马氏珠母贝肠道及其养殖水体可培养细菌群落结构

【目的】了解马氏珠母贝(Pinctada fucata martensii)肠道及其养殖水体可培养细菌的群落组成。【方法】采用2216E平板涂布法研究海区养殖马氏珠母贝肠道与养殖水体的可培养菌群种类及丰度。【结果与结论】马氏珠母贝肠道及其养殖水体的可培养细菌归属于2门(变形菌门和厚壁菌门)3纲7目10科23属56种。属水平上,肠道中以弧菌属(74.7%)和假交替单胞菌属(18.7%)为主;养殖水体中α-变形菌纲的FJ943236_g属(40.7%)丰度最大,弧菌属(16.7%)相对肠道丰度较低。样品共有菌属为弧菌属、假交替单胞菌属、发光杆菌属和芽孢杆菌属;肠道特有菌属为希瓦氏菌属和盐单胞菌属;养殖水体特有菌属主要为FJ943236_g、鲁杰氏菌属和Nautella。在种水平上,7个种为二者共有;马氏珠母贝肠道和养殖水体特异性菌种分别为18个和31个。虽然门水平上马氏珠母贝肠道中可培养细菌群落与其养殖水体中的细菌群落大致相似,但在属、种水平上二者差异明显。     

工厂化养殖暗纹东方鱼屯致病菌的分离和鉴定

从不同养殖场患病暗纹东方鱼屯(Takifaguobscurus)的鳃、肠、肝、肌肉及溃疡部位分离到9株菌株,经人工感染致病性试验,其T-22、T-11、J-2、H-3、H-5表现出较强的致病性,出现症状与自然发病鱼相同。通过对菌体形态特征、培养特性和API20E细菌生化快速鉴定试条的测定,分别鉴定为温和气单胞菌(Aeromonassobria)、豚鼠气单胞菌(Aeromonascaviae)、嗜水气单胞菌(Aeromonashydrophila)、类志贺邻单胞菌(Plesiomonasshigelloides)、霍乱弧菌(Vibriocholerae)。证实这5株菌为致病菌。药敏试验结果表明9种药物对温和气单胞菌、豚鼠气单胞菌、霍乱弧菌均有较好的抑菌效果;嗜水气单胞菌表现出明显的耐药性。氟嗪酸、环丙沙星、复合磺胺对类志贺邻单胞菌有一定抑菌效果。     

酵母培养物对凡纳滨对虾肠道菌群结构的影响

在基础饲料中分别添加质量分数0(对照)和0.30%的酵母培养物,配制2种等氮等脂饲料(分别记为Y0、Y0.3组),投喂初始体质量为(1.20±0.01)g的凡纳滨对虾(Litopenaeus vannamei)56 d。提取凡纳滨对虾肠道总DNA,采用Illumina Mi Seq高通量测序比较样品菌群组成和多样性,研究饲料中添加酵母培养物对凡纳滨对虾肠道菌群结构的影响。结果表明,Y0.3组对虾肠道分类单元(OTUs)数量、丰富度指数(Chao1、ACE值)、多样性指数(Shannon、np Shannon指数)与Y0组差异无统计学意义(P0.05);从物种分类树发现,两组的主要细菌类群均为变形菌门(Proteobacteria),其中,γ-变形菌纲是对虾肠道的最主要菌群,所占比例达69.48%;从科分类水平看,Y0.3组对虾肠道菌群链球菌科(Streptococcaceae)比Y0组提高4.98%;从优势属水平来看,Y0.3组对虾肠道的乳球菌属(Lactococcus)高于Y0组,差异有统计学意义(P0.01);Y0.3组对虾肠道的发光杆菌属(Photobacterium)低于Y0组,差异有统计学意义(P0.05)。饲料中添加质量分数0.30%酵母培养物可改善凡纳滨对虾肠道菌群多样性。     

