罗氏沼虾源产气肠杆菌分离鉴定及耐药性分析

2017至2018年春季罗氏沼虾育苗期间,江苏省高邮市多家育苗场幼体和仔虾持续发生死亡。从发病幼体和仔虾分离到大量优势生长的细菌,人工感染试验证明其对罗氏沼虾幼体和仔虾有较强的致病性。对分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等表型生物学性状检验,测定了代表菌株的16S rRNA和gyrB基因序列,分析了16S rRNA和gyr B基因序列的同源性。结果显示:引起罗氏沼虾幼体大量死亡的病原之一为产气肠杆菌(Enterobacter aerogenes),代表菌株XL2-1对罗氏沼虾仔虾和蚤状幼体的半数致死量(LD50)分别为2. 0×106和7. 0×105CFU/m L,胞外酶活性及溶血活性检测表明分离菌不溶血,不产生蛋白酶、卵磷脂酶、淀粉酶、尿酶及DNA酶;分离菌的耐药谱测定结果发现,分离菌对头孢噻肟等7种药物敏感,对青霉素G等9种药物耐药,对头孢曲松等19种药物存在株间差异。     

食品中克罗诺杆菌(原阪崎肠杆菌)双重PCR检测方法研究

根据克罗诺杆菌(Cronobacter spp.)16S rRNA基因以及局部大分子合成(MMS)操纵子特异序列设计2对引物,经反应体系和条件优化,建立了双重PCR检测方法。特异性检测结果显示,Cronobacter spp.菌株PCR扩增均可见2条特异性条带,而其他菌株PCR扩增均为阴性。纯菌检测双重PCR的灵敏度为6.3×103 CFU.mL-1,而相对应单重PCR的灵敏度分别为6.3×101 CFU.mL-1和6.3×103 CFU.mL-1;人工污染的食品样品(奶粉、牛奶、鸡肉)在经过24 h增菌后,检测限均可达100 CFU.mL-1或100 CFU.g-1。在鼠伤寒沙门氏菌(Salmonella typhimurium)存在的条件下,双重PCR的检测限没有受到影响。表明本试验建立的双重PCR检测方法具有很好的特异性和灵敏度,能克服食品样品基质及杂菌的干扰,可应用于食品中Cronobacter spp.的检测。     

Multidrug-resistance plasmid of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis>: transfer to <Emphasis Type="Italic">Erwinia herbicola</Emphasis> on the phylloplane of mulberry and weeds

A mulberry epiphytic Enterobacter cloacae MUL1 harbors plasmid pMUL1 encoding five drug-resistance genes. This plasmid was examined upon its conjugal transfer into epiphytic Erwinia herbicola on the phylloplane of mulberry and 12 species of weeds. The plasmid was transferred into Er. herbicola at a frequency of 10^–5–10^–3/recipient in mulberry and Lolium multiflorum LAM. 1–8 days after wound inoculation with 10⁶–10⁸/ml suspensions. In Chenopodium album L. and C. album L. var. centrorubrum, however, it was transferred only after wound inoculation with a 10⁸/ml suspension, but not with 10⁷/ml or 10⁶/ml suspensions, owing to the weak epiphytic fitness of Ent. cloacae on these weeds. Transconjugants were also obtained for seven other species of weeds in the case of inoculation with a 10⁸/ml suspension. In contrast, when bacterial suspensions were sprayed on mulberry leaves with or without fresh wounds, transconjugants were obtained only in wounded leaves, which were considered suitable for bacterial conjugation. These findings suggest that epiphytic bacteria, including Ent. cloacae and Er. herbicola, may be carriers of drug-resistance genes distributed among plant pathogenic bacteria in nature.     

