效果研究,可为生物法降解焦化废水提供新的理论依据和数据支撑。JWJ-Y6降解萘的优化条件选择以柠檬酸钠为共代谢碳源、碳氮比2∶1、pH为8.0,与优化前相比,在此优化条件下JWJ-Y6能够在144 h内完全降解100 mg/L的萘,降解时间缩短了24 h;在168 h内对40 mg/L蒽的降解率为77.04%,提高了7.73%。在处理焦化废水方面,JWJ-Y6以柠檬酸钠为共代谢基质,对COD和蒽的降解率分别为77.28%和97.35%。JWJ-Y6降解萘和蒽的代谢途径均为邻苯二甲酸途径。JWJ-Y6降解焦化废水、JWJ-Y6与柠檬酸钠共代谢降解焦化废水、干菌剂与柠檬酸钠共代谢降解焦化废水3种情况都符合二级动力学模型。

1例血液透析患者并发导管相关性感染与肺部感染的抗感染治疗药学监护

目的:分析1例血液透析患者并发导管相关性感染与肺部感染的抗感染治疗药学监护过程,为临床透析患者的抗感染治疗提供参考。方法与结果:该患者因“透析过程中出现寒战1 h”入院治疗,入院检查显示,导管口皮肤红肿,白细胞计数16.71×109/L、中性粒细胞百分比93.40%,考虑为导管相关性感染,遂予头孢唑林抗感染治疗;第2天,患者CT检查提示存在肺部感染,临床药师会诊后,考虑导管相关性感染、肺部感染的常见病原菌谱和患者的肾功能基础,建议改用哌拉西林-他唑巴坦钠,结合患者的肌酐清除率,其用法用量宜为“2.25 g,q12h”,医生采纳了;第5天,患者血培养回报嗜麦芽窄食单胞菌,且其对哌拉西林-他唑巴坦钠敏感。结论:针对血液透析患者所并发的感染,临床在制定经验性抗感染治疗方案时应首先考虑相关感染的常见病原菌谱,并结合患者的肝肾功能,待病原学结果明确后再优化调整,以保证患者的治疗效果。

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。

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。

光叶苕子(Vicia villosa var.)内生细菌Sz-2的特性及其对香蕉枯萎病的防治效果

【目的】从云南绿肥主推品种光叶紫花苕根内筛选出对香蕉具有促生和拮抗作用的菌株,并对其特性和香蕉枯萎病的防治效果进行研究,以丰富生防促生菌株资源库。【方法】从光叶紫花苕根中筛选出高效拮抗菌株Sz-2,使用平板对峙试验测定菌株抑菌效果;并测定其体外解磷、解钾、固氮及产铁载体能力;通过形态学观察、生理生化测定和16S rDNA序列分析,对该菌株进行了分类鉴定。布置盆栽试验,设4个处理:无抗生菌对照和接种病原菌TR4、Sz-2+TR4、Sz-2,测定香蕉叶片和球茎的发病指数以及促生效果。【结果】光叶紫花苕根内分离得到的菌株Sz-2对香蕉枯萎病菌具有较好的拮抗效果,平板径向抑菌率达74.3%。菌株Sz-2为革兰氏阴性细菌,菌落形态呈浅白色、放射状,接触酶、氧化酶、赖氨酸脱羧酶、β-半乳糖苷酶、精氨酸双水解酶、明胶水解、硝酸还原反应、纤维二糖、柠檬酸反应、葡萄糖发酵反应、酪素水解等均为阳性,吲哚反应、淀粉水解、产H2S反应、鸟氨酸脱羧酶、脲酶反应为阴性。结合形态学和分子生物学鉴定菌株Sz-2为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)。盆栽结果显示,接种拮抗菌Sz-2后的香蕉球茎和叶片枯萎病发生较轻,球茎纵切面和叶片颜色正常且植株生长健康,对球茎和叶片的防效达69.66%和84.60%;香蕉茎粗、单株鲜重和株高与CK相比分别提高了6.01 mm、83.3 g和11.82 cm。【结论】菌株Sz-2对香蕉枯萎病具有较好的防治作用,加之其解磷、解钾和产铁载体功能,对香蕉植株具有显著的促生效果,可结合绿肥研制具有防控香蕉枯萎病的功能型绿肥,为香蕉促生抗病菌株的进一步田间开发应用提供理论依据。

