Phage Therapy and Photodynamic Therapy: Low Environmental Impact Approaches to Inactivate Microorganisms in Fish Farming Plants

Owing to the increasing importance of aquaculture to compensate for the progressive worldwide reduction of natural fish and to the fact that several fish farming plants often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria, more environmentally-friendly strategies to control fish infections are urgently needed to make the aquaculture industry more sustainable. The aim of this review is to briefly present the typical fish farming diseases and their threats and discuss the present state of chemotherapy to inactivate microorganisms in fish farming plants as well as to examine the new environmentally friendly approaches to control fish infection namely phage therapy and photodynamic antimicrobial therapy.     

Oleic acid produced by a marine Vibrio spp. acts as an anti-Vibrio parahaemolyticus agent

It is known that some strains of Vibrio parahaemolyticus are responsible for gastroenteric diseases caused by the ingestion of marine organisms contaminated with these bacterial strains. Organic products that show inhibitory activity on the growth of the pathogenic V. parahaemolyticus were extracted from a Vibrio native in the north of Chile. The inhibitory organic products were isolated by reverse phase chromatography and permeation by Sephadex LH20, and were characterized by spectroscopic and spectrometric techniques. The results showed that the prevailing active product is oleic acid, which was compared with standards by gas chromatography and high-performance liquid chromatography (HPLC). These active products might be useful for controlling the proliferation of pathogenic clones of V. parahaemolyticus.     

螺旋粉虱体内共生菌的研究

采用变性梯度凝胶电泳技术[denaturing gradient gel electrophoresis（DGGE）]、16S rDNA文库构建技术,以及使用Wolbachia菌特异引物研究在不同寄主上取食繁殖的螺旋粉虱自然种群体内共生菌。结果显示,在不同寄主植物取食的新入侵螺旋粉虱体内共生菌均稳定存在有初级共生菌（Candidatus Portiera aleyrodidarum）,属于变形菌门（Proteobacteria）,γ-变形菌纲,共生菌多样性较少;研究同时发现取食不同寄主植物的螺旋粉虱个体间共生菌群落多样性有一定差异,一些螺旋粉虱个体内还生存有葡萄球菌（Staphylococcus sp.）;此外,研究还发现新入侵的螺旋粉虱体内尚未检测到Wolbachia菌。表明取食不同寄主的螺旋粉虱体内均稳定存在有共生菌,同时取食不同的寄主其共生菌群落也会发生变化;Wolbachia菌尚未在新入侵海南的螺旋粉虱中建立共生关系。     

The Role of Biophysical Parameters in the Antilipopolysaccharide Activities of Antimicrobial Peptides from Marine Fish

Numerous antimicrobial peptides (AMPs) from marine fish have been identified, isolated and characterized. These peptides act as host defense molecules that exert antimicrobial effects by targeting the lipopolysaccharide (LPS) of Gram-negative bacteria. The LPS-AMP interactions are driven by the biophysical properties of AMPs. In this review, therefore, we will focus on the physiochemical properties of AMPs; that is, the contributions made by their sequences, net charge, hydrophobicity and amphipathicity to their mechanism of action. Moreover, the interactions between LPS and fish AMPs and the structure of fish AMPs with LPS bound will also be discussed. A better understanding of the biophysical properties will be useful in the design of AMPs effective against septic shock and multidrug-resistant bacterial strains, including those that commonly produce wound infections.     

Ecological and Pharmacological Activities of Polybrominated Diphenyl Ethers (PBDEs) from the Indonesian Marine Sponge Lamellodysidea herbacea

Two known Polybrominated Diphenyl Ethers (PBDEs), 3,4,5-tribromo-2-(2′,4′-dibromophenoxy)phenol (1d) and 3,4,5,6-tetrabromo-2-(2′,4′-dibromophenoxy)phenol (2b), were isolated from the Indonesian marine sponge Lamellodysidea herbacea. The structure was confirmed using ¹³C chemical shift average deviation and was compared to the predicted structures and recorded chemical shifts in previous studies. We found a wide range of bioactivities from the organic crude extract, such as (1) a strong deterrence against the generalist pufferfish Canthigaster solandri, (2) potent inhibition against environmental and human pathogenic bacterial and fungal strains, and (3) the inhibition of the Hepatitis C Virus (HCV). The addition of a bromine atom into the A-ring of compound 2b resulted in higher fish feeding deterrence compared to compound 1d. On the contrary, compound 2b showed only more potent inhibition against the Gram-negative bacteria Rhodotorula glutinis (MIC 2.1 μg/mL), while compound 1d showed more powerful inhibition against the other human pathogenic bacteria and fungi. The first report of a chemical defense by compounds 1d and 2b against fish feeding and environmental relevant bacteria, especially pathogenic bacteria, might be one reason for the widespread occurrence of the shallow water sponge Lamellodysidea herbacea in Indonesia and the Indo-Pacific.     