噬菌弧菌N1对淡水和海水养殖水体细菌群落影响PCR-DGGE分析

【目的】研究噬菌弧菌Bacteriovorax sp. N1在一个投放周期内对淡水和海水养殖环境中细菌、弧菌总数及细菌群落结构的影响。【方法】自市售微生态制剂分离蛭弧菌N1并进行分子鉴定,测定其裂解效果,制备高浓度N1菌液分别投放至淡水红鲤鱼(red carp)和海水仿刺参(Apostichopus japonicus)养殖水体,采用细菌平板计数法及PCR-变性梯度凝胶电泳(PCR-DGGE)技术分析48 h内水体环境中细菌、弧菌总数及细菌群落结构变化。【结果】经鉴定蛭弧菌N1为噬菌弧菌,其对大肠杆菌(Escherichia coli)、溶藻弧菌(Vibrio alginolyticus)、黄海希瓦氏菌(Shewanella smarisflavi)、灿烂弧菌(Vibrio splendidus)、哈氏弧菌(Vibrio harveyi)和金黄色葡萄球菌(Staphylococcus aureus)均有裂解效果。将噬菌弧菌N1投放进淡水和海水养殖环境的12～24 h,其能显著降低两种环境中弧菌含量(P 0.05)。DGGE分析显示添加噬菌弧菌N1后淡水组优势菌群弧菌属(Vibrio,a1)和不可培养杆菌属(Uncultured bacterium, a5)在12 h以后含量有明显的减少,假单胞菌属(Pseudomonas, a3)和红杆菌科Shimia属(Shimia, a6)菌含量增加。海水组优势菌群不可培养杆菌属(c2)菌在12 h时变成非优势菌群,而白杆菌属(Albirhodobacter,c1)增加成为优势菌群。噬菌弧菌N1在水中含量在24 h时降至最低。【结论】噬菌弧菌N1对海水和淡水环境中的弧菌属和不可培养杆菌属菌群有明显的裂解作用导致其含量下降,但也使假单胞菌属(Pseudomonas),红杆菌科Shimia属和白杆菌属(Albirhodobacter)菌群含量增加,但生物效应不明。为维持噬菌弧菌N1对弧菌的控制,需要24 h左右重新补充投放。     

工厂化养殖暗纹东方鲀致病菌的分离和鉴定

从不同养殖场患病暗纹东方鲀（Takifagu obscurus）的鳃、肠、肝、肌肉及溃疡部位分离到9株菌株，经人工感染致病性试验，其T-22、T-11、J-2、H-3、H-5表现出较强的致病性，出现症状与自然发病鱼相同。通过对菌体形态特征、培养特性和API20E细菌生化快速鉴定试条的测定，分别鉴定为温和气单胞菌（Aeromonas sobria）、豚鼠气单胞菌（Aeromonas calyioe）、嗜水气单胞菌（Aeromonas hydrophila）、类志贺邻单胞菌（Plesiomonas shigelloides）、霍乱弧菌（Vrbrio cholerae）。证实这5株菌为致病菌。药敏试验结果表明9种药物对温和气单胞菌、豚鼠气单胞菌、霍乱弧菌均有较好的抑菌效果；嗜水气单胞菌表现出明显的耐药性。氟嗪酸、环丙沙星、复合磺胺对类志贺邻单胞菌有一定抑菌效果。     

近江牡蛎肠道细菌及其产酶能力

从近江牡蛎肠道中分离出21株菌,其中, 9株为革兰阳性菌, 12株革兰阴性菌。为寻找能分泌多种酶的有益微生物,研究了它们蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明,有61 .9%的菌株能分泌蛋白酶或淀粉酶(各有13株菌);有52. 4%的菌株能分泌脂肪酶(11株菌);有33. 3%的菌株能产纤维素酶(7株菌)。在这21株菌中,产4种酶的有5株菌,产3种酶的3株菌,产两种酶的5株菌,产1种酶的3株菌,不产酶的仅有5株菌。不产酶的菌株仅占细菌总数的23. 8%,而各种产酶菌则高达76. 2%,由此表明细菌在近江牡蛎对食饵消化过程的重要性。     

