微孔板杂交法测定郴毒假单胞酵米面亚种的ＤＮＡ—ＤＮＡ同源性

采用一种新的ＤＮＡ－ＤＮＡ发校方法－微孔板法对郴毒假单胞菌酵米面亚种及伯克霍尔德氏菌属中１１个的ＤＮＡ－ＤＮＡ同源性进行测定。结果发现郴毒假单胞菌酵米面亚种与唐菖蒲伯克霍尔德氏菌及椰毒伯克霍尔德氏菌的ＤＮＡ－ＤＮＡ同源性均大于７５％,建议这３个种应合并为同一个种,命名为唐菖蒲的伯克霍尔德氏菌（Brukholderia gladioli)。     

微孔板杂交法测定椰毒假单胞酵米面亚种的DNA-DNA同源性

采用一种新的DNADNA杂交方法—微孔板法对椰毒假单胞菌酵米面亚种及伯克霍尔德氏菌属中11个种的DNADNA同源性进行测定。结果发现椰毒假单胞菌酵米面亚种与唐菖蒲伯克霍尔德氏菌及椰毒伯克霍尔德氏菌的DNADNA同源性均大于75%,建议这3个种应合并为同一个种,命名为唐菖蒲伯克霍尔德氏菌(Burkholderia gladioli)。     

椰毒假单胞菌的电镜观察

酵米面假单胞菌(Pseudomonas farinofermentans)系从食物中毒的变质酵米面中分离到的病原菌,它与揶毒假单胞菌(P.cocovenenans)为同种不同生物型,与洋葱假单胞菌(P.cepaia)有许多共同点。电镜观察表明,此3株假单胞菌在形态和结构上有以下共同特点：菌体呈短杆状,0．6-0．8×1．5—2．0μm。细胞壁由肽聚糖层和外膜(outer membrane)构成。有时可见丝状体(filaments) 甚至畸形细胞。无荚膜,无菌毛(pili)一端有多根鞭毛。细胞质内有电子透明的聚β-羟基丁酸盐(PHB)颗粒。核区内有电子致密体(e|ectron-densebodies)或层状体(laminar bodies)。细胞表面可见到wei7细胞(mlncells)。均能产生细胞外“丝状物质”,这一特点尚未见报道。     

大连地区耐亚胺培南铜绿假单胞菌金属β-内酰胺酶检测及耐药性分析

目的了解大连地区分离的耐亚胺培南铜绿假单胞菌的耐药特征,金属β-内酰胺酶携带情况,提供大连地区院内控制铜绿假单胞菌感染的依据。方法选取2013年1月至2014年9月临床分离的400株铜绿假单胞菌,进行菌种鉴定和药物敏感试验;金属酶检测采用E-test试验;PCR法扩增产金属β-内酰胺酶的基因,并对扩增阳性产物进行测序确认。结果 400株铜绿假单胞菌的标本以呼吸道分泌物最多,占81.5%;分布以呼吸内科和ICU病房最高,占总数的19.8%和25.5%;药敏结果发现有89株铜绿假单胞菌对亚胺培南耐药,且多数为多重耐药菌株;400株铜绿假单胞菌经E-test试验进行金属酶表型筛查,有17株金属酶阳性,检出率为19.1%;经PCR扩增金属酶基因阳性的有11株,其中8株为IMP-1,3株为VIM-2,其他几种基因均未检出。结论大连地区耐亚胺培南的89株铜绿假单胞菌多数是多重耐药菌株;产金属β-内酰胺酶在大连地区铜绿假单胞菌对亚胺培南耐药中发挥重要作用,酶的基因型主要为IMP-1和VIM-2。     

