An outbreak of Shewanella putrefaciens group in wild eels Anguilla anguilla L. favoured by hypoxic aquatic environments

Microbiological analyses were conducted on wild eels from the L′Albufera Lake (Spain). A total of 174 individuals were collected in two surveys (i.e. year 2008 and autumn–winter 2014) among those caught by local fishermen into the lagoon. The prevalence of Shewanella putrefaciens group was 1.7% in 2008 and rose above 32% in 2014. It was due to an outbreak of shewanellosis that presented a morbidity rate of 64%. S. putrefaciens group strains were isolated as pure cultures from the sick eels that showed white ulcers surrounded by a reddish inflammation, damage of the mouth, extensive skin discoloration, exophthalmia, ascites and bad odour. The S. putrefaciens group was recovered from freshwater samples taken at the L′Albufera system, along autumn–winter 2015. Its counts significantly increased in freshwater parallel to hypoxia and temperature rising. Shewanellae strains were identified as S. putrefaciens and S. xiamenensis by 16S rRNA gene sequencing. These isolates recovered from sick eels or freshwater were virulent for European eel by IP challenge (LD₅₀ 10⁶ CFU g^?1 body weight). They also caused 30–38% cumulative mortality, in European eels challenged by a 2‐h bath (10⁷ CFU mL^?1). These results suggest that shewanellosis could be transmitted through water highlighting the fact that hypoxic conditions increase this bacterium levels in water.     

抗菌药联用对海藻希瓦菌的体外抗菌效果

为评价硫酸庆大霉素和头孢噻肟钠对水产致病性海藻希瓦菌SFH3的体外联合抗菌效果,以6种渔用抗菌药作为对照,比较测定硫酸庆大霉素、头孢噻肟钠对海藻希瓦菌SFH3的体外抗菌作用,在此基础上进一步采用试管二倍稀释法测定分析硫酸庆大霉素和头孢噻肟钠在不同复配比例下对海藻希瓦菌SFH3的体外联合抑菌作用,并通过时间-杀菌曲线法测定其体外联合杀菌效果。试验结果显示,硫酸庆大霉素和头孢噻肟钠对海藻希瓦菌SFH3的最小抑菌质量浓度为80 mg/L和160 mg/L,抗菌活性明显优于甲砜霉素、多西环素、噁喹酸、磺胺间甲氧嘧啶钠、磺胺甲噁唑、盐酸恩诺沙星等渔用抗菌药。硫酸庆大霉素和头孢噻肟钠在复配比例8∶2和7∶3时,对海藻希瓦菌SFH3的体外联合抑菌效果表现为协同作用,对海藻希瓦菌SFH3的最小抑菌质量浓度达40 mg/L。此外,时间-杀菌曲线表明,40 mg/L硫酸庆大霉素和80 mg/L头孢噻肟钠对海藻希瓦菌SFH3的体外联合杀菌作用表现为协同效果。本研究结果证实,硫酸庆大霉素和头孢噻肟钠联用有利于提升对海藻希瓦菌SFH3的体外抗菌效果,可为研发水产致病性海藻希瓦菌的潜在控制药物提供理论参考。     

一株腐败希瓦氏菌的筛选鉴定及其染料脱色研究

[目的]研究一株腐败希瓦氏菌对染料的脱色效果。[方法]从印染废水中筛选到一株对偶氮染料活性艳红X-3B和蒽醌染料活性艳蓝KN-R均具有高效降解作用的菌株,对其进行了菌种鉴定,并分析了该菌在不同培养条件下对两种染料的脱色效果。[结果]生理生化鉴定和16S r DNA序列鉴定结果表明,该菌为希瓦氏菌属腐败希瓦氏菌,命名为S.putrefaciens 4H。该菌在pH=8、37℃下静置培养时,对两种染料的脱色效果最好。该菌6 h内可将50 mg/L活性艳红X-3B完全降解脱色,在含有活性艳红X-3B的LB平板上可形成水解圈,推测其能产生水解活性艳红X-3B的胞外酶;该菌对50 mg/L活性艳蓝KN-R的脱色率48 h时可达81.84%,且脱色时染料先被细胞吸附沉淀后再随着细胞的生长代谢逐渐降解,其脱色机制还有待研究。[结论]该研究为印染废水的处理提供了理论依据。     

