蜜蜂幼虫粪肠球菌的分离与鉴定

在对患有欧洲幼虫腐臭病的蜜蜂幼虫进行细菌分离与培养时,获得1株未知菌株。从分离培养特性、形态学、生理生化特性和分子生物学等方面对该菌株进行了鉴定,得知该菌株为欧洲幼虫腐臭病的次生菌——粪肠球菌,属于肠球菌属,并暂定名为Enterococcus faecalis FB102。同时,得知根据该菌的分离培养特性可与欧洲幼虫腐臭病病原区分,并且将其单独接种蜜蜂幼虫后未能使幼虫患病。根据粪肠球菌较欧洲幼虫腐臭病病原易于分离培养的特性,得知通过对粪肠球菌的鉴定可以简化欧洲幼虫腐臭病的诊断过程,而且推测该菌株可能对人体健康有一定的影响。     

中华蜜蜂的欧洲幼虫腐臭病病原研究

采用中华蜜蜂Apis cerana cerana F.临床自然发病的欧洲幼虫腐臭病(European Foulbrood, EFB)幼虫,对其病原体进行了分离鉴定。结果表明：中华蜜蜂EFB的病原系蜂房蜜蜂球菌Melissococcus pluton。该菌为革兰氏阳性的兼性厌氧菌,形态学及染色特性、致病性试验、血清学试验和细菌DNA G+C mol%试验均证明中华蜜蜂和西方蜜蜂的EFB病原属于同属的蜂房蜜蜂球菌。生理生化特性结果与国外的早期研究结果相近似。该试验为不同地理位置、不同种寄主间蜂房蜜蜂球菌的遗传差异的研究,以及中华蜜蜂EFB病的防治学研究打下基础。     

患病大鲵中弗氏柠檬酸杆菌的分离与鉴定

【目的】确定导致大鲵(Andrias davidianus)细菌性感染死亡的病原。【方法】从大鲵肝脏中分离细菌,通过Biolog微生物自动鉴定系统及分子生物学方法对纯培养的细菌进行鉴定,再用大鲵和鲫鱼分别进行人工感染试验,以确定分离菌的致病性,同时对分离到的病原菌进行药物敏感试验。【结果】从患病大鲵肝脏中分离到一株致病菌JZ01,经人工感染健康大鲵,可复制与自然发病相同的症状,且从人工感染病鲵体内再次分离到相同的病原菌。该致病菌对健康鲫鱼也有致病性。经Biolog微生物自动鉴定系统的鉴定,以及进一步的16S rDNA基因序列和系统发育分析都表明,此致病菌为弗氏柠檬酸杆菌。药物敏感性试验表明,该菌株对氨曲南、头孢三嗪、先锋噻肟等9种药物高度敏感。【结论】弗氏柠檬酸杆菌是大鲵的一种致病菌。本文在国内外首次报道了该菌对大鲵具有致病性。     

雏鸡源致病性粪肠球菌的分离鉴定及其耐药性分析

【背景】粪肠球菌(Enterococcus faecalis)是人和动物肠道正常菌群之一,也是一种条件性致病菌。近年来,粪肠球菌引起人和动物感染的报道越来越多。【目的】探明引起某养鸡场雏鸡发病死亡的病原及其致病性和有效治疗药物。【方法】结合临床症状和病理剖检,开展病原菌分离、生理生化特性检测和16S rRNA基因序列分析、致病性试验、耐药分析和对患病鸡群的药物治疗。【结果】患病鸡有昏睡、瘫痪或共济失调等临床症状;肝、脾肿大,肝脏发黄、少量出血点、质脆易碎,肠道粘膜增厚、出血,脑轻微水肿;从肝脏组织分离得到一株革兰氏阳性球菌,经纯化培养后命名为CJ517;依据该菌株形态特征、生理生化特性和16S rRNA基因序列分析鉴定为粪肠球菌;致病性试验显示CJ517菌株能致死小鼠,致死率为66.67%;该菌对头孢噻肟、磷霉素、丁胺卡那等药物敏感,对多西环素、卡那霉素、新霉素、氟苯尼考等药物耐药;经用敏感药物和提高免疫力结合治疗后,鸡群病情得到控制。【结论】研究结果可为临床诊断和治疗动物粪肠球菌感染提供参考。     

