鲟致病性类志贺邻单胞菌的鉴定及药物敏感性

【目的】2012年夏季北京地区多地养殖的鲟鱼发病,主要临床症状为肛门红肿、伴有黄色分泌物,腹腔内有大量腹水,腹腔内壁有出血点,肝脏点状出血,脾脏肿大等,累计死亡率达60%。本文目的为研究其病原。【方法】从具有临床症状的濒死鱼中分离病原菌,分析病原菌的形态特征、理化特性、分类地位及药物敏感性等特性,经过人工感染及引起的组织病理确认致病性。【结果】结果显示病原菌的16S rDNA序列构建的进化树,与类志贺邻单胞菌同源性最高,在99%以上;结合其生理生化特征和API细菌鉴定系统的结果,确认为类志贺邻单胞菌。该菌对鲟鱼的半致死量LD50为1.0×105.8CFU/mL,引起肝、肾和脾组织病变。胞外产物不具有淀粉酶、蛋白酶、脂肪酶和明胶酶活性,也无溶血性,推测其毒性可能来源于内毒素。该菌对恩诺沙星、盐酸多西环素、氟苯尼考和甲枫霉素敏感,药物敏感浓度均小于2μg/mL;而对试验的其它抗菌药物不敏感。【结论】确认类志贺邻单胞菌是引起北京地区鲟鱼发生上述临床症状疾病的主要致病菌,可优选盐酸多西环素、氟苯尼考和恩诺沙星进行防治。     

草鱼致病性类志贺邻单胞菌的分离与鉴定

【目的】分离鉴定引起草鱼(Ctenopharyngodon idellus)肌肉糜烂的类志贺邻单胞菌(Plesiomonas shigeloide)菌株JX-09。【方法】从患病草鱼分离致病菌,经形态学观察、人工感染试验、生理生化特性测定和16S rRNA基因序列分析对其进行分类鉴定和致病性检验;同时对分离到的菌株做药物敏感性试验。【结果】从回归感染后的草鱼体内再次分离到菌株JX-09,表明该菌株为致病菌;菌株JX-09对草鱼的半致死量为6.4×104cfu/g。通过生化特征和分子系统学分析,将菌株JX-09鉴定为类志贺邻单胞菌。药敏试验表明该菌株对氨曲南、头孢唑林、头孢噻吩、头孢曲松等头孢类药物敏感,对卡那霉素、麦迪霉素、万古霉素和哌拉西林等耐药。【结论】类志贺邻单胞菌是草鱼肌肉糜烂病的致病菌,这是首次报道了该菌对草鱼有致病性。     

草鱼致病性类志贺邻单胞菌的分离与鉴定

摘要:【目的】分离鉴定引起草鱼(Ctenopharyngodon idellus)肌肉糜烂的类志贺邻单胞菌(Plesiomonas shigeloide)菌株JX-09。【方法】从患病草鱼分离致病菌,经形态学观察、人工感染试验、生理生化特性测定和16S rRNA基因序列分析对其进行分类鉴定和致病性检验;同时对分离到的菌株做药物敏感性试验。【结果】从回归感染后的草鱼体内再次分离到菌株JX-09,表明该菌株为致病菌;菌株JX-09对草鱼的半致死量为6.4×104cfu/g。通过生化特征和分子系统学分析,将菌株JX-09鉴定为类志贺邻单胞菌。药敏试验表明该菌株对氨曲南、头孢唑林、头孢噻吩、头孢曲松等头孢类药物敏感,对卡那霉素、麦迪霉素、万古霉素和哌拉西林等耐药。【结论】类志贺邻单胞菌是草鱼肌肉糜烂病的致病菌,这是首次报道了该菌对草鱼有致病性。     

