基于全基因组关联分析研究粪肠球菌发酵食品分离株的耐药性

【目的】研究15株分离自自然发酵食品的粪肠球菌(Enterococcus faecalis)和1株粪肠球菌模式株对15种(1种β-内酰胺类和14种非β-内酰胺类)抗生素的耐药性,并通过菌株耐药表型与基因型的关联分析找到粪肠球菌潜在的耐药基因。【方法】利用微量肉汤稀释法检测试验菌株对15种抗生素的耐药性,运用Scoary软件进行表型与基因型的关联分析。【结果】16株粪肠球菌对卡那霉素、万古霉素、利奈唑胺和红霉素100%敏感;对其余11种抗生素均表现出不同程度的耐药,其中对克林霉素为100%耐药。基因组关联分析发现了9个功能基因与5种抗生素(氯霉素、环丙沙星、甲氧苄啶、新霉素和四环素)存在显著相关关系,其中基因FAM000296和FAM005768与氯霉素、甲氧苄啶和环丙沙星的耐药性有关。进一步分析发现基因FAM000296和FAM005768同被注释为Sec G,但基因FAM005768与基因FAM000296相比在3′端丢失了21个碱基。【结论】分离自自然发酵食品的粪肠球菌对多种抗生素具有耐药性,其基因组中携带有潜在的耐药基因。因此,对分离自自然发酵食品的粪肠球菌需要进行全面的安全性评估后方可考虑应用。     

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

Genetic Organization of Plasmid pXF51 from the Plant Pathogen Xylella fastidiosa

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.     

Degradation of tannic acid and purification and characterization of tannase from Enterococcus faecalis

Tannins, present in various foods, feeds and forages, have anti-nutritional activity; however, presence of tannase in microorganisms inhabiting rumen and gastrointestinal tract of animals results in detoxification of these tannins. The present investigation was carried out to study the degradation profile of tannins by Enterococcus faecalis and to purify tannase. E. faecalis was observed to degrade tannic acid (1.0% in minimal media) to gallic acid, pyrogallol and resorcinol. Tannase from E. faecalis was purified up to 18.7 folds, with a recovery of 41.7%, using ammonium sulphate precipitation, followed by DEAE-cellulose and Sephadex G-150. The 45 kDa protein had an optimum activity at 40 °C and pH 6.0 at substrate concentration of 0.25 mM methyl gallate.     

Sequence and Gene Expression Analyses of Plasmid pHPM8 from Helicobacter pylori Reveal the Presence of Two Operons with Putative Roles in Plasmid Replication and Antibiotic Activity

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.     

Characterization and Sequence Analysis of the Replicator Region of the Novel Plasmid pALC1 from Paracoccus alcaliphilus

The replicator region of a low-copy-number plasmid, pALC1, of Paracoccus alcaliphilus JCM 7364 was cloned in a form of the minireplicon pALC100 (3.6 kb). The host range of the minireplicon embraces several species of genus Paracoccus, as well as Agrobacterium tumefaciens, Rhizobium leguminosarum, and Rhodobacter sphaeroides (all belonging to alpha-Proteobacteria), but not Escherichia coli. The complete nucleotide sequence of the replicator region (2276 bp) revealed the presence of one complete open reading frame coding for the 28.4-kDa protein (RepA) with similarity to replication proteins of plasmid pSW500 of Erwinia stewartii and pVS1 of Pseudomonas fluorescens. The iteron-like region was identified upstream of the repA gene and consisted of two clusters of repeated sequences (17 bp long) separated by a putative DnaA box. Analysis of the predicted amino acid sequence of two adjacent incomplete ORFs suggests the localization of repA between genes involved in conjugation (traG) and partitioning (parA) within the pALC1 genome.     