耐亚胺培南铜绿假单胞菌金属酶的筛选及基因型研究

目的研究重庆医科大学附属第一医院分离的29株耐亚胺培南铜绿假单胞菌中金属酶（Metallo-β-Lactamase,MBL）的基因型分布情况。方法用亚胺培南-EDTA纸片法筛选29株铜绿假单胞菌中产MBL的铜绿假单胞菌,用PCR扩增法检测29株菌中金属酶VIM和IMP基因。结果29株耐亚胺培南的铜绿假单胞菌中,亚胺培南-EDTA纸片法筛选出MBL阳性菌株5株,阳性率为17％。PCR扩增出IMP基因型有4株,阳性率为14％,均为金属酶IMP-9型,未扩增出VIM基因。以PCR法结果为判定标准,亚胺培南-EDTA纸片法敏感性100％,特异性96％。结论IMP-9型为该院分离的这29株耐亚胺培南的铜绿假单胞菌中主要MBL基因型。本实验中所用的亚胺培南一EDTA纸片法能简单有效的筛选出产MBL的铜绿假单胞菌。     

617株耐亚胺培南铜绿假单胞菌耐药性及其产超广谱β-内酰胺酶基因型研究

探讨耐亚胺培南铜绿假单胞菌的耐药性及其产超广谱β-内酰胺酶基因型。收集2011年7月至2013年12月上海市中医药大学附属曙光医院临床分离的铜绿假单胞菌共1 125株,筛选亚胺培南耐药株,常规纸片法检测其耐药性,并用E-test检测金属β-内酰胺酶（MBL）,采用PCR法检测耐药基因型。结果显示,1 125株铜绿假单胞菌中耐亚胺培南铜绿假单胞菌共计617株,占54.8%;亚胺培南敏感铜绿假单胞菌共计508株,占45.2%。617株亚胺培南耐药铜绿假单胞菌100%为多重耐药,而亚胺培南敏感铜绿假单胞菌的多重耐药率仅为13.78%,明显较前者低（χ²=871.15,P<0.05）;亚胺培南耐药的铜绿假单胞菌中MBL表型阳性共126株,阳性率为15.4%,94株(74.60%)表现为VIM-2阳性,10株（7.94%）表现为IMP-1阳性,1株检出OXA-10,〖WTBZ〗且该例菌株同时表达VIM-2。临床分离的耐亚胺培南的铜绿假单胞菌多为多重耐药,其产MBL的主要基因型是VIM-2。     

铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌的抑菌活性研究

目的观察铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)的体外抑菌活性。方法用交叉划线接种方法进行铜绿假单胞菌对32株耐甲氧西林金葡菌的体外抗菌活性的测定。结果铜绿假单胞菌抗菌物质对MRSA的体外抑菌活性良好,产生色素的菌株的抗菌活性最好,15株铜绿假单胞菌中,7株产蓝绿色色素的铜绿假单胞菌,对MRSA的抑制率均达到了100%,平均抑菌带的宽度为37.7 mm。结论铜绿假单胞菌抗菌物质对32株MRSA具有较强的抗菌活性,无疑对MRSA感染的抗菌药物研制方面开辟了一条新的途径。这是国内的首次研究报道。     

铜绿假单胞菌体外抑菌活性研究

目的 观察铜绿假单胞菌抗菌物质对鲍曼不动杆菌等细菌的体外抑菌效果.方法 用交叉条带实验方法检测了铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌的体外抑制活性.结果 铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌体外抑菌活性良好,10株铜绿假单胞菌中,有8株对鲍曼不动杆菌的抑制率均达到了100％.另外有8株对耐甲氧西林表皮葡萄球菌的抑菌率均为100％;有6株对粪肠球菌的抑菌率为100％.结论 铜绿假单胞菌对上述3种致病菌具有较强的抗菌活性,具有开发前景.     