草鱼(Ctenopharyngodon idellus)烂鳃病的研究 Ⅰ.细菌性病原的研究

本文主要是报导1972年从患细菌性烂鳃的草鱼和其他鱼的鳃上分离到的病原粘细菌。通过多次人工感染(包括再分离和再感染),证明G4菌株是草鱼细菌性烂鳃病的病原菌,它能引起草、鳙、鲢、草鳙杂种、团头鲂、鲤等鱼的烂鳃病。对G4菌株的菌体、子实体、小孢子的形态、培养特性、生理生化特性作了较详细的观察和试验;根据其特征,定名为鱼害粘球菌——新种(Myxococcus piscicola Lu,Nie Ko,sp.nov.)。较详细地观察了G4菌株在胰胨液体培养中的群集习性,“柱子”形成、繁殖散发和衰老死亡的全过程。认为“柱子”形成是G4菌株,也可能是所有鱼类寄生粘细菌所共有的一种运动、繁殖的表现形式,它是不会转化为真正的子实体的。草鱼细菌性烂鳃病是危害很大的鱼病之一。其病原菌分离的成功,对有效地防治该病,控制该病的流行,提供了有利条件,同时也丰富了研究鱼类寄生粘细菌的学术内容。       

浙江1株水稻细菌性条斑病菌株的分离鉴定

细菌性条斑病(简称细条病)是水稻的重要病害之一,随着气候的变暖,某些水稻品种有逐年发生加重的趋势,对产量影响较大。2013年浙江省金华市部分水稻种植区域发生细条病,发病严重的田块减产30%以上。采用组织分离法从发病水稻叶片分离获得6株细菌菌株,选择典型菌株JH01回接水稻幼苗,进行柯赫法则验证。接种后发病症状与自然发病症状一致,并重新分离得到此菌株,证明菌株JH01为水稻细条病的致病菌。通过形态学观察、常规生理生化指标测定、16S rDNA 序列测定和同源性分析,鉴定菌株JH01为稻黄单胞菌水稻致病变种（Xanthomonas oryzae pv. oryzicola,Xoc）。     

草鱼（Ctenopharyngodon idellus）烂鳃病的研究——Ⅰ．细菌性病原的研究

本文主要是报导1972年从患细菌性烂鳃的草鱼和其他色的鳃上分离到的病原粘细菌。通过多次人工感染(包括再分离和再感染),证明G4菌株是草鱼细菌性烂鳃病的病原菌,它能引起草、鳙、鲢、草鳙杂种、团头鲂、鲤等色的烂鳃病。对G4菌株的菌体、子实体、小孢子的形态、培养特性、生理生化特性作了较详细的观察和试验;根据其特征,定名为色害粘球菌——新种(Myxococcus piscicola Lu,Nie ＆ Ko,sp.nov）。较详细地观察了G4菌株在胰胨液体培养中的群集习性,“柱子”形成、繁殖散发和衰老死亡的全过程。认为“柱子”形成是G4菌株,也可能是所有色类寄生粘细菌所共有的一种运动、繁殖的表现形式,它是不会转化为真正的子实体的。草鱼细菌性烂鳃病是危害很大的鱼病之一。其病原菌分离的成功,对有效地防治该病,控制该病的流行,提供了有利条件,同时也丰富了研究鱼类寄生粘细菌的学术内容。     

鲍氏不动杆菌——鳜鱼暴发性死亡的新病原

１９９５年广东某地人工养殖的鳜鱼暴发传染病,大量病鱼急性死亡,引致严重经济损失,本试验从已接种嗜水气单胞菌和柱状噬纤维菌疫苗的病于脏器中分离到一种细菌,经腹腔注射和腮部接种人工感染健康鳜鱼及罗非鱼,均复制出与自然发病相似的病例,主要特点为肝脏严重变性,从上述人工感染的病鱼体内再次分离与自然发病相似的病例,主要特点为肝脏严重变性,从上述人工感染的病鱼体内再次分离到与自然发病相同的细菌。该菌为革兰氏阴     

