In vitro antibacterial effect of berberine hydrochloride and enrofloxacin to fish pathogenic bacteria

Berberine the main antibacterial substance in the rhizome of Coptis chinensis Franch, is used as an antimicrobial agent for a long time. This study was carried out to investigate the potential of berberine alone or in combination with enrofloxacin against six common fish pathogens for use in fish disease management in aquaculture. The minimum inhibitory concentrations (MICs) of berberine hydrochloride against Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio vulnificus, Edwardsiella ictaluri, Escherichia coli and Streptococcus agalactiae were determined and to be >500, >500, >500, 300, 400 and 100 μg mL^?1 respectively. The minimal bactericidal concentrations (MBCs) of berberine hydrochloride to E. coli, E. ictaluri and S. agalactiae were 300–500 μg mL^?1. In combination with enrofloxacin, the MICs and MBCs of berberine hydrochloride significantly decreased against E. coli and E. ictaluri but not against Streptococcus dysgalactiae. These results demonstrated that the berberine hydrochloride enhanced the bactericidal effect of enrofloxacin and vice versa. The synergistic bactericidal effect of berberine hydrochloride and enrofloxacin suggest its potential use in fish disease management in aquaculture. This is the first study on the inhibitory effect of berberine hydrochloride against fish pathogenic bacteria.     

In vitro and in vivo antibacterial activity of shrimp chitosan against some intestinal bacteria

ABSTRACT:   The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus , and six strains of Bifidobacterium were evaluated. In vitro , the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C .  perfringens . The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

海水鱼类细菌性疾病病原及其检测、疫苗研究概况

概述了海水鱼类常见细菌性疾病包括弧菌病、冬季溃疡病、巴斯德氏菌病、疖病、海水屈挠杆菌病、假单胞菌病、链球菌病、细菌性肾病的病原研究及其检测、疫苗研究概况,为及时了解当前细菌性疾病的研究进展及发展趋势以采取更有效的措施防治疾病提供参考依据。     

Bactericidal activity of cecropin B and cecropin P1 expressed in fish cells (CHSE-214): application in controlling fish bacterial pathogens

Cecropins are a group of antimicrobial peptides which have bactericidal activity against a broad range of bacteria. To date, the cecropins used in a variety of studies were either purified from their natural source or obtained by chemical synthesis. The present study was conducted to test whether bactericidally active cecropins could be expressed in a fish cell line. For this purpose, Chinook salmon embryo cells (CHSE-214) were transfected with cecropin transgene constructs: Hyalophora cecropia preprocecropin B, procecropin B, cecropin B, and porcine P₁ cecropin. From the transfected cells, single cell clones were selected and screened for the presence of cecropin gene constructs by PCR amplification. The expression of the cecropin transgene in the PCR positive clones was determined by RT-PCR reaction. Southern blot hybridization results showed that the cecropin gene constructs were integrated into the genome in a multiple integration pattern. Bactericidal activity of the cecropins, synthesized from transgene constructs, was detected using inhibition zone assay for fish pathogenic bacteria: Aeromonas hydrophila, Pseudomonas fluorescens, and Vibrio anguillarum. Cecropin antimicrobial peptides produced in CHSE-214 cells possess bactericidal activity against these three fish pathogenic bacteria.     

苍术和番石榴的不同炮制方法及部位对水产病原菌抑制效果的影响

采用微量稀释法,以溶珊瑚弧菌（Vibrio coralliilyticus）、哈维弧菌（旷harveyi）、美人鱼发光杆菌杀鱼亚种（Photobacterium damselae subsp piscicida）、副溶血弧菌（V.parahaemolyticus）、摩氏摩根氏菌（Morganellafulton）、创伤弧菌（V.vulnificus）6种病原菌共7株为受试菌,研究苍术（Atractylodeslancea）不同炮制品和番石榴（Psidiumguajava）不同部位的抑菌作用,并进一步测定其最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。结果显示,番石榴叶和枝果部位以及苍术不同炮制品对溶珊瑚弧菌、美人鱼发光杆菌杀鱼亚种和哈维弧菌抑菌效果较明显,MIC和MBC分别为10^-3g·mL^-1和10^-1g·mL^-1,抑菌效果由高至低依次为番石榴叶〉番石榴果〉番石榴枝,麸炒苍术〉盐炙苍术〉苍术〉醋炙苍术。     