粪肠球菌MG 2108对小鼠结肠炎症的缓解作用及机制研究

【目的】为了筛选能抑制鼠类柠檬酸杆菌(Citrobacter rodentium)诱发的小鼠结肠炎的益生菌,并研究其干预机制。【方法】对4株筛选的菌株进行人工模拟胃肠液耐受试验,并体外测试它们对鼠类柠檬酸杆菌的抑制能力,最终筛选出粪肠球菌(Enterococcus faecalis)MG 2108。72只雄性7周龄ICR小鼠经过适应性饲养7d后,被随机分为2组：正常对照组(MC组,24只,生理盐水)和炎症对照组(IC组,48只,1×10¹⁰CFU/mL灌胃鼠类柠檬酸杆菌),7d后各采12只小鼠,通过结肠组织HE染色和炎症因子检测实验,判断炎症模型建成。原MC组(剩下12只小鼠)更名为NC组,用以区别建模前后的正常对照组,IC组随机分成3组：自然恢复组(IR组,12只,生理盐水)、环丙沙星组(CF组,12只,4mg/mL环丙沙星)和粪肠球菌MG 2108组(EF组,12只,1×10⁸CFU/mL粪肠球菌MG 2108)。18d后结束灌胃,所有小鼠麻醉后眼球取血,解剖。【结果】粪肠球菌MG 2108可以缓解和修复鼠类柠檬酸杆菌引发的小鼠结肠和肝脏损伤,并且通过降低炎症细胞因子的表达水平和增加紧密连接蛋白的表达水平,促进了结肠炎症组织的修复。它改变了肠道微生物菌群结构,EF组的肠杆菌属(Enterorhabdus)和阿克曼菌属(Akkermansia)等有益菌群的丰度增加,同时短链脂肪酸也显著增加(P<0.05),并且优于CF组和IR组。【结论】粪肠球菌MG2108是一株有利于肠道健康的益生菌,治疗鼠类柠檬酸杆菌诱导的小鼠结肠炎效果优于环丙沙星,自然恢复组效果明显差于EF组。     

Sequence Analysis of the Plasmid pRRI2 from the Rumen Bacterium Prevotella ruminicola 223/M2/7 and the Use of pRRI2 in Prevotella/Bacteroides Shuttle Vectors

pRRI2 is a small cryptic plasmid from the rumen bacterium Prevotella ruminicola 223/M2/7 which has been used for the construction of shuttle vectors (pRH3 and pRRI207) that replicate in many Bacteroides/Prevotella strains as well as in Escherichia coli. Sequence analysis of pRRI2 reveals that it is a 3240-bp plasmid carrying two clear open reading frames. Rep, encoded by ORF1, shows 48 and 47% amino acid sequence identity with RepA proteins from Bacteroides vulgatus and Bacteroides fragilis, respectively. ORF2, named Pre, shares 34% amino acid sequence identity with a putative plasmid recombination protein from the Flavobacterium spp. plasmid pFL1 and 30% amino acid sequence identity with BmpH from B. fragilis Tn5520. Disruption of ORF1 with HindIII prevents replication and maintenance in Bacteroides spp. hosts, but shuttle vectors carrying pRRI2 interrupted within ORF2, by EcoRI*, are able to replicate. pRRI2 shows no significant similarity with the only other P. ruminicola plasmid to have been studied previously, pRAM4.     

High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation

Summary A highly efficient electroporation system for Enterococcus faecalis was developed by systematically optimizing different parameters. One parameter found to be particularly critical for electroporation was cultivation of E. faecalis in medium containing a high glycine concentration, prior to electroporation. Osmotic stabilization of cells with 0.5 M sucrose was also found to be critical during glycine treatment. 10⁶ transformants per microgram of plasmid DNA were consistently obtained within 48 h. Electrocompetent preparations of E. faecalis could be stored at –70° C without loss of competence.     

Nucleotide Sequence and Analysis of pBL1, a Bacteriocin-Producing Plasmid from Lactococcus lactis IPLA 972

The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp. lactis IPLA 972 has been determined. Thirteen ORFs were encountered, of which 5 were incomplete. pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp. The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences. As in other plasmids, the product of orfX was not necessary for plasmid replication. However, it was shown to be involved in plasmid stability. Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region. Upstream of the origin of replication a nicking site (oriT) was found. This oriT sequence proved to be functional by mobilization of plasmids wearing it. One complete and several partial IS elements were identified on pBL1.     