卵形鲳鲹结节病病原的分离与鉴定

从湛江港、阳江闸坡海水网箱养殖的患结节病卵形鲳鲹(Trachinotus ovatus)中分离一株细菌,回归感染结果证明该菌是引起卵形鲳鲹结节病的病原,对卵形鲳鲹半致死浓度为45 g-1。该菌株呈革兰阳性,好氧,具有弱抗酸性,菌体呈短杆状或细长分枝状,过氧化氢酶阳性,氧化酶阴性,还原硝酸盐,不水解酪素、黄嘌呤、酪氨酸、淀粉、明胶,以柠檬酸盐为唯一碳源生长。对其16S rDNA序列作Blast分析,构建系统进化树。结果表明,该菌株与诺卡氏菌属的菌株亲缘关系最近,且与鰤鱼诺卡氏菌(Nocardia seriolea JCM 3360T)的16S rDNA序列同源性高达99%。综合患病鱼的症状及病原菌形态、生理生化特性及16S rDNA序列的结果,该菌被鉴定为鰤鱼诺卡氏菌(N.seriolea)。     

凡纳滨对虾烂尾病病原的分离鉴定及药敏分析

【目的】研究广东湛江地区凡纳滨对虾(Litopenaeus vannamei)烂尾病主要病原及其药敏特征。【方法】从对虾病灶部位分离纯化细菌,用2株代表菌株对健康虾进行创伤浸浴感染试验,分析菌株形态特征、生理生化特征以及16S rRNA和HSP60基因序列,以哈维氏弧菌(Vibrio harveyi)和坎氏弧菌(V. campbelli)溶血素基因特异引物扩增菌株基因组DNA,通过纸片扩散法测试菌株对17种药物的敏感性。【结果】从患病亲虾和养殖对虾分别分离获得编号2018B22和2018MZ1的代表菌,两株菌16Sr RNA和HSP60基因序列均与哈维氏弧菌最相似,且均可被哈维氏弧菌溶血素基因引物特异扩增,表型特征亦与哈维氏弧菌相近;两株菌均可引起凡纳滨对虾烂尾病,其中菌株2018B22的致病力较2018MZ1更强,而后者的耐药谱更广。【结论】湛江地区凡纳滨对虾烂尾病主要病原为哈维氏弧菌。     

鰤鱼诺卡氏菌与绛红色诺卡氏菌的融合菌研究

鰤鱼诺卡氏菌(Nocardia seriolae)为鱼类诺卡氏菌病的主要病原,绛红色诺卡氏菌(N.purpurea)是分离自土壤中一种无毒诺卡氏菌。以鰤鱼诺卡氏菌和绛红色诺卡氏菌为亲本进行原生质体融合,获取融合菌。经生长特征观察、革兰染色、16SrRNA测序分析,对融合菌进行初步鉴定,并通过斑马鱼模型评估融合菌毒力检测和免疫保护效果。结果表明,融合菌的菌落形态与亲本菌株不同,为诺卡氏菌属革兰阳性菌,融合菌的毒力较鰤鱼诺卡氏菌野生株降低70%,免疫保护率约为50%。     

近江牡蛎肠道细菌及其产酶能力

从近江牡蛎肠道中分离出21株菌，其中，9株为革兰阳性菌，12株革兰阴性菌。为寻找能分泌多种酶的有益微生物，研究了它们蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明，有61．9％的菌株能分泌蛋白酶或淀粉酶(各有13株菌)；有52．4％的菌株能分泌脂肪酶(11株菌)；有33．3％的菌株能产纤维素酶(7株菌)。在这21株菌中，产4种酶的有5株菌，产3种酶的3株菌，产两种酶的5株菌，产1种酶的3株菌，不产酶的仅有5株菌。不产酶的菌株仅占细菌总数的23．8％，而各种产酶菌则高达76．2％，由此表明细菌在近江牡蛎对食饵消化过程的重要性。     

Antifouling activities of marine bacteria associated with sponge (Sigmadocia sp.)