水产品中4种常见致病菌多重PCR检测方法的建立及评价

根据金黄色葡萄球菌的耐热核酸酶基因nuc、沙门氏菌的侵袭蛋白基因invA、志贺氏菌的侵染性质粒抗原H基因ipaH和副溶血性弧菌的毒力表达调控基因toxR设计引物,建立一种快速检测水产品中上述4种常见食源性致病菌的多重PCR方法。结果显示,沙门氏菌、金黄色葡萄球菌、志贺氏菌及副溶血性弧菌的多重PCR扩增片段产物大小分别为549、426、348和243 bp,该检测方法具有良好的特异性和灵敏度。设计优化了可用于水产样品中4种目标致病菌的共增菌培养基,该方法应用于35份实际水产样品的检测,并与国标方法对比验证,4种致病菌的整体符合率达94%以上,两种检测方法无显著性差异(P>0.05)。可见,该多重PCR方法可应用于水产品中沙门氏菌、金黄色葡萄球菌、副溶血性弧菌及志贺氏菌4种食源性致病菌的快速检测和分子流行病学调查。     

Lactic acid bacteria in fish: a review

Fish are continuously exposed to a wide range of microorganisms present in the environment, and the microbiota of fish have been the subject of several reviews. This review evaluates lactic acid bacteria in fish, and focuses on the several investigations that have demonstrated that Streptococcus, Leuconostoc, Lactobacillus, and Carnobacterium belong to the normal microbiota of the gastrointestinal tract in healthy fish. However, it is well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress and salinity. Pathogenic lactic acid bacteria such as Streptococcus, Enterococcus, Lactobacillus, Carnobacterium and Lactococcus have been detected from ascites, kidney, liver, heart and spleen. Some antibiotic treatments and vaccinations have been proposed to cure or prevent these diseases that seem, however, to spread with the development of fish culture. It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotics. These candidates are able to colonise the gut, and act antagonistic against Gram-negative fish pathogens. These harmless bacteriocin-producing strains may reduce the need to use antibiotics in future aquaculture.     

斑点叉尾鮰病原细菌的分离鉴定及系统发育分析

用传统的微生物学分离培养方法从斑点叉尾鮰的鳃、肠和肝等器官共分离到16株可疑病原菌（编号为WJ01~WJ16）,以提取的基因组DNA为模版,扩增出约600bp大小的16S rDNA片段,测序分析表明：与GenBank数据库中的已知序列相比,16株细菌中有9株同源性在98%以上,5株在95%以上,2株为92%。系统发育分析表明这16株细菌明显的聚为3支。     

Lactic acid bacteria vs. pathogens in the gastrointestinal tract of fish: a review

Intensive fish production worldwide has increased the risk of infectious diseases. However, before any infection can be established, pathogens must penetrate the primary barrier. In fish, the three major routes of infection are the skin, gills and gastrointestinal (GI) tract. The GI tract is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit a regional variation in structure and function. In the last two decades, our understanding of the endocytosis and translocation of bacteria across this mucosa, and the sorts of cell damage caused by pathogenic bacteria, has increased. Electron microscopy has made a valuable contribution to this knowledge. In the fish‐farming industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp. salmonicida) and vibriosis [agent, Vibrio (Listonella) anguillarum]. This article provides an overview of the GI tract of fish from an electron microscopical perspective focusing on cellular damage (specific attack on tight junctions and desmosomes) caused by pathogenic bacteria, and interactions between the ‘good’ intestinal bacteria [e.g. lactic acid bacteria (LAB)] and pathogens. Using different in vitro methods, several studies have demonstrated that co‐incubation of Atlantic salmon (Salmo salar L.) foregut (proximal intestine) with LAB and pathogens can have beneficial effects, the cell damage caused by the pathogens being prevented, to some extent, by the LAB. However, there is uncertainty over whether or not similar effects are observed in other species such as Atlantic cod (Gadus morhua L.). When discussing cellular damage in the GI tract of fish caused by pathogenic bacteria, several important questions arise including: (1) Do different pathogenic bacteria use different mechanisms to infect the gut? (2) Does the gradual development of the GI tract from larva to adult affect infection? (3) Are there different infection patterns between different fish species? The present article addresses these and other questions.     