嗜麦芽窄食单胞菌CA1定殖及促生长特性相关基因挖掘

【目的】挖掘嗜麦芽窄食单胞菌CA1定殖和促生长特性相关基因,为探究其促生长作用机制提供理论参考。【方法】利用Nanopore测序技术平台对CA1菌株进行全基因组测序,将其基因组序列与GO、NR、KEGG、COG等数据库进行比对,获得基因功能注释信息,挖掘分析其功能特异性基因;使用基质培法收集甘蔗根系分泌物,并分析根系分泌物对菌株CA1的趋化性、生物膜形成和定殖相关基因表达的影响。【结果】嗜麦芽窄食单胞菌CA1基因组大小为4371327 bp,携带6个质粒,GC含量64.58%,其中4696个基因被注释预测,包括4525个蛋白编码基因、87个tRNA、13个rRNA和1个tmRNA。GO、COG、KEGG和NR数据库分别成功注释2795、2078、2494和2584个基因。该菌基因组含有细菌趋化性(cheA、cheV、cheY)、生物膜形成(flhA、flk、flgG)、细菌黏附(pgaA、mrkD)、嗜铁(fes、fepA、fecA)、合成氨(gltP、gltB、gltD)和磷酸盐代谢(phoR、phoB、pstA)等促进植物生长和定殖相关的基因。趋化性试验结果显示,与对照组相比,根系分泌物处理下的菌落数显著增加(P<0.05,下同),且cheA、cheV和cheY基因的相对表达量显著升高,CA1生物膜形成显著增加。【结论】甘蔗根系分泌物有助于菌株CA1的趋化性及生物膜形成,提高与趋化性相关基因的表达量。菌株CA1基因组含有多个与促生长和定殖特性相关的基因,是其促生长特性的分子基础,细菌的趋化性和生物膜的形成是其定殖的关键步骤,根系分泌物有利于菌株的定殖。

2016—2021年河北省血培养嗜麦芽窄食单胞菌分离株的临床分布及耐药性变迁

目的了解河北省血标本分离嗜麦芽窄食单胞菌的临床分布及耐药性变迁,为临床治疗及合理应用抗生素提供依据。方法收集2016年1月—2021年12月河北省血标本分离嗜麦芽窄食单胞菌数据,采用WHONET 5.6和SPSS 24.0软件对细菌分布及耐药性进行分析。结果河北省2016—2021年血标本分离嗜麦芽窄食单胞菌471株,主要来自老年人(34.4%)、中年人(23.6%)和儿童(21.9%),其中男性分离292株,女性分离179株;重症医学科、内科和儿科为最常见的分离科室,分别占27.8%、25.7%和24.2%。2016—2021年该菌对米诺环素、左氧氟沙星、甲氧苄啶-磺胺甲唑、氯霉素、替卡西林-克拉维酸和头孢他啶的耐药率保持稳定,六年平均耐药率分别为2.3%、7.6%、9.8%、15.8%、31.4%和37.5%;在不同科室间该菌对甲氧苄啶-磺胺甲唑和氯霉素的耐药率差异存在统计学意义(均P<0.05),外科分离菌株对甲氧苄啶-磺胺甲唑耐药率高于其他科室;在不同年龄组间该菌对左氧氟沙星、氯霉素、替卡西林-克拉维酸和头孢他啶的耐药率差异存在统计学意义(均P<0.05)。结论2016—2021年河北省血标本分离嗜麦芽窄食单胞菌主要来自重症医学科,以中老年人群为主。六年间,该菌对常用抗菌药物的耐药率保持稳定,对部分抗菌药物的耐药率在不同科室和年龄组间存在差异。临床可根据药敏结果、患者科室和年龄合理选择抗菌药物。

餐厨垃圾高效油脂降解菌的筛选及其降解条件研究

以葵花籽油作为唯一碳源,采用中性红培养基从厨余垃圾中筛选到5株降解菌株,再通过产脂肪酶培养基复筛得到2株降解效果较好的菌株,将其命名为QT1和QT3,其油脂降解率分别达到54.0%和65.5%,培养10 d内脂肪酶活性均在17.0 U·mL^(-1)以上。在10 g·L^(-1)的油脂含量下,QT1菌株在温度35℃、pH 7、转速200 r·min^(-1)、盐含量20 g·L^(-1)时降解效率最佳,QT3在温度40℃、pH 8、转速180 r·min^(-1)、盐含量30 g·L^(-1)时降解效率最佳。通过形态学观察和16S rDNA序列分析,QT1菌株为腐生葡萄球菌(Staphylococcus saprophyticus),QT3菌株为嗜麦芽窄食胞菌(Stenotrophomonas maltophilia)。