饮食从业人员健康体检分离到类志贺邻单胞菌

我们于 2 0 0 2年 3月从一名饮食从业人员健康体检粪便中分离到 1株类志贺邻单胞菌 (Plesiomonas shigelloid es) ,现将结果报告如下。1　材料与方法1.1　标本来源　我市一名猪肚米线馆从业人员粪便。1.2　培养基　麦康凯琼脂、三糖铁琼脂和 L B肉汤 ,购自北京陆桥技术有限责任公司。1.3　诊断用品　肠杆菌科鉴定系统 API 2 0 E试条 ,全自动微生物分析系统 VITEK3 2 NFC(非发酵菌鉴定卡 )和 GNS-50 6(革兰阴性菌药敏卡 ) ,购自法国生物梅里埃 (bio Merieux)公司 ;志贺菌诊断血清为卫生部兰州生物制品研究所生产。1.4　方法　按全国…     

类志贺邻单胞菌肠道感染（附8例临床分析）

本文通过对邻单胞菌肠道感染的临床分析,对其生物性状及致病性作了详细观察,结果１２株菌性状基本相同,符合邻单胞菌特征。药敏提示除丁胺卡那霉素和氟哌酸有较强的抗菌活性外,对其它抗生素均有不同程度耐药,动物实验表明,该菌具有较强致病性,提示本菌是临床感染中不可忽视的菌种。     

类志贺邻单胞菌7-63-5株及其多糖的研究

类志贺邻单胞菌O17血清型与宋内氏痢疾志贺氏菌的脂多糖结构一致,类志贺邻单胞菌7-63-5株属于O17血清型。实验中通过对该菌株培养特性、生化特征、免疫学特性的研究,证明其完全符合类志贺邻单胞菌特性。多糖的研究证明,类志贺邻单胞菌7-63-5株的O-特异性多糖无论从化学结构,还是免疫学特性上,都与宋内氏I相菌的一致。因此,类志贺邻单胞菌7-63-5株可以用以研究宋内氏痢疾多糖蛋白质结合疫苗。     

中华绒螯蟹组织中一株类志贺邻单胞菌的分离与特性分析

从江苏省启东市某水产养殖场患病的中华绒螯蟹组织中,分离到一株类志贺邻单胞菌,定名为JE－1株。该菌株为革兰阴性短杆菌,具端生单鞭毛,主要生化特性为氧化酶阳性,发酵葡萄糖产酸不产气,发酵麦芽糖,乳糖,肌醇等。不发酵甘露醇,鼠李糖,阿拉伯糖等;触酶,精氨酸双水解酶,鸟氨酸脱羧酶,赖氨酸脱羧酶阳性,对小鼠,鹌鹑,鲫鱼血细胞无溶血性,最适生长条件为温度28℃,pH7,含1％NaCl,经健康蟹回感,未见发病。     

急性腹泻患者类志贺邻单胞菌流行及药物敏感性分析

目的了解类志贺邻单胞菌在急性腹泻患者中的分布及对常用抗菌药物的敏感性,为疾病的预防和临床治疗提供依据。方法收集2012年6月至2013年12月期间浙江省4家医院急性腹泻患者病例,采用常规微生物检验程序对常见肠道致病菌进行分离培养及检测。分离到的类志贺邻单胞菌采用K-B法进行药物敏感性试验。结果收集1 640例病例,共分离到类志贺邻单胞菌共41株,检出率为2.5%,占所有分离菌株的7.5%,居细菌性病原第四。41例病例中,男女检出率分别为2.6%和2.4%,差异无统计学意义;患者半数以上分布在18~44岁;每月均能够分离到类志贺邻单胞菌,其中夏季(6-8月)分离率最高,达3.9%。类志贺邻单胞菌对氨苄西林耐药率高达89.3%,对阿米卡星、复方新诺明、庆大霉素耐药率分别在30%~50%,未出现对头孢哌酮-舒巴坦、哌拉西林-他唑巴坦、亚胺培南及美罗培南耐药株。结论类志贺邻单胞菌是沿海地区夏秋季腹泻较为常见的病原菌之一,是临床肠道感染中不可忽视的病原。     

一株类志贺邻单胞菌噬菌体生物学特性及全基因组分析

[目的]多重耐药菌株的出现给食品安全带来严重威胁.噬菌体是不同于抗生素的一类重要杀菌因子,对其生物学特性及基因组的研究和分析可为噬菌体的抗菌应用提供依据.[方法]对噬菌体phiP4-7的生物学特性、基因组学、分类学进行研究.[结果]经透射电子显微镜观察,确定phiP4-7头部直径为(50.59±1.68) nm,尾部长...     