The present study aimed at assessing the antifouling activity of bacteria associated with marine sponges. A total of eight bacterial strains were isolated from the surface of sponge Sigmadocia sp., of them, SS02, SS05 and SS06 showed inhibitory activity against biofilm-forming bacteria. The extracts of these 3 strains considerably affected the extracellular polymeric substance producing ability and adhesion of biofilm-forming bacterial strains. In addition to disc diffusion assay, microalgal settlement assay was carried out with the extracts mixed with polyurethane wood polish and coated onto stainless steel coupons. The extract of strain SS05 showed strong microalgal settlement inhibitory activity. Strain SS05 was identified as Bacillus cereus based on its 16S rRNA gene. Metabolites of the bacterial strains associated with marine invertebrates promise to be developed into environment-friendly antifouling agents.     

Isolation and characterization of AHL-degrading bacteria from fish and pond sediment

Quorum sensing(QS)disruption is considered as a potential alternative strategy to combat bacterial diseases in aquaculture.In this study,we isolated and identified bacteria degrading QS molecules from pond sediment and fish intestine.A total of 132 strains were obtained in the enrichment culture,of which two strains were identified as Enterobacter sp.f003 and Staphylococcus sp.sw120,being isolated from the fish intestine and pond sediment,respectively.We found that strains f003 and sw120 could degrade acyl-homoserine lactones(AHLs)and cause no hemolysis of sheep red blood cells.The AHL lactonase(aiiA)homologous gene in the two strains was detected in PCR amplification and the high-degrading activity to N-hexanoyl-L-homoserine lactone(C6-HSL)and AHLs secreted from pathogenic Aeromonas hydrophila was assessed.Meanwhile,the artificial infection of cyprinid Carassius auratus gibelio with intraperitoneal injection showed that the two strains were avirulent.Therefore,the obtained indigenous bacteria are candidate probiotics against pathogenic A.hydrophila in aquaculture.     

Lytic Characteristics and Identification of Two Alga-lysing Bacterial Strains

Lytic Characteristics and Identification of Two Alga-lysing Bacterial StrainsBeaulieu, S. E., M. R. Sengco, and D. M. Anderson, 2005. Using clay to control harmful algal blooms: deposition and resuspension of clay/algal flocs. Harmful Algae . (4): 123-138…     

Contribution of trimethylamine N-oxide on the growth and pressure tolerance of deep-sea bacteria

Trimethylamine N-oxide (TMAO) is widely dispersed in marine environments and plays an important role in the biogeochemical cycle of nitrogen. Diverse marine bacteria utilize TMAO as carbon and nitrogen sources or as electron acceptor in anaerobic respiration. Alteration of respiratory component according to the pressure is a common trait of deep-sea bacteria. Deep-sea bacteria from different genera harbor high hydrostatic pressure (HHP) inducible TMAO reductases that are assumed to be constitutively expressed in the deep-sea piezosphere and facilitating quick reaction to TMAO released from fish which is a potential nutrient for bacterial growth. However, whether deep-sea bacteria universally employ this strategy remains unknown. In this study, 237 bacterial strains affiliated to 23 genera of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were isolated from seawater, sediment or amphipods collected at different depths. The pressure tolerance and the utilization of TMAO were examined in 74 strains. The results demonstrated no apparent correlation between the depth where the bacteria inhabit and their pressure tolerance, regarding to our samples. Several deep-sea strains from the genera of Alteromonas, Halomonas, Marinobacter, Photobacterium, and Vibrio showed capacity of TMAO utilization, but none of the isolated Acinebacter, Bacillus, Brevundimonas, Muricauda, Novosphingobium, Rheinheimera, Sphingobium and Stenotrophomonas did, indicating the utilization of TMAO is a species-specific feature. Furthermore, we noticed that the ability of TMAO utilization varied among strains of the same species. TMAO has greater impact on the growth of deep-sea isolates of Vibrio neocaledonicus than shallow-water isolates. Taken together, the results describe for the first time the TMAO utilization in deep-sea bacterial strains, and expand our understanding of the physiological characteristic of marine bacteria.