复合生物保鲜剂对腐败希瓦氏菌的抑菌机理

为了研究复合生物保鲜剂对冷藏带鱼优势腐败菌(腐败希瓦氏菌)的抑菌作用机理,通过牛津杯法测定了复合生物保鲜剂对腐败希瓦氏菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并测定了复合生物保鲜剂对腐败希瓦氏菌的抑菌活力、细菌生长曲线、细胞膜完整性和细胞的稳定性,对细菌超微结构进行了观察。结果显示:复合生物保鲜剂对腐败希瓦氏菌的MIC与MBC分别为1.6 mg/ml与3.3 mg/ml,且随着作用时间的延长,复合生物保鲜剂对腐败希瓦氏菌的生长有明显抑制作用;尤其经2倍最小杀菌浓度的复合生物保鲜剂处理后,细菌未出现对数生长期;菌液在260 nm处的吸光值明显升高,菌体细胞膜完整性受到破坏;菌体细胞中的碱性磷酸酶升高,菌体细胞壁通透性增大;菌悬液的电导率值明显增大,细胞膜稳定性受损。细菌超微结构观察发现,复合生物保鲜剂作用于菌体细胞后,菌体细胞开始出现皱缩、扭曲变形、表面粗糙和布满泡状物及细胞壁塌陷等现象。表明复合生物保鲜剂可以破坏细菌的细胞内环境和细胞膜稳定性,影响了细菌的正常生长;并且通过影响细菌对营养物质的吸收和代谢物的排出,最终导致菌体死亡。     

Phenotypical and genotypical characterization of Shewanella putrefaciens strains isolated from diseased freshwater fish

Between 2007 and 2012, a variety of disease outbreaks most often characterized by skin disorders were observed among different species of freshwater fish in Poland. In most cases, the clinical signs included focally necrotized gills, necrotic skin lesions or ulcers. Internally, haemorrhages, oedematous kidney and abnormal spleen enlargement were generally noted. The disorders were accompanied by increased mortality. Most of the problems concerned cultured common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss (Walbaum). Fish have been examined from a number of these farms, and additionally, the wild and ornamental fish with similar clinical signs of diseases were also tested. Bacteria were isolated consistently from lesions and internal organs. They had characteristic orange-pigmented colonies which grew in pure culture or constituted 55–95% of total bacterial flora. One hundred and eighteen isolates were collected and biochemically identified as Shewanella putrefaciens group, and this was confirmed by sequencing. Challenge tests confirmed the pathogenicity of these bacteria. This is the first report characterizing and describing S. putrefaciens as a pathogen of different species of freshwater fish in Europe.     

冷藏大黄鱼SSO希瓦氏菌致腐能力差异机制初探

为探讨特定腐败菌(SSO)希瓦氏菌(Shewanella)致腐能力的差异机制,采用生化和16S rDNA鉴定冷藏大黄鱼货架期终点的产H2S菌,在灭菌鱼汁和无菌鱼块筛选4株致腐差异的希瓦氏菌,扩增氧化三甲胺(TMAO)还原酶基因及分析其表达量,并预测其蛋白质的理化性质。结果显示,22株产H2S菌均为希瓦氏菌属,其中 S.baltica占54.5%,为S. putrefaciens占40.9%,S. hafniensis占4.5%。希瓦氏菌在灭菌鱼汁中致腐能力存在显著差异,其中S. balticaXH2和XH8的缺点评分和TVB-N最高,S. putrefaciensXH14和XH17菌最低。接种无菌鱼块的4株希瓦氏菌中XH2的样品在72h出现腐败气味,48h细菌高于107cfu/g,产生较高的TMA、TVB-N、尸胺和腐胺和,XH8菌次之,XH14和XH17菌最慢。四株希瓦氏菌都扩增出2490bp的torA基因,且torA基因的表达量与致腐能力密切相关,S.balticaXH2最高。预测的TorA蛋白中S.balticaXH2的分子量和不稳定指数最大,理论等电点和总平均疏水性最小。可见,S. baltica XH2为冷藏大黄鱼的SSO,其强致腐能力与torA基因高表达量和TorA蛋白理化性质有关。研究为阐明希瓦氏菌致腐机制奠定了良好的基础。     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