126株铜绿假单胞菌的临床分布及耐药性分析

目的调查分析象山县中医医院铜绿假单胞菌的临床分布及药敏情况,为临床合理选用抗生素提供可靠的依据。方法采集疑似患者的标本,进行分离、培养与鉴定。采用自动微生物鉴定／药敏分析仪进行鉴定及药敏试验,对2012年7月至2013年10月分离出的126株铜绿假单胞菌（包括21株黏液型铜绿假单胞菌）的分布及耐药性进行回顾性分析。结果126株铜绿假单胞菌临床主要分布情况：痰占80．2％,尿液占11．1％,脓液占7．1％,以呼吸道感染为主。对铜绿假单胞菌保持活性较强同时耐药率〈20％的抗生素有阿米卡星、妥布霉素、亚胺培南、哌拉西林／他唑巴坦,其中碳青霉烯类耐药率升至5％,原来被认为抗铜绿假单胞菌较为有效的喹诺酮类抗生素的耐药率也有了很大提升,左氧氟沙星耐药率升至33％。黏液型铜绿假单胞菌的体外抗菌药物敏感试验耐药性较弱,且明显弱于非黏液型铜绿假单胞菌的耐药性。结论铜绿假单胞菌为医院呼吸道感染的常见致病菌,对多种抗菌药物呈不同程度耐药。加强动态监测,合理使用抗菌药物,对铜绿假单胞菌感染的预防和药物治疗具有重要指导意义。     

基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用

为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。     

Synergistic degradation of linuron by a bacterial consortium and isolation of a single linuron-degrading variovorax strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Cloning and nucleotide sequence analysis of xylE gene responsible for meta-cleavage of 4-chlorocatechol from Pseudomonas sp. S-47

Pseudomonas sp. S-47 expresses catechol 2,3-dioxygenase (C230) catalyzing the conversion of 4-chlorocatechol (4CC) as well as catechol to 5-chloro-2-hydroxymuconic semialdehyde and 2-hydroxymuconic semialdehyde, respectively, through meta-ring cleavage. The xylE gene encoding C230 for meta-cleavage was cloned from strain S-47 and its nucleotide sequence was analyzed. The pRES101 containing the xylE gene exhibited high C230 activity toward catechol and 4CC without altering the substrate specificity from natural strain. The xylE gene was composed of 924 bp and encoded polypeptide of molecular mass 35 kDa containing 307 amino acids. A deduced amino acid sequence of the C230 from strain S-47 exhibited over 80% identity with those of Pseudomonas putida mt-2, Pseudomonas putida G7, and Pseudomonas sp. CF600. However, it shows below 45% identity with those of Pseudomonas cepacia LB400 and Pseudomonas sp. KKS102. The C230 of strain S-47 was conserved in the amino acids (His150, His214, Glu261) for metal binding ligands and those (His199, His242, and Tyr251) for catalytic sites. Therefore, Pseudomonas sp. S-47 can be explained as acting by degrading catechol as well as 4CC by xylE-encoding C230 which was fused by N domain of nahH and C domain of dmpB from other Pseudomonas strains.     

牛蒡根际可培养细菌多样性和镉耐性初步分析

从牛蒡根际土壤中分离可培养细菌,进行多样性分析,并对镉耐受性菌株进行筛选及其抗性和种群多样性进行了分析。限制性内切酶多态性分析显示,分离的菌株可分为9个操作分类单元(OUT),分别属于变形菌门、厚壁菌门和放线菌门,分属于6个科,9个属,其中隶属于肠杆菌属、芽胞杆菌属和假单胞菌属的是优势物种。分离到的耐镉菌株分别属于Bacillus subtilis、Enterobacter aerogenes、Enterobacter ludwigi、Klebsiellasp.、Pectobacterium carotovorum、Pseudomonassp.,而Pectobacterium carotovorumNP22、Enterobacter ludwigii NP23、Pseudomonassp.NP39三菌株可在Cd2+浓度为400 mg/L固体培养基上生长。     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Respiration of 2,4,6-trinitrotoluene by Pseudomonas sp. strain JLR11