尼罗罗非鱼和萨罗罗非鱼遗传生殖隔离的初步证据(英文)

罗非鱼类(Tilapiini)含3个属70余种,种间和属间颇易人工杂交,但尼罗罗非鱼(Oreochromis niloticus)和萨罗罗非鱼(Sarotherodon melanotheron)人工杂交难度大,产苗概率甚低,要获得数量足够的可用于生产的杂交子代相当困难。该文对这两种鱼及其正交(O.niloticus♀×S.melanotheron♂)和反交(S.melanotheron♀×O.niloticus♂)子代的头肾细胞的核型进行了比较。此外,采用同工酶电泳方法检测肾、肝、眼、肌肉、心中乳酸脱氢酶等4种同工酶的表型差异。4种遗传型罗非鱼具有相同的染色体二倍数(2n=44)和总臂数(NF=50),但各具不同的染色体类型,尼罗罗非鱼为3对近中着丝点染色体(sm)、12对近端着丝点染色体(st)和7对端着丝点染色体(t);萨罗罗非鱼为1对中间着丝点染色体(m)、2对sm、12对st和7对t;正反杂交子代表现为介于双亲之间的混合类型,为0.5对m、2.5对sm、12对st和7对t。在同工酶中,仅见肾脏乳酸脱氢酶电泳结果有清晰差异,尼罗罗非鱼出现5条谱带,萨罗罗非鱼3条,而杂交子代6条,且所有谱带的迁移率和活性均表现出多态性。据此初步认为,核型和同工酶方面的差异可能是导致这两种不同属罗非鱼生殖隔离的遗传原因,这些差异也可能为这两种(属)鱼的分类学提供新的遗传背景资料。     

Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.     

Invasive phenotype and apoptosis induction of Plesiomonas shigelloides P-1 strain to Caco-2 cells

AIMS: The mechanism of the host cell invasion of Plesiomonas shigelloides and its capability to induce apoptosis were investigated. METHODS AND RESULTS: We performed a time course experiment on the bacterial adherence and invasion of the P. shigelloides P-1 strain into Caco-2 cells using an invasion assay and flow cytometry. The adherence of P. shigelloides to the Caco-2 cells was almost completed within 10 min after the infection. Thereafter, P. shigelloides starts internalization within the Caco-2 cells, which was completed within 60 min after the infection. Based on the invasion assay using nocodazole, cytochalasin D, and genistein, it became clear that the mechanism of the internalization depended on the signal transduction followed by the rearrangement of the cytoskeletal protein. Based on the DNA laddering and TUNEL methods, the cytotoxicity of the Caco-2 cells by the invasion of P. shigelloides occurred through the induction of apoptosis. CONCLUSIONS: This work demonstrated that the mechanism of invasion of P. shigelloides into Caco-2 cells and the invasion of P. shigelloides induces apoptotic cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the virulence factor, which may be important for understanding of the pathogenesis of P. shigelloides.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Primary structure and function of a cytotoxic outer-membrane protein (ComP) of Plesiomonas shigelloides

