半滑舌鳎源美人鱼发光杆菌美人鱼亚种的分离鉴定

2016年5月河北省秦皇岛市昌黎县某海水养殖基地半滑舌鳎突发大批死亡,为了确定引起死亡的病原菌,无菌采集心血进行病原菌分离鉴定,16S rDNA基因序列分析与GenBank中公布的美人鱼发光杆菌参考株序列同源性为99.6%,被鉴定为美人鱼发光杆菌;进一步通过生化特性分析,被确认为美人鱼发光杆菌美人鱼亚种。动物回归试验结果表明,其对舌鳎鱼具有很强的致病性,其LD_(50)为3.1×10~4CFU/mL,且对哺乳动物具有致病性。药敏试验结果显示,分离菌对诺氟沙星、恩诺沙星、环丙沙星,乙酰甲喹高度敏感;对多西环素、阿米卡星中敏感;对庆大霉素、头孢曲松、替米考星等不敏感。     

Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.     

条石鲷(Oplegnathus fasciatus)源美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp. piscicida)的分离鉴定

2013年浙江省舟山市某网箱养殖条石鲷(Oplegnathus fasciatus)暴发了一种严重的疾病,病鱼主要症状为脾、肾出现1-2 mm的白色类结节.从患病鱼内脏处分离得到1株优势菌OF-1,经人工感染实验证实为此次引起条石鲷死亡的致病菌,半数致死量为5.93×104 CFU/g.形态学观察结果显示,菌株OF-1为革兰氏阴性、短杆状,在TCBS培养基上不生长.API 20E细菌鉴定系统、16S rRNA系统发育树分析结果证实,该菌株为美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.piscicida).该菌对庆大霉素、青霉素、氟哌酸、氧氟沙星、氨苄青霉素等药物高度敏感,对红霉素、链霉素、卡那霉素、苯唑青霉素等药物具有抗性.     

Fish photobacteriosis—The importance of rapid and accurate identification of Photobacterium damselae subsp. piscicida

MALDI‐TOF MS was tested for the identification of Photobacterium damselae subsp. piscicida on isolates grown on two media, cultured at three incubation times and applied on the target plate by the direct sample spotting (DS), by the on‐target extraction (OTE) and by the full extraction (FE) method, in triplicates. The identification of samples grown on blood agar (BA) outperformed identification on tryptic soya agar (TSA) by 0.64% for DS and OTE. The OTE gave the highest scores in both culture media, all incubation times and replicates. Reliable 24‐hr species identification was 61.54%, 84.61% and 53.85% for samples grown on TSA and identified by DS, OTE and FE, respectively. For isolates grown on BA, they were 76.92%, 96.15% and 30.77%, respectively. When identified by OTE, the 48‐hr identification was 93.58%, but for 72 hr declined to 71.79%. The reliable identification with the highest score from the first measurement was 100% only for OTE from BA (24 hr), whereas OTE from TSA gave 84.61% (24 hr), 76.92% (48 hr) and 84.61% (72 hr). The reliable MALDI‐TOF MS identification of Ph. damselae subsp. piscicida is incubation time, media, target plate preparation and replicate‐dependent.     

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida

Cobalamin (vitamin B₁₂) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B₁₂. A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B₁₂-binding protein precursor of P. profundum , 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida , but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida , the recombinant protein was expressed with a C-terminal His₆-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 ± 2 μ m .     

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus)

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour‐long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan–Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan–Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent.     

Superoxide dismutase and catalase activities in Photobacterium damselae ssp. piscicida

The ability of a set of Photobacterium damselae ssp. piscicida strains isolated from different fish species to produce different superoxide dismutase (SOD) and catalase enzymes was determined. Unlike other bacterial pathogens, P. damselae ssp. piscicida is not able to produce different isoforms of SOD or catalase containing different metal cofactors when cultured under oxidative stress induced by hydrogen peroxide or methyl viologen, or under iron depleted conditions. However, iron content of the growth medium influenced the levels of SOD and catalase activity in cells, these levels decreasing with iron availability of the medium. Comparison of virulent and non-virulent strains of P. damselae ssp. piscicida showed similar contents of SOD, but higher levels of catalase were detected in cells of the virulent strain. Incubation of bacteria with sole, Solea senegalensis (Kaup), phagocytes has shown that survival rates range from 19% to 62%, these rates being higher for the virulent strain. The increased levels of catalase activity detected in the virulent strain indicates a possible role for this enzyme in bacterial survival.     

水体常见喹诺酮类抗生素对发光菌的毒性作用

以明亮发光杆菌(Photobacterium phosphoreum)为受试生物,研究3种水体常见的喹诺酮类抗生素(QNs)对其的单一毒性和联合毒性作用,并基于毒性单位法、相加指数法和混合毒性指数法等联合作用评价方法,对混合体系的联合毒性作用类型进行分析。结果表明,不同的评价方法对3种QNs的联合效应评价结果具有较好的一致性,联合毒性作用类型均表现为不同程度的拮抗作用。根据发光菌发光原理和QNs分子结构不同,可对联合毒性作用的机理进行初步探讨。多种QNs混合体系的联合毒性作用呈现出以拮抗效应为主,揭示了此类医药品在环境中联合使用时可导致药效的降低或引发微生物耐药性的传播。     

Antibacterial compounds from marine Vibrionaceae isolated on a global expedition

On a global research expedition, over 500 bacterial strains inhibitory towards pathogenic bacteria were isolated. Three hundred of the antibacterial strains were assigned to the Vibrionaceae family. The purpose of the present study was to investigate the phylogeny and bioactivity of five Vibrionaceae strains with pronounced antibacterial activity. These were identified as Vibrio coralliilyticus (two strains), V. neptunius (two strains), and Photobacterium halotolerans (one strain) on the basis of housekeeping gene sequences. The two related V. coralliilyticus and V. neptunius strains were isolated from distant oceanic regions. Chemotyping by LC-UV/MS underlined genetic relationships by showing highly similar metabolite profiles for each of the two V. coralliilyticus and V. neptunius strains, respectively, but a unique profile for P. halotolerans. Bioassay-guided fractionation identified two known antibiotics as being responsible for the antibacterial activity; andrimid (from V. coralliilyticus) and holomycin (from P. halotolerans). Despite the isolation of already known antibiotics, our findings show that marine Vibrionaceae are a resource of antibacterial compounds and may have potential for future natural product discovery.