Marine Pseudovibrio sp. as a Novel Source of Antimicrobials

Antibiotic resistance among pathogenic microorganisms is becoming ever more common. Unfortunately, the development of new antibiotics which may combat resistance has decreased. Recently, however the oceans and the marine animals that reside there have received increased attention as a potential source for natural product discovery. Many marine eukaryotes interact and form close associations with microorganisms that inhabit their surfaces, many of which can inhibit the attachment, growth or survival of competitor species. It is the bioactive compounds responsible for the inhibition that is of interest to researchers on the hunt for novel bioactives. The genus Pseudovibrio has been repeatedly identified from the bacterial communities isolated from marine surfaces. In addition, antimicrobial activity assays have demonstrated significant antimicrobial producing capabilities throughout the genus. This review will describe the potency, spectrum and possible novelty of the compounds produced by these bacteria, while highlighting the capacity for this genus to produce natural antimicrobial compounds which could be employed to control undesirable bacteria in the healthcare and food production sectors.     

Identification of the antibacterial compound produced by the marine epiphytic bacterium Pseudovibrio sp. D323 and related sponge-associated bacteria

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens.     

Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease

American foulbrood disease has a major impact on honeybees (Apis melifera) worldwide. It is caused by a Gram-positive, spore-forming bacterium, Paenibacillus larvae. The disease can only affect larval honeybees, and the bacterial endospores are the infective unit of the disease. Antibiotics are not sufficient to combat the disease due to increasing resistance among P. larvae strains. Because of the durability and virulence of P. larvae endospores, infections spread rapidly, and beekeepers are often forced to burn beehives and equipment. To date, very little information is available on the use of bacteriophage therapy in rescuing and preventing American foulbrood disease, therefore the goal of this study was to test the efficacy of phage therapy against P. larvae infection. Out of 32 previously isolated P. larvae phages, three designated F, WA, and XIII were tested on artificially reared honeybee larvae infected with P. larvae strain NRRL B-3650 spores. The presence of P. larvae DNA in dead larvae was confirmed by 16S rRNA gene-specific polymerase chain reaction amplification. Survival rates for phage-treated larvae were approximately the same as for larvae never infected with spores (84%), i.e., the phages had no deleterious effect on the larvae. Additionally, prophylactic treatment of larvae with phages before spore infection was more effective than administering phages after infection, although survival in both cases was higher than spores alone (45%). Further testing to determine the optimal combination and concentration of phages, and testing in actual hive conditions are needed.     

玉米秸秆还田对盐碱地土壤细菌多样性的影响

以通辽典型的盐碱地玉米田为研究对象,在玉米吐丝期利用高通量测序技术对盐碱地土壤细菌进行测序,结合相关生物信息学分析,研究未开垦盐碱地(ZH1)、非盐碱地连作玉米田(ZH2)和玉米秸秆还田盐碱地种植玉米田(ZH3)3个处理下耕层土壤细菌群落丰富度、多样性和群落结构的变化。结果表明,3个处理土壤细菌群落结构和丰度差异大,已知菌属中鞘氨醇单胞菌属(Sphingomonas)和pontibacter是ZH1与ZH3的优势菌属;ZH2的优势菌属为链霉菌属(Streptomyces)、鞘氨醇单胞菌属和类诺卡氏属(Nocardioides)。玉米秸秆还田对盐碱地土壤细菌多样性的影响较大,细菌菌群Alpha及样品群落组成丰度分析可知,两年玉米秸秆还田盐碱地土壤细菌多样性和物种丰度与未开垦盐碱地比较均有所上升,并出现能降解有机物料和农药的功能菌属。     