一株猪源屎肠球菌HDRsEf1益生特性研究

本研究旨在筛选一株具有优良生物特性的屎肠球菌,作为微生态制剂候选菌株;重点研究其与肠道相关的抗逆性能和对病原菌的抑菌活性。以自健康通城猪直肠内容物分离得到的屎肠球菌HDRsEf1为研究对象,通过耐受试验、抑菌试验研究该菌的益生特性。发现屎肠球菌HDRsEf1能够耐受浓度为0.5%的胆盐,并能在pH为2.0的酸中存活;对粘蛋白有显著的粘附作用(P0.05);能耐受70℃的温度。该菌发酵上清液能够抑制食源致病菌和畜禽临床主要致病菌的生长,结果表明屎肠球菌HDRsEf1可作微生态制剂菌株。     

徐闻方斑东风螺暴发性疾病病原分离鉴定以及防治手段探索

为了明确引起方斑东风螺急性死亡症的病原,对2015年6月广东省徐闻县发生的方斑东风螺(Babylonia areolata)急性死亡症进行了病原分离纯化,获得1株优势细菌,命名为XW-01。将XW-01人工感染方斑东风螺,表现出自然发病症状,证实分离菌株为致病菌。分离的菌株经形态学观察、生理生化鉴定和病原16S r RNA序列分析,结果显示该病原菌为哈维氏弧菌(Vibrio harveyi)。XW-01对方斑东风螺半致死剂量(LD50)测定值为6.3×106 cfu/m L。药敏试验结果显示,该病原菌对常见的7种抗菌药物氟哌酸、氟苯尼考、氨苄西林、恩诺沙星、头孢三嗪、左旋氧氟沙星和妥布霉素敏感。添加不同剂量的三联生物噬菌王产品到水族箱中,对方斑东风螺用浸泡法进行人工感染哈维氏弧菌试验,观察东风螺发病及死亡情况。试验结果表明,添加1%的此产品可以明显降低东风螺的死亡率,表明三联生物噬菌王产品对东风螺感染哈维氏弧菌所引起的急性坏死病有明显的预防作用。     

The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.     

Speciation in bacteria: comparison of the 16S rRNA gene for closely related Enterococcus species

Sequencing of the 16S rRNA genes from enterococcal strains used as starters suggested the existence of specialized taxa of lactic acid enterococci within the species Enterococcus durans and E. faecium and a new species, E. lactis. Comparisons showed that the 16S rRNA genes of closely related species have the same sets of variable positions with different combinations of nucleotides. The presence of identical combinations of nucleotide substitutions in different species was assumed to result from a transfer of genetic information via gene conversion between different rRNA operons. Such events were presumably associated with speciation in bacteria.     

不同抗药性水平二化螟幼虫中肠细菌群落多样性分析

为探讨二化螟Chilo suppressalis （Walker）肠道微生物多样性与抗药性的关系, 本研究采用基于16S rDNA 的变性梯度凝胶电泳(DGGE)和16S rDNA文库序列分析方法, 检测和分析了二化螟4个抗药性水平不同的种群幼虫中肠细菌群落多样性。生测结果表明, 以二化螟黑龙江种群（HLJ种群）作为相对敏感品系, 连云港种群（LYG种群）对杀虫单、 毒死蜱、 三唑磷的抗性为低抗至中抗水平, 瑞安种群（RA种群）和诸暨种群（ZJ种群）的抗性为中抗至极高抗水平, 这3个种群对阿维菌素均为敏感水平。16S rDNA文库序列分析表明, PCR 扩增得到的16S rDNA基因代表了二化螟幼虫中肠内21种细菌系统发育型, 其中大多数属于链球菌属Streptococcus。在不同抗药性水平二化螟种群中, 幼虫中肠微生物群落除ZJ种群的Lactococcus garvieae, L. lactissubsplactis和Ochrobactrum anthropic等3种菌较丰富外, 其余均以肠球菌属Enterococcus为主。DGGE 图谱显示, HLJ种群条带较为单一, LYG种群条带最为丰富, ZJ种群与RA种群条带丰富度相似。4个种群均出现Enterococcus faecium, E. hirae. 和Arthrobacter sp.等细菌, 且以肠球菌属Enterococcus为主。结果显示了不同抗药性水平的二化螟种群中肠细菌群落的丰富度存在差异, 推测可能与二化螟不同抗药性差异有关。     

Elucidation of the taxonomic status of industrial strains of thermophilic lactic acid bacteria by sequencing of 16S rRNA genes

Phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.     