A Novel Conjugative Plasmid Conferring Multiple-antibiotic Resistance Detected in Epiphytic Strains of Enterobacter cloacae

Several epiphytic strains of Enterobacter cloacae isolated from mulberry leaves were resistant to antibiotics such as streptomycin, kanamycin, ampicillin, tetracycline and chloramphenicol, and harbored a 100-kb plasmid designated pMUL1. Plasmid profile analysis of spontaneous mutants derived from Ent. cloacae MUL1 and MUL1 (RSF1010) suggested that pMUL1 confers resistance to multiple antibiotics. Southern blot analysis using probes of five antibiotic-resistance genes against EcoRI-digested DNA from pMUL1 and defective pMUL1s (mutants) revealed that all these genes were located within a 24-kb region of pMUL1 and that some genes were assigned to the defective plasmids. A similar antibiotic-resistance plasmid was detected in several orchid-pathogenic strains of Ent. cloacae, but not in the type-culture strain (JCM 1232) or strains of Ent. cloacae of insect-origin. Strains MUL1 and MUL1 (RSF1010) were then mated with epiphytic Erwinia spp. and Pseudomonas spp. on filters, respectively. Several recipient strains of epiphytic Er. herbicola simultaneously acquired plasmid pMUL1 and the phenotype of multiple-antibiotic resistance. Thus, pMUL1 was verified to be conjugative and to encode genes for multiple-antibiotic resistance, including genes homologous to the strA-strB of the nonconjugative IncQ plasmid RSF1010. These findings suggest that epiphytic Ent. cloacae may play a role in the dissemination of antibiotic-resistance genes among epiphytic bacteria and plant pathogenic bacteria. Received 10 January 2002/ Accepted in revised form 29 March 2002     

蟾酥对产气肠杆菌抑制作用的方差分析和相关分析

把采自中华大蟾蜍的蟾酥用蒸馏水稀释成4、6、8、10mg／mL共4种浓度，作为因素A，用LB液体培养基将活化的产气肠杆菌原液按1：10^4和1：10^5稀释，作为因素B，采用二因子（A和B）方差分析观察了蟾酥对产气肠杆菌抑菌圈直径的影响，并分析了蟾酥浓度与抑菌圈直径之间的相关关系。结果显示，蟾酥浓度与抑菌圈直径之间存在显著正相关（r=0．988～0．997）；不同浓度的蟾酥溶液对产气肠杆菌均有抑制作用；且随着蟾酥浓度的升高，抑制作用加强。     

Biocontrol of Phytophthora Root Rot of Angelica Trees by Enterobacter cloacae and Serratia ficaria Strains

Bacterial strains isolated from the rhizosphere of angelica trees were evaluated for their antagonistic activity against Phytophthora cactorum, a causal agent of Phytophthora root rot. Of these, three bacterial strains, designated as T-1-8, T-1-14 and T-1-23, strongly inhibited mycelial growth of P. cactorum ARE-862 in a dual-culture plate assay. Biocontrol activity of these strains was then examined by dipping root of young seedlings of angelica trees into a bacterial suspension. The incidence of Phytophthora root rot was markedly suppressed for at least 79 days in pot tests when treated seedlings were planted in naturally infested soil. The suppression was maintained through June of the next year. In addition, these strains significantly reduced the development of Phytophthora root rot up to 47 days in naturally infested field and up to 63 days (the last day of testing) in an artificially (moderately) infested field. Based on their main bacteriological properties, strain T-1-14 was identified as Enterobacter cloacae and T-1-8 and T-1-23 were identified as Serratia ficaria. Received 5 July 1999/ Accepted in revised form 25 October 1999     

一种基于TaqMan探针法的阪崎肠杆菌qPCR全自动检测技术体系的建立

[目的]建立快速准确地检测食物样品中食源性致病菌的体系。[方法]配套湖州市微生物制剂与农产品安全重点实验室自主研发的微生物分子检测仪,建立一种基于Taq Man探针法的阪崎肠杆菌q PCR检测体系。[结果]体系建立成功,标准曲线斜率为-3.44,R~2=0.995 5,扩增效率为95.3%。[结论]该q PCR体系特异性强,灵敏度高,明显缩短了检测时间,并具有良好的稳定性与重复性,为食品中阪崎肠杆菌的高通量和自动化检测提供了一个新的途径。     