常温贮藏软烤扇贝品质及潜在病原菌分析

对软烤扇贝制品在常温贮藏期间的感官、化学和微生物品质进行分析。结果表明,样品感官品质良好,水分含量40.65±2.38%,Aw0.92±0.02,符合产品标准要求;但细菌菌落总数和耐热菌落总数分别为4.16±1.54 log10cfu/g和3.98±1.02 log10cfu/g,pH6.77±0.14,不符合产品标准要求。对潜在病原菌金黄色葡萄球菌、蜡样芽孢杆菌进行测试鉴定。结果表明,样品中检出5组菌,2组为葡萄球菌属,但不是金黄色葡萄球菌;3组为芽孢杆菌属。探究其是否具有致病性,需要增加样本并与产品加工过程关联,作进一步研究分析。     

海带对褐藻酸降解菌的抗性与其抗氧化能力的相关性分析

采用生物化学的方法测试 2个品系的海带 (Laminariajaponica)对褐藻酸降解菌抗性的差异性 ,并对其抗氧化能力进行研究 ,旨为海带烂病的防治提供生物学基础依据。结果表明 ,1)海带荣城 1号对褐藻酸降解菌的抗性远高于海带 90 1;2 )海带荣城 1号细胞内 2种抗氧化酶 -超氧化物歧化酶 (SOD)和过氧化物酶 (POD)的活性明显高于海带 90 1;而另一种抗氧化酶—过氧化氢酶 (CAT)的活性与海带 90 1相比没有明显的差别 ;3 )海带荣城 1号的脂溶性抗氧化能力同样明显高于海带 90 1。说明海带的抗氧化能力与其对褐藻酸降解菌的抗性有一定的相关性。     

Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus . Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas , Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas , Bacillus , coryneforms, Moraxella , Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae , Pasteurella piscicida , Vibrio anguillarum and V. vulnificus . In particular, as much as 53.3% of Vibrio spp. other than Vibrio -swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida . These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.     

Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel

Cysteine proteases, cathepsins L and B, in Japanese eel epidermis are suspected bacteriolysins against aquatic pathogens. This work examines the lysis of three species of gram-negative pathogenic bacteria by the eel epidermal extract according to the assay method for cysteine proteases. The optimum pH for the lysis of the three species of bacteria were commonly determined to be 5.0, where both of eel epidermal cathepsins L and B caused active proteolysis. Four kinds of specific inhibitors for cysteine proteases strongly inhibited these catheptic proteolyses and bacteriolyses. The activities for the lysis of three types of bacteria were at a similar level, but the effects on the bacterium which infects predominantly eel skin largely varied with individual fish. These results suggest that epidermal cathepsins L and B are responsible for lysis of any pathogenic bacteria in the nonspecific defense of Japanese eel.     

Evaluating the use of Lactobacillus acidophilus as a biocontrol agent against common pathogenic bacteria and the effects on the haematology parameters and histopathology in African catfish Clarias gariepinus juveniles

This study was carried out to evaluate the use of Lactobacillus acidophilus as a biocontrol agent against some common fish pathogenic bacteria (Staphylococcus xylosus, Aeromonas hydrophila gr.2 and Streptococcus agalactiae) in African catfish, Clarias gariepinus. Eight treatments were designed inclusive of 10 C. gariepinus juveniles (mean weight 190 g) per tank, each in triplicate. Four groups of fish were fed a diet supplemented with L. acidophilus, comprising about 3.01 × 10⁷ colony‐forming units per gram of diet (the probiotics diet), while the other four groups were fed a diet not supplemented with probiotics (the non‐probiotics diet). In the first group, fish were injected with 1 mL physiological saline and fed the non‐probiotic diet (non‐probiotic control); in the second, third and fourth groups, fish were injected with 1 mL each of S. xylosus, A. hydrophila gr.2 and S. agalactiae, respectively, and were all fed the non‐probiotic diet (designated as non‐probiotic treatments; NPsx, NPah and NPsa respectively). In the fifth group, fish were injected with 1 mL physiological saline but fed the probiotic diet (probiotic control), while fish in the sixth, seventh and eighth groups were each injected with 1 mL of S. xylosus, A. hydrophila gr.2 and S. agalactiae, respectively, and were all fed the probiotic diet (and designated as probiotic treatments; Psx, Pah and Psa respectively). Blood samples were collected for haematology analysis, while samples of the liver and kidney were examined for pathohistology after 7 and 21 days of infection. The results showed that the haematology parameters, packed cell volume, haemoglobin, erythrocyte sedimentation rate, red blood cell, white blood cell, total serum protein, Mg²⁺, Ca²⁺, Cl^?, glucose, cholesterol and total immunoglobulin concentrations and the pathohistology of the liver and kidney were better in the challenged fish (infected) maintained on the probiotic diet than those in the groups fed the non‐probiotic diet. It is concluded, based on these results, that L. acidophilus is useful as a probiotic agent in C. gariepinus against these pathogenic bacteria (S. xylosus, A. hydrophila gr.2 and S. agalactiae).     