化肥减量配施有机肥下芒果园土壤细菌多样性及群落结构特征

为了探究化肥减量配施有机肥对芒果园土壤细菌多样性和群落结构的影响。本研究以海南省乐东黎族自治县某芒果园为研究对象,采用Illumina Miseq测序平台,对化肥减量配施有机肥和常规施用化肥两种施肥方式处理的芒果园土壤进行16S rRNA高通量测序分析。结果表明:通过化肥减量配施有机肥处理的芒果园土壤的有机质含量、碱解氮含量和有效磷含量比常规施肥增加了54.87%、40.73%、64.17%,镉和铅含量分别比常规施肥减少了65.09%和68.81%,而汞和铜含量分别比常规施肥增加了74.19%和32.21%。基于97.00%的相似度对所得序列进行聚类分析,2种处理的芒果园土壤中共有2702个operational taxonomic units(OTUs)。其中化肥减量处理的土壤平均有1784个OTUs,常规施肥处理的土壤平均有1568个OTUs。化肥减量配施有机肥的施肥方式能显著改善土壤质量,提高芒果园土壤细菌丰富度和多样性,并在一定程度上改变土壤细菌的群落结构。施肥方式的改变对土壤细菌的分布有较大的影响,化肥减量处理的芒果园土壤中变形菌门、放线菌门的含量较高;两种施肥方式下的土壤细菌群落结构有了明显的变化,但土壤中主要细菌的种类没有改变,土壤细菌群落结构仍保持一定的相似性;相关性分析结果显示土壤细菌群落受有机质、有效氮、有效磷和铜含量的影响较大。     

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.     

Use of Natural Antimicrobial Peptides and Bacterial Biopolymers for Cultured Pearl Production

Cultured pearls are the product of grafting and rearing of Pinctada margaritifera pearl oysters in their natural environment. Nucleus rejections and oyster mortality appear to result from bacterial infections or from an inappropriate grafting practice. To reduce the impact of bacterial infections, synthetic antibiotics have been applied during the grafting practice. However, the use of such antibiotics presents a number of problems associated with their incomplete biodegradability, limited efficacy in some cases, and an increased risk of selecting for antimicrobial resistant bacteria. We investigated the application of a marine antimicrobial peptide, tachyplesin, which is present in the Japanese horseshoe crab Tachypleus tridentatus, in combination with two marine bacterial exopolymers as alternative treatment agents. In field studies, the combination treatment resulted in a significant reduction in graft failures vs. untreated controls. The combination of tachyplesin (73 mg/L) with two bacterial exopolysaccharides (0.5% w/w) acting as filming agents, reduces graft-associated bacterial contamination. The survival data were similar to that reported for antibiotic treatments. These data suggest that non-antibiotic treatments of pearl oysters may provide an effective means of improving oyster survival following grafting procedures.     

Microbial Diversity and Phage–Host Interactions in the Georgian Coastal Area of the Black Sea Revealed by Whole Genome Metagenomic Sequencing

Viruses have the greatest abundance and highest genetic diversity in marine ecosystems. The interactions between viruses and their hosts is one of the hot spots of marine ecology. Besides their important role in various ecosystems, viruses, especially bacteriophages and their gene pool, are of enormous interest for the development of new gene products with high innovation value. Various studies have been conducted in diverse ecosystems to understand microbial diversity and phage–host interactions; however, the Black Sea, especially the Eastern coastal area, remains among the least studied ecosystems in this regard. This study was aimed at to fill this gap by analyzing microbial diversity and bacteriophage–host interactions in the waters of Eastern Black Sea using a metagenomic approach. To this end, prokaryotic and viral metagenomic DNA from two sampling sites, Poti and Gonio, were sequenced on the Illumina Miseq platform and taxonomic and functional profiles of the metagenomes were obtained using various bioinformatics tools. Our metagenomics analyses allowed us to identify the microbial communities, with Proteobacteria, Cyanobacteria, Actinibacteria, and Firmicutes found to be the most dominant bacterial phyla and Synechococcus and Candidatus Pelagibacter phages found to be the most dominant viral groups in the Black Sea. As minor groups, putative phages specific to human pathogens were identified in the metagenomes. We also characterized interactions between the phages and prokaryotic communities by determining clustered regularly interspaced short palindromic repeats (CRISPR), prophage-like sequences, and integrase/excisionase sequences in the metagenomes, along with identification of putative horizontally transferred genes in the viral contigs. In addition, in the viral contig sequences related to peptidoglycan lytic activity were identified as well. This is the first study on phage and prokaryote diversity and their interactions in the Eastern coastal area of the Black Sea using a metagenomic approach.     