絮凝剂产生菌KLE-1的特性研究

从土壤中筛选出1株产絮凝剂的菌种KLE-1,采用BIOLOG菌种鉴定仪鉴定该菌株为产气肠杆菌(Enterobactet aerogenes)。该菌株培养产生的絮凝物质主要存在于发酵液中;絮凝剂以粉末状态存在时,在室温下放置50 d以后絮凝活性仪下降14.1％;絮凝剂对高岭土悬浊液絮凝的最适宜pH值为7.0~8.0,即中性偏碱性条件,Ca2+,Al3+,Mg2,Na和Fe3+等金属阳离子均有助于絮凝作用。     

耐铜苏丹草根内生细菌的分离筛选及其生物学特性研究

从生长在铜矿废弃地土壤中的Cu耐性苏丹草根中分离筛选到二株产1-氨基环丙烷-1-羧酸(ACC)脱氨酶内生细菌K1-6和K3-9菌株,并对菌株生物学特性进行了研究。菌株K1-6和K3-9具有较强的Cu抗性和多种抗生素抗性,菌株K1-6和K3-9能够溶磷和分泌吲哚乙酸(IAA),另外,菌株K3-9还能够产生铁载体和精氨酸脱羧酶,菌株K1-6和K3-9对温度、p H和盐浓度具有一定的耐受性,经16S r DNA序列分析,菌株K1-6和K3-9分别被鉴定为根瘤菌属(Rhizobium sp.K1-6)和肠杆菌属(Enterobacter aerogenes K3-9)。采用平皿培养试验研究了菌株K1-6和K3-9对生长在不同浓度Cu(0、4 mg/L)环境中的苏丹草的生长和吸收Cu的影响。结果表明,接菌处理苏丹草根部和地上部干重分别比对照增加了10.6%~45.5%和13%~40%,差异达显著水平(P0.05);接菌株K1-6处理苏丹草根部和地上部Cu含量比对照增加了46%和85%(P0.05),而接菌株K3-9处理苏丹草根部和地上部Cu含量与对照相比没有显著差异。另外,接菌株K1-6处理苏丹草根部和地上部总Cu吸收量比对照增加了88%和114%(P0.05),接菌株K3-9处理苏丹草根部总Cu吸收量比对照增加了44%(P0.05)。另外,接菌株K1-6和K3-9处理的苏丹草根部吸收的Cu是地上部吸收Cu的16~23倍。研究表明,分离自耐铜苏丹草根部的内生细菌具有多种植物促生特性,能够显著促进苏丹草的生长、提高苏丹草对Cu的耐受性,并强化苏丹草根部对Cu的富集能力。另外,不同的内生细菌对苏丹草的生长、富集和耐受Cu的影响不同。     

Effect of phosphate-solubilizing bacteria and vesicular-arbuscular mycorrhizae on tomato growth and soil microbial activity

The interaction of vesicular-arbuscular mycorrhizae (VAM) and phosphate-solubilizing bacteria (PSB) on plant growth, soil microbial activities, and the production of organic acids was studied in non-sterile soil containing hydroxyapatite and glucose. Glomus etunicatum (VAM), a fungus, and Enterobacter agglomerans, a bacterium able to solubilize insoluble phosphate, were used as inocula. Three treatments and a control were used: inoculation with E. agglomerans (treatment E), inoculation with G. etunicatum (treatment G), inoculation with E. agglomerans+G. etunicatum (treatment E+G) and the control (C). Inoculation with E, G, or E+G had increased plant growth by days 35, 55, and 75 compared with the control. Microbial biomass carbon (C) and alkaline phosphatase activity in the rhizosphere generally increased with time. Alkaline phosphatase activity was higher in treatments G and E+G compared with the control at 35 and 55 days. The highest acid phosphatase activity was observed in treatment E at 35 days; however, this markedly decreased with time. A significantly higher soluble phosphorus (P) concentration was observed in treatments E and E+G on day 55 compared with C. However, there was no significant difference in soluble P concentration in the rhizosphere between treatments with time. The P concentration was greatest in all treatments on day 55. The highest oxalic acid concentration was observed in the rhizosphere of the non-sterile soil in E+G on day 35. Total N and P uptake in plants from treatments E and G were higher compared with the control. However, the highest N and P uptake was observed in treatment E+G. This study suggests a synergistic interaction between E. agglomerans and G. etunicatum. Received: 3 January 1997