Conjugative transfer of plasmid pTd33 in agrobacteria

Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy. The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein. Pretreatment of agrobacterial cells with acetosyringone resulted in a six-to tenfold increase in the transfer frequency of plasmid pTd33 at 19–25°C and had almost no effect at 30°C. The transfer of plasmid pTd33 fromA. tumefaciens strain GV3101 to plasmid-freeA. tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells. The transfer efficiency of plasmid pTd33 fromA. tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-freeA. tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-typeA. tumefaciens strain GV3101. Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26. The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

香坊肠球菌和罗伊氏乳杆菌在无菌猪肠道中的定殖

【背景】已有研究表明,微生物在宿主肠道中的定殖受宿主、肠道环境、微生物物种特性和菌株来源等多个因素的影响。一般认为,来源于同类宿主的微生物菌株,在该类宿主肠道中具有定殖优势,但缺乏在物种和菌株水平上研究微生物自身特性在宿主肠道中定殖的研究报道。【目的】将不同来源(同类宿主肠道、非同类宿主肠道和非肠道环境)、具有不同生物学特性的3株香坊肠球菌(Enterococcus xiangfangensis)和4株罗伊氏乳杆菌(Lactobacillusreuteri)对无菌猪肠道进行定殖,在物种和菌株2个水平上探究物种特性和菌株来源对宿主肠道定殖的偏好性,揭示影响微生物定殖效率的关键因素。【方法】在本项研究中,将从藏猪(Tibetan pigs)、小鼠(ob/ob mice)、食蟹猴(Macaca fascicularis)和发酵食品中分离得到的多株香坊肠球菌和罗伊氏乳杆菌,制成混合菌剂对无菌巴马香猪(Bama miniature pig)进行为期4周的饲喂,并通过实时荧光定量PCR方法检测这7株菌在无菌猪肠道中的定殖情况。【结果】在物种水平上,香坊肠球菌和罗伊氏乳杆菌在无菌猪体内具有相近的定殖...     

污水厂产超广谱β内酰胺酶大肠杆菌通过接合水平传递耐药性

【目的】研究废水中产超广谱β-内酰胺酶大肠杆菌中可移动质粒在耐药基因水平传播机制中的作用。【方法】对污水厂分离所得的50株产ESBLs大肠杆菌进行接合试验,并对所得的接合子采用纸片扩散法测定其对15种常见药物的耐药表型,针对质粒介导的产ESBLs菌株的耐药基因设计7对特异性引物对接合子进行PCR扩增。【结果】研究结果显示,80份水样分离得50株产ESBLs大肠杆菌,共接合成功35株细菌,接合成功率高达70%。接合子与供体菌相比,均发生耐药谱型的改变,且存在丢失一种或几种药物耐药性且产生另一种或几种药物耐药性的现象。PCR扩增结果显示,接合子与供体菌相比,耐药基因型有所减少或不变,bla_(TEM)、bla_(CTX-M)基因全部接合成功,bla_(SHV)基因仅1株未接合成功,耐氟喹诺酮类基因未发生转移。【结论】本研究表明,不同的耐药基因可能位于不同的可移动质粒上,可移动质粒在大肠杆菌耐药性水平传播的过程中起到了十分重要的作用。     

Complete Sequence of Plasmid pLH1 from Lactobacillus helveticus ATCC15009: Analysis Reveals the Presence of Regions Homologous to Other Native Plasmids from the Host Strain

The complete sequence for plasmid pLH1 from Lactobacillus helveticus ATCC15009 has been determined. Analysis of the 19,360-bp primary sequence revealed a putative replication origin and initiation protein, information that could provide the basis for the construction of cloning vectors for L. helveticus. Evidence that pLH1 is theta-replicating could be deduced from the plasmid size, from the homology to the replication protein of the Bacillus natto theta-replicating plasmid pLS32, and from the identification of a putative resolvase gene (orf-195). Although 14 open reading frames capable of encoding polypeptides longer than 100 amino acids were identified, none, on the basis of homology with known sequences, appeared to encode a well-characterized trait relevant to milk fermentation. Plasmid pLH1 revealed regions of identity with the smaller cryptic plasmids (pLH2 and pLH3) from the same strain and with other tracts of DNA, including insertion sequence elements, from a variety of other lactic acid bacteria. The presence of such regions provides a basis for developing an explanation of the phenotypic variability observed in these bacteria. The plasmid also appears to possess a number of genetic elements present in other lactic acid bacterial plasmids, conservation of which would be consistent with an important functional or evolutionary role. It could be argued that the plasmid complement of L. helveticus ATCC15009 consists of parasitic entities concerned only with their own replication and survival.     