Improved Stability and Activity of a Marine Peptide-N6NH2 against Edwardsiella tarda and Its Preliminary Application in Fish

Edwardsiella tarda can cause fatal gastro-/extraintestinal diseases in fish and humans. Overuse of antibiotics has led to antibiotic resistance and contamination in the environment, which highlights the need to find new antimicrobial agents. In this study, the marine peptide-N6 was amidated at its C-terminus to generate N6NH2. The antibacterial activity of N6 and N6NH2 against E. tarda was evaluated in vitro and in vivo; their stability, toxicity and mode of action were also determined. Minimal inhibitory concentrations (MICs) of N6 and N6NH2 against E. tarda were 1.29–3.2 μM. Both N6 and N6NH2 killed bacteria by destroying the cell membrane of E. tarda and binding to lipopolysaccharide (LPS) and genomic DNA. In contrast with N6, N6NH2 improved the stability toward trypsin, reduced hemolysis (by 0.19% at a concentration of 256 μg/mL) and enhanced the ability to penetrate the bacterial outer and inner membrane. In the model of fish peritonitis caused by E. tarda, superior to norfloxacin, N6NH2 improved the survival rate of fish, reduced the bacterial load on the organs, alleviated the organ injury and regulated the immunity of the liver and kidney. These data suggest that the marine peptide N6NH2 may be a candidate for novel antimicrobial agents against E. tarda infections.     

Antibacterial activity of marine and black band disease cyanobacteria against coral-associated bacteria

Black band disease (BBD) of corals is a cyanobacteria-dominated polymicrobial disease that contains diverse populations of heterotrophic bacteria. It is one of the most destructive of coral diseases and is found globally on tropical and sub-tropical reefs. We assessed ten strains of BBD cyanobacteria, and ten strains of cyanobacteria isolated from other marine sources, for their antibacterial effect on growth of heterotrophic bacteria isolated from BBD, from the surface mucopolysaccharide layer (SML) of healthy corals, and three known bacterial coral pathogens. Assays were conducted using two methods: co-cultivation of cyanobacterial and bacterial isolates, and exposure of test bacteria to (hydrophilic and lipophilic) cyanobacterial cell extracts. During co-cultivation, 15 of the 20 cyanobacterial strains tested had antibacterial activity against at least one of the test bacterial strains. Inhibition was significantly higher for BBD cyanobacteria when compared to other marine cyanobacteria. Lipophilic extracts were more active than co-cultivation (extracts of 18 of the 20 strains were active) while hydrophilic extracts had very limited activity. In some cases co-cultivation resulted in stimulation of BBD and SML bacterial growth. Our results suggest that BBD cyanobacteria are involved in structuring the complex polymicrobial BBD microbial community by production of antimicrobial compounds.     

Inhibition of virulence gene expression in Staphylococcus aureus by novel depsipeptides from a marine photobacterium

During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA). To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.     

花生根际土壤细菌群落对干旱和盐胁迫的响应

为明确旱、盐及旱盐双重胁迫对花生根际土壤细菌群落的影响，本研究采用盆栽试验，通过16S rRNA基 因测序技术，研究了花生开花期干旱、盐胁迫及旱盐双重胁迫下花生根际土壤细菌群落结构的变化。结果表明，花 生根际土壤细菌群落均以放线菌纲（Actinobacteria）、α-变形菌纲（Alphaproteobacteria）、未分类菌目（norank_p__Sac⁃ charibacteria）、蓝藻纲（Cyanobacteria）、酸杆菌纲（Acidobacteria）、芽单胞菌纲（Gemmatimonadetes）和β-变形菌纲 （Betaproteobacteria）7个优势菌纲为主。干旱和盐胁迫处理均不同程度提高了α-变形菌纲和蓝藻纲的含量，且对蓝 藻纲的诱导效果较显著，推测蓝藻纲在提高花生胁迫耐受性方面具有重要功能。非生物胁迫影响根际土壤微域环 境，对花生根际土壤细菌群落结构具有调控作用。调节微生物群落结构，改良土壤微域环境，是提高植物胁迫耐受 性的有效途径。     