紫菜腐霉拮抗菌的筛选和鉴定

生物防治广泛用于农作物的病害防治,该方法在藻类病害防控方面尚未有相关的报道。腐霉(Pythium sp.)是引起紫菜(Neopyropia)赤腐病(red rot disease)的主要病原,本研究的目的是筛选和鉴定对紫菜腐霉有拮抗能力的细菌。从养殖藻类及其养殖环境中分离鉴定了385株细菌,通过平板对峙法筛选到9株对腐霉有拮抗作用的细菌,进一步通过含毒介质法筛选到3株胞外产物对腐霉具有抑制活性的细菌(P3、P6和P19)。3株拮抗菌对8株腐霉均有拮抗活性,对腐霉生长的抑制率分别为52.09%~97.95% (P3)、26.81%~78.04% (P6)、10.47%~41.91% (P19)。通过16S rRNA鉴定和多位点序列进化分析(16S rRNA-dnaA-dnaN-recA),将P3和P6鉴定为杀鱼假交替单胞菌(Pseudoalteromonas piscicida),P19鉴定为解肽假交替单胞菌(Pseudoalteromonas peptidolytica)。本研究筛选得到的拮抗菌为下一步建立紫菜赤腐病的生物防治方法奠定了基础。     

The ability of carnobacteria isolated from fish intestine to inhibit growth of fish pathogenic bacteria: a screening study

In the present study a microtitre plate assay was used to evaluate antagonistic activity of 157 intestinal bacteria belonging to Carnobacterium isolated from Arctic charr (Salvelinus alpinus L.), Atlantic salmon (Salmo salar L.) and wolf fish (Anarhichas lupus L.) against fish pathogenic bacteria. One hundred and forty‐nine strains isolated from Arctic charr fed; (a) different lipid levels and (b) different fatty acids were screened for their ability to inhibit growth of the fish pathogen Aeromonas salmonicida ssp. salmonicida strain AL 2020 (the causative agent of furunculosis). Carnobacterium maltaromaticum and Carnobacterium mobile isolated from fish fed a low‐lipid diet inhibited growth of the pathogen, while none of the Carnobacterium divergens isolated from fish fed the high‐lipid diet had this ability. When Arctic charr was fed different fatty acids, was the frequency of antibacterial ability of C. maltaromaticum highest in strains isolated from fish fed 4%α‐linolenic acid (18:3 n‐3) and lowest in strains isolated from fish fed 4% linoleic acid (18:2 n‐6). Extracellular growth inhibitory compounds harvested in exponential and stationary growth phase from eight carnobacteria strains isolated from three fish species were tested for their ability to inhibit growth of six fish pathogens [A. salmonicida, Vibrio splendidus strain VS11, Vibrio salmonicida strain LFI 315, Vibrio anguillarum strain LFI 317, Moritella (Vibrio) viscosa strain LFI 5000 and C. maltaromaticum (piscicola) CCUG 34645]. The highest antibacterial activity was found when cellular extracts of the producer isolate were harvested in stationary growth phase. Scanning electron microscopy (SEM) investigations of A. salmonicida showed that cell morphology was affected by the inhibitory substance produce by strain 8M851, a Carnobacterium inhibens‐like bacteria.     

冰冻对硝化细菌制剂活性的影响

在实验室模拟条件下,研究冰冻对淡水型硝化细菌和海水型硝化细菌制剂中氨氧化细菌和亚硝酸盐氧化细菌活性的影响。结果表明,冰冻对淡水型硝化细菌和海水型硝化细菌制剂活性均有较为明显的抑制作用,且10d处理组的抑制作用高于5d处理组,对淡水型硝化细菌制剂活性的抑制作用大于海水型硝化细菌制剂。液体硝化细菌制剂在冬季运输和保存过程中要采取保温措施,以避免由于冰冻导致的菌剂活性降低。