大黄鱼腐败菌腐败能力分析与特定腐败菌鉴别

探讨了分别接种腐败希瓦氏菌和假单胞菌的无菌大黄鱼鱼块在5℃贮藏中的生长速率和腐败代谢产物的生成速率,以产生异臭味时的腐败菌数和腐败代谢产物的产量因子作为腐败菌腐败能力的定量指标。接种鱼块腐败时(产生明显异臭味)腐败希瓦氏菌与假单胞菌的菌数即最小腐败菌数(N_s)的对数值分别为9.14lg cfu/g和9.25lgcfu/g。由Gompertz方程得到腐败希瓦氏菌和假单胞菌的生长动力学参数:最大比生长速率(μ_max)分别为0.0714和0.0700,最大菌数(N_max)的对数值分别为9.45lg cfu/g和9.37lg cfu/g。结果表明,两种腐败菌在无菌大黄鱼鱼块中的μ_max基本一致,且N_max都超过N_s,这两种腐败菌都能引起大黄鱼的腐败。接种鱼块中腐败希瓦氏菌的腐败代谢产物(TVBN和TMA)的产量因子Y_TVBN/cfu和Y_TMA/cfu分别为1.38×10^-10mg-N TVBN/cfu和1.31×10^-10mg-N TMA/cfu,假单胞菌的腐败代谢产物(TVBN)的产量因子Y_TVBN/cfu为1.18×10^-10mg-N TVBN/cfu,比较产量因子可得,两菌的腐败能力相当。因为大黄鱼低温贮藏中的优势腐败菌是腐败希瓦氏菌,其次是假单胞菌,根据特定腐败菌的概念,大黄鱼低温贮藏过程中的特定腐败菌是腐败希瓦氏菌,其次是假单胞菌。     

微生物燃料电池对磺胺间甲氧嘧啶的降解研究

为探究微生物燃料电池(Microbial fuel cells, MFCs)对抗生素磺胺间甲氧嘧啶(Sulfamonomethoxine,SMM)的降解效果及MFCs的产电性能,通过构建单室MFCs,比较不同电极材料、菌种种类、抗生素初始浓度以及腐植酸存在条件下MFCs对SMM的降解效果及其产电性能。结果表明:3种阳极材料下MFCs对SMM的降解率及产电性能的高低为碳毡碳纸石墨棒;在SMM初始浓度为10 mg·L~(-1)的条件下,以Shewanella putrefaciens为菌种的MFCs对SMM的降解率达到58.92%,高于Shewanella oneidensis MR-1的降解率46.48%,MFCs的最大输出功率前者比后者约高6.51 mW·m~(-2);随着抗生素初始浓度的增加,SMM的降解效果逐渐减弱,MFCs的电压输出逐渐降低;随着腐植酸浓度增加,SMM的降解率逐渐提高,MFCs的产电性能逐渐增强。研究表明,MFCs可以利用SMM作为燃料,在实现降解的同时输出电能,这为水体环境中磺胺类抗生素的高效低耗处理提供了科学依据。     

Genomic and phylogenetic characterization of Shewanella xiamenensis isolated from giant grouper (Epinephelus lanceolatus) in Taiwan

Shewanella xiamenensis is an emerging pathogen causing intra‐abdominal infection and intestinal colonization. Epidemiologic clues suggest its role as a potential food‐borne zoonotic agent. To date, four genome sequences of S. xiamenensis have been made publicly available. All of them were isolated from water samples. In this study, we characterized the genome of a S. xiamenensis strain isolated from a giant grouper in Taiwan. The genome of S. xiamenensis ZYW1 is 4,827,717 bp in length and encodes 4,239 open reading frames. Its genomic sequence shares high homology with other S. xiamenensis strains. bla_OXA‐416 was identified. This is the first detection of S. xiamenensis in Taiwan. These genomic data and analyses contribute to our understanding of S. xiamenensis and may help to elucidate disease‐causing mechanisms in future studies.