     

Bacteriological Analysis of the Digestive Tube of the Mud Snail (Bullacta exarata Philippi) and Its Rearing Shoal

The bacterial flora in the digestive tube of Bullacta exarata Philippi and its rearing shoal were investigated. A to-tal of 157 strains of heterotrophic bacteria, isolated from crop, stomach intestine and other parts of the digestive tube,mainly belong to the genera Photobacterium, Bacillus, Pseudomonas Vibrio and some genera of the family Enterobacteri-aceae. There are significantly more varieties of bacteria in crop than in stomach and intestine. A total of 173 strains of bacte-ria were isolated from the rearing shoal, belonging to 13 genera. The 5 predominant genera, such as Bacillus and Photobac-terium, are the same as those in the digestive tube, but greatly differ in percentages. The number of heterotrophic bacteriaand Vibrio in rearing shoal change in line with the alteration of the temperature, and are significantly affected by the use ofpesticides.     

Bacteriological analysis of the digestive tube of the mud snail (Bullacta exarata Philippi) and its rearing shoal

The bacterial flora in the digestive tube of Bullacta exarata Philippi and its rearing shoal were investigated. A total of 157 strains of heterotrophic bacteria, isolated from crop, stomach intestine and other parts of the digestive tube, mainly belong to the genera Photobacterium, Bacillus, Pseudomonas Vibrio and some genera of the family Enterobacteriaceae. There are significantly more varieties of bacteria in crop than in stomach and intestine. A total of 173 strains of bacteria were isolated from the rearing shoal, belonging to 13 genera. The 5 predominant genera, such as Bacillus and Photobacterium, are the same as those in the digestive tube, but greatly differ in percentages. The number of heterotrophic bacteria and Vibrio in rearing shoal change in line with the alteration of the temperature, and are significantly affected by the use of pesticides.     

异育银鲫气单胞菌病原菌鉴定和药敏试验

从患病异育银鲫体内分离到5株可疑菌株,经API 20NE细菌生化鉴定,YCS07-03和YZ07-11株鉴定率为99.2%和99.3%。注射感染中5×108 CFU.mL-1剂量组表现出较强的致病性,12 d死亡率100%。菌株在营养肉汤培养液中呈均匀混浊状;在营养琼脂平皿中呈灰白色半透明菌落;在血琼脂培养基中呈光滑灰白色菌落,YZ07-11株出现透明的β溶血环;在TCBS培养基呈黄色菌落;在SS和麦康凯培养基中形成无色透明的扁平菌落。经负染电镜观察,菌株两端圆,呈直杆状,无芽孢,极生单鞭毛,菌株大小(短径×长径)分别为1.53μm×2.71μm和0.80μm×2.12μm。菌株经生化实验鉴定为温和气单胞菌(Aeromonas sobria)和嗜水气单胞菌(Aeromonas hydrophila)。试验结果表明:菌株对丁胺卡那霉素、头孢噻肟、庆大霉素、左氟沙星、复合磺胺、氟嗪酸、洛美沙星、复方新诺明、链霉素和新霉素均产生高度敏感;对万古霉素为中度敏感;氨苄青霉素、羧苄青霉素、强力霉素和青霉素产生耐药。     

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes(protease,amylase,and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16 S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus,which can produce protease,amylase,and cellulase. Virgibacillus,however,can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.