南方红壤丘陵区土壤细菌对土壤水分和温度的响应差异

红壤丘陵区稻田不同时期进行的水分管理导致土壤水分和温度同时变化,继而影响土壤微生物群落结构,二者对土壤微生物的影响机制目前还不清楚。本研究以中国科学院桃源农业生态实验站长期定位试验的稻田作为研究对象,同时以旱地农田作为对照,利用末端限制性酶切片段长度多态性分析（T-RFLP）和荧光定量PCR（qPCR）方法,研究不同水分管理时期细菌群落结构与丰度的变化特征,进而分析其变化的影响因素,阐明水分和温度的相对贡献。结果表明,稻田土壤细菌受土壤水分状况的显著影响,淹水期土壤细菌群落结构明显区别于其他时期,且淹水期土壤细菌丰度和多样性指数均显著低于晒田期。稻田土壤细菌群落结构和多样性指数均受到土壤含水量和土壤温度的显著影响,但土壤含水量的相关性大于土壤温度。而作为对照的旱地农田土壤含水量在不同时期没有差异,土壤细菌群落结构也没有差异,仅土壤细菌丰度和多样性指数发生一定变化,但这种变化并不与土壤温度呈现相关性。因此,本研究认为,南方丘陵区农田土壤细菌对土壤水分的响应比对土壤温度更敏感。     

茶树根际土壤物质的自毒潜力及其对土壤微生物多样性的影响

为了分析茶树根际土壤物质对茶树根际土壤微生物多样性的影响,本研究以植茶年限0、3、9、25 a的铁观音茶树根际土壤为材料,采用不同极性树脂吸附茶树根际土壤物质并洗脱,探讨不同植茶年限茶树根际土壤物质的自毒潜力及其对土壤微生物多样性的影响。结果表明,不同极性树脂吸附洗脱液以ADS-7树脂洗脱液对受体根长的抑制作用最强。ADS-7树脂吸附洗脱液处理重新种植的茶树后,随着土壤植茶年限的增加,茶树根际土壤细菌数量呈现下降趋势。相关性分析结果表明,与土壤年限呈正相关的细菌T-RFs片段15个,涉及8个纲,31种细菌,按照其功能可分为4类,其中病原菌19种,占比61.29%;负相关细菌T-RFs片段18个,涉及11个纲,31种细菌,按照其功能可分为6类,其中与抑制病原菌、碳素循环、氮素循环、硫素循环、土壤质地改善相关的细菌总占比达到83.87%。综上表明,ADS-7树脂洗脱液处理重新种植的茶树后,随着土壤植茶年限的增加,茶树根际土壤病原菌数量大幅上升,益生菌与土壤养分循环相关的细菌数量显著下降,土壤微生物生态系统平衡失调。     

多效唑对芒果园土壤细菌多样性的影响及PICRUSt基因功能预测分析

为了探究多效唑对芒果园土壤微生物多样性和群落结构的影响,本研究以海南乐东某芒果园为研究对象,设置施用和不施用多效唑的土壤分别为处理组和对照组,通过Illumina Miseq测序平台对芒果园土壤进行16S rRNA高通量测序分析。2组样品通过高通量测序共得到3586个OTUs(operational taxonomic units),可注释到38个门、89个纲、195个目、378个科、673个属、1353个种。Alpha多样性分析表明,施用多效唑后,土壤细菌丰富度指数显著高于同时期未施用多效唑的土壤;但施药后土壤的细菌多样性显著降低。主成分分析表明,施用多效唑对土壤细菌的群落结构产生影响。与对照组相比,施用多效唑的土壤中变形菌门和浮霉菌门等含量显著增高,而放线菌门和厚壁菌门等含量显著降低。PICRUSt功能预测分析结果表明,芒果园土壤细菌主要涉及细胞生长与死亡、碳水化合物代谢、次生产物代谢的生物合成、氨基酸代谢等43个子功能,表现出功能上的丰富性。多效唑处理后,会降低土壤细菌的整体代谢能力。由此可见,施用多效唑会降低土壤细菌的多样性,改变土壤细菌的相对丰度,且对土壤细菌的功能会有一定的影响。