We previously isolated and characterized a 40-kDa cytotoxic outer-membrane protein (ComP) produced by Plesiomonas shigelloides strain P-1 (P-1). Sequence analysis of the comP gene revealed a coding region of 1068 bp, with a predicted mature protein composed of 335 amino acids and a molecular mass of 38.597 kDa. Three-dimensional structural modeling of ComP suggests that it has a beta-barrel structure with 16 transmembrane strands, eight short periplasmic turns and eight external loops. blast search results and protein modeling suggest that ComP may be a novel porin protein of P. shigelloides. In order to understand the role of ComP during P. shigelloides infection, we constructed a deletion mutant strain (P. shigelloides DeltacomP; P-1201), and compared the pathogenicity of P-1201 vs. the wild-type strain P-1 in Caco-2 cells. Unlike P-1, the deletion strain P-1201 was not cytotoxic to Caco-2 cells and did not lead to apoptosis. These data indicate that ComP may be the predominant virulence factor that triggers cell death in the host cells following infection.     

Structure of the lipid A-inner core region and biological activity of Plesiomonas shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of beta-d-GlcpN4PPEtn-(1-->6)-alpha-d-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.     

Homogeneously sized groups increase aggressive interaction and affect social stress in Thai strain Nile tilapia (Oreochromis niloticus)

Social fish raised in farms are usually kept in groups of similar-sized individuals. However, social animals of similar size typically have similar fighting ability, which increases aggressive interaction for social rank establishment, as well as social stress. We compared Thai strain Nile tilapia fish, Oreochromis niloticus (L.), held under two treatments: (1) The Homogeneous one, with five adult male fish of similar size and (2) the Heterogeneous treatment with five adult males of different sizes. We recorded the frequency of aggressive interactions and checked social stability and stress levels (cortisol) after five days in the groups. Grouping similar sized Thai Nile tilapia increased the aggressive interactions and delayed rank stability with increased body injuries as a consequence. Homogeneous-sized individuals showed a similar level of stress while heterogeneous-sized individuals showed different stress levels with dominants being more stressed than subordinates. The data indicate that the practice of selecting fish of similar size in aquaculture management could reduce the welfare of social fish and that the effect is observed in different lineages.     

Effects of the pyriproxyfen and fenthion on the gonadal morphology of Nile tilapia (Oreochromis niloticus: Perciformes,Cichlidae)

Commercially available insecticides present acute toxicity to the health of fish and other aquatic organisms, which may impair the local aquaculture. This study evaluated the gonadal morphology of freshwater fish exposed to pyriproxyfen and fenthion. Forty-five juvenile male Nile tilapias (Oreochromis niloticus) were divided into control, pyriproxyfen-exposed (0.01 g/L), and fenthion-exposed (0.001 g/L) groups. They were evaluated in three moments (30, 60, and 90 days). The variables analyzed were the gonadosomatic index (GSI), weight to length ratio, seminiferous tubules morphometry (diameter and height), tissue damage, and immunohistochemical analysis for caspase-3, tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF). Pyriproxyfen and fenthion injured the seminiferous tubule tissue, and the damage progressed according to the exposure time. In addition, the GSI gradually reduced over time in all groups compared with the first moment (30 days), while caspase-3, TNF-α, and VEGF values increased only in the fenthion-exposed group. Therefore, pyriproxyfen and fenthion changed the gonadal morphology of male Oreochromis niloticus, which may affect their reproduction in the wild or captivity.     

Bone development in the jaw of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae)

East African cichlids have evolved feeding apparatus morphologies adapted to their diverse feeding behaviors. The evolution of the oral jaw morphologies is accomplished by the diversity of bone formation during development. To further understand this evolutionary process, we examined the skeletal elements of the jaw and their temporal and sequential emergence, categorized by developmental stages, using the Nile tilapia Oreochromis niloticus as a model cichlid. We found that chondrogenesis started in Stage 17. The deposition of osteoid for the dermal bones commenced in Stage 18. The uptake of calcium dramatically shifted from the surface of larvae to the gills in Stage 20. The bone mineralization of the skeleton began in Stage 25. These data provide important information regarding the sequential events of craniofacial development in East African cichlids and lay the groundwork for studying the molecular mechanisms underlying adaptation of jaw structure to feeding behavior.