屎肠球菌对斑节对虾肠道代谢组学及炎性因子的影响

【背景】屎肠球菌(Enterococcus faecium)作为乳酸菌属的潜在益生菌,因其具有良好的生物学特性在动物养殖中应用广泛,但是屎肠球菌对肠道代谢组学的研究很少。【目的】以斑节对虾为试验动物,初步探究屎肠球菌R8对肠道代谢组学及炎性因子的影响,为屎肠球菌作为益生菌在对虾养殖中的应用提供理论基础。【方法】将400条斑节对虾随机分配,设计饲料中屎肠球菌添加量分别为107、108、109 CFU/g的3个试验组,不添加屎肠球菌为对照组进行试验,养殖周期为28 d。养殖周期结束后测定斑节对虾免疫球蛋白M (immunoglobulin M,IgM)、对虾酚氧化酶(phenoloxidase,PO)、白介素6 (interleukin 6,IL-6)、补体片段3a (complement fragment 3a,C3a)的活性含量并运用LC-MS代谢组学研究肠道内源代谢物的变化,分析差异代谢物和相关的代谢通路。【结果】添加屎肠球菌对斑节对虾炎性因子有积极的影响,增加了斑节对虾体内免疫球蛋白IgM、对虾酚氧化酶、补体片段3a的含量,降低了其体内IL-6的含量,对肠道差异代谢物的分析发现,对...     

克雷伯氏菌属(Klebsiella)细菌比较基因组学分析揭示多复制子抗性质粒介导广泛的抗性基因传播

[目的] 研究克雷伯氏菌与多复制子抗性质粒间的关系,分析细菌携带多复制子质粒对抗生素环境的响应机制。[方法] 以2018-2020年分离的56株不同来源克雷伯氏菌（Klebsiella sp.）分离株为研究对象,利用微量肉汤稀释法评估其多重耐药表型,对分离菌株进行全基因组测序（WGS）,通过细菌全基因组关联分析（BGWAS）技术和比较基因组学方法深入解析多复制子抗性质粒形成的机制。[结果] 耐药表型分析发现野生动物来源的菌株具有更广的耐药谱系,总体Klebsiella sp.对氨苄西林表现出很高的耐药率（80.36%）,尤其是马来穿山甲来源菌株对头孢类抗生素高度耐受,同时对氯霉素、左氧氟沙星和复方新诺明等药物耐受,基因组分析发现这些菌株携带了抗性质粒和更多的抗生素抗性基因。进一步对69个质粒序列分析,发现有28个质粒为多复制子质粒,主要携带bla_CTX-M-15、bla_CTX-M-14、bla_CTX-M-55、bla_OXA-1和bla_TEM-1等β-内酰胺酶基因。细菌携带质粒类型分析认为Klebsiella pneumoniae可能是多复制子质粒的重要宿主,质粒骨架与结构分析发现多复制子质粒多由2个或2个以上单个质粒融合而成,携带此类质粒的菌株不仅获得了更广的耐药表型,而且在全球传播扩散分布逐年增加,因此产生对抗生素环境更强的适应性。[结论] 多重耐药性细菌呈现的表型与携带的多复制子质粒有关,相比较下多复制子质粒比非多复制子质粒有更强的抗性基因携带能力,或许是细菌在强大的抗生素压力下产生的重要响应机制。本研究对于未来探索细菌抗性基因的传播扩散机制具有重要意义。     

食品中蜡样芽孢杆菌耐药性及其机制研究进展

蜡样芽孢杆菌是常见的食源性致病菌之一,其产生的毒素会引起食物中毒。蜡样芽孢杆菌主要引起2种类型的食物中毒,即呕吐和腹泻综合征,并可造成各种局部和全身感染。随着抗生素的广泛、大量使用,蜡样芽孢杆菌的耐药性不断增强,现已有报道出现多重耐药性。本文对蜡样芽孢杆菌的耐药现状及耐药性机制进行了综述,以期正确理解蜡样芽孢杆菌耐药性的特点及其规律,从而为防治蜡样芽孢杆菌耐药性的产生及合理用药提供理论依据。