Under anoxic conditions Pseudomonas sp. strain JLR11 can use 2,4, 6-trinitrotoluene (TNT) as the sole N source, releasing nitrite from the aromatic ring and subsequently reducing it to ammonium and incorporating it into C skeletons. This study shows that TNT can also be used as a terminal electron acceptor in respiratory chains under anoxic conditions by Pseudomonas sp. strain JLR11. TNT-dependent proton translocation coupled to the reduction of TNT to aminonitrotoluenes has been observed in TNT-grown cells. This extrusion did not occur in nitrate-grown cells or in anaerobic TNT-grown cells treated with cyanide, a respiratory chain inhibitor. We have shown that in a membrane fraction prepared from Pseudomonas sp. strain JLR11 grown on TNT under anaerobic conditions, the synthesis of ATP was coupled to the oxidation of molecular hydrogen and to the reduction of TNT. This phosphorylation was uncoupled by gramicidin. Respiration by Pseudomonas sp. strain JLR11 is potentially useful for the biotreatment of TNT in polluted waters and soils, particularly in phytorhizoremediation, in which bacterial cells are transported to the deepest root zones, which are poor in oxygen.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

假单胞菌S-2降解甲胺磷性能的研究

从甲胺磷生产车间分离到一株假单胞菌编号为S-2。S-2可利用甲胺磷为唯一氮源,但不能利用甲胺磷为唯一磷源。该文对S-2体内具有的降解甲胺磷的酶类进行了研究,初步断定：S-2可代谢产生酸性磷酸酶,主要在胞外降解甲胺磷。S-2在甲胺磷诱导的情况下,这些降解酶类可大量聚积。用诱导过的菌液降解甲胺磷比未经诱导的快了2d左右。     

Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na(2)-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to approximately 35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.     

脱氮除磷假单胞菌的筛选及定量检测

【目的】筛选具有较强脱氮除磷能力的细菌,建立结合S1酶保护分析的分子探针技术,以分析该菌在发酵过程中的数量变化情况。【方法】采用缺磷培养基厌氧培养、富磷培养基好氧培养和硝酸盐还原产气实验进行脱氮除磷菌筛选。通过16S rRNA基因序列分析及同源性比对,结合菌株的生理生化鉴定试验,鉴定筛选株。设计相应的16S rRNA探针组,建立结合S1酶保护分析的分子探针技术。【结果】筛选的菌株被鉴定为假单胞菌Pseudomonas sp.,命名为LY10。菌株LY10在富磷培养基中好氧培养24 h,总磷去除率达90.01%。在反硝化聚磷培养基中培养48 h,总氮和总磷去除率分别为84.71%和89.37%。针对假单胞菌16S rRNA基因序列设计了一组用于结合S1酶保护分析的分子探针Probe-P.sp,该探针具有很高的甄别灵敏度,能够将LY10与丛毛单胞属(Commonas)等5种细菌区分开;分子探针定量分析假单胞菌LY10,其细胞量与吸光值呈线性关系,检测的线性范围为103~106 cells/mL,线性方程为:y=-0.967 87+0.372 99x(R2=0.996 7,n=5)。【结论】新筛的假单胞菌LY10的脱氮除磷能力较强,具有生物脱氮除磷的工业化应用潜质。所建立的结合S1酶保护分析的分子探针技术的特异性和灵敏度良好,有望应用于混菌体系中的假单胞菌的定性定量分析。     

Aerobic denitrifying bacteria that produce low levels of nitrous oxide

Most denitrifiers produce nitrous oxide (N(2)O) instead of dinitrogen (N(2)) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N(2)O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO(3)(-)) to N(2) at rates of 0.9 and 0.03 micro mol min(-1) unit of optical density at 540 nm(-1) at dissolved oxygen (O(2)) (DO) concentrations of 39 and 38 micro mol liter(-1), respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N(2) but evolved more than 10-fold more N(2)O than strains TR2 and K50 evolved. The isolates denitrified NO(3)(-) with concomitant consumption of O(2). These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the beta subclass of the class Proteobacteria and were identified as P. stutzeri TR2 and Pseudomonas sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N(2)O emission, the single-stage process for nitrogen removal, and microbial N(2)O emission into the ecosystem.