粪肠、屎肠球菌及相近种部分持家基因的系统发育分析

【目的】利用16S rRNA、clpX和recA基因分子标记研究Enterococcus faecalis、Enterococcus faecium及相近种间的种系发育关系,并比较这些基因序列对E.faecalis、E.faecium及相近种的区分能力。【方法】以分离自传统乳制品中的9株E.faecium和1株E.durans分离株为研究对象,以clpX和recA基因片段为标记,通过PCR扩增、测序,结合已公布的近缘种相应序列构建系统发育树并与16S rRNA基因进行比较。【结果】在基于clpX和recA基因的进化树中,10株试验菌株与E.faecalis始终处于同一分支。与该物种这两个基因的平均相似性为99.6%和98.6%,与另一分支的Faecium-group(E.durans和E.faecium)的平均相似性仅为61.5%和33.5%。相近种E.durans和E.hirae间这两个基因的差异性为20.3%和39.0%;在基于16S rRNA基因的进化树中,试验菌株与Faecium-group(E.lactis、E.faecium、E.durans、E.hirae)处于同一分支。与这些成员间该基因的相似性大于99.6%,与E.faecalis基因的平均相似性可达98.4%。相近种间该基因相似性无明显差异。【结论】按照10株试验菌株clpX和recA基因的分析结果可将由传统生理生化和16S rRNA基因序列鉴定的9株E.faecium和1株E.durans归类为E.faecalis,clpX和recA基因可用于部分相近种的分类鉴定。     

Molecular and probiotic characterization of bacteriocin-producing Enterococcus faecium strains isolated from nonfermented animal foods

Aims:  The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as probiotics in raw and processed foodstuffs.
Methods and Results:  16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3·0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents.
Conclusions:  In our study, DNA sequencing of the 16S rRNA gene and RAPD-PCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract.
Significance and Impact of the Study:  This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.     

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.     

Lactic acid bacteria isolated from fermented flour of finger millet,its probiotic attributes and bioactive properties

This study aims to isolate and identify lactic acid bacteria from fermented flour of selected finger millet varieties grown in Sri Lanka and to evaluate their probiotic attributes and bioactive properties in vitro. Fifteen lactic acid bacteria were isolated from three varieties of fermented finger millet flour namely ravi, raavana and oshadha. These isolates were screened for phenotypical and biochemical characteristics. The selected isolates were identified by 16 S rRNA sequencing as Bacillus cereus (five strains), Streptococcus lutetiensis, Lactobacillus plantarum, Lactobacillus fermentum (two strains), Brevibacillus borstelensis, Paenibacillus species, Lactococcus lactis subspecies lactis, Enterococcus faecium, Pediococcus acidilactici, and Enterococcus lactis, and their partial sequences were deposited in GenBank. Among them, five isolates including two isolates, L. plantarum MF405176.1 and L. fermentum MF033346.1 isolated from ravi; two isolates, L. lactis MF480428.1 and E. faecium MF480431.1 isolated from raavana; and P. acidilactici MF480434.1 isolated from oshadha varieties respectively, exhibited in vitro safety attributes and could tolerate acid, gastric juice, bile, salt, phenol, and temperature under simulated gastric conditions, and also were susceptible to antibiotics tested. Further, they demonstrated bactericidal activity against both drug-sensitive and multidrug-resistant pathogens. Among the selected isolates, L. plantarum MF405176.1 demonstrated highest hydrophobicity and adhesion to both colon colorectal adenocarcinoma and colon colorectal carcinoma cell lines. L. lactis subspecies lactis MF480428.1 exhibited the highest auto-aggregation and 2, 2, diphenyl-1-pricrylhydrazyl free radical scavenging activity. P. acidilactici MF480434.1 demonstrated the lowest IC50 values against HCT-116 and HT-29 cells. None of the LAB isolates could assimilate > 10% cholesterol in vitro.     

Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci

The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.