Disruption of bacterial quorum sensing: an unexplored strategy to fight infections in aquaculture

Disease outbreaks—some of them caused by pathogenic bacteria—are considered to be one of the largest constraints to development of the aquaculture sector. So far, antibiotics and disinfectants have only had limited success in the prevention or cure of aquatic disease. Moreover, the frequent use of biocides, especially in subtherapeutic doses, is leading to the rapid development of resistance. Therefore, there is an urgent need to develop alternative ways to control infections caused by bacterial pathogens in aquaculture. Many of these pathogens are found to control virulence factor expression by a cell-to-cell communication system. Hence, disruption of bacterial quorum sensing has been proposed as a new anti-infective strategy and several techniques that could be used to disrupt quorum sensing have been investigated. These techniques comprise (1) the inhibition of signal molecule biosynthesis, (2) the application of quorum sensing antagonists (including natural occurring as well as synthetic halogenated furanones, antagonistic quorum sensing molecules and undefined exudates of higher plants and algae), (3) the chemical inactivation of quorum sensing signals by oxidised halogen antimicrobials, (4) signal molecule biodegradation by bacterial lactonases and by bacterial and eukaryotic acylases and (5) the application of quorum sensing agonists. The few reports that deal with disruption of quorum sensing of aquatic pathogens, together with the results obtained with human and plant pathogens, indicate that this new approach has potential in fighting infections in aquaculture. However, before this new strategy can be applied in aquaculture, the impact of quorum sensing disruption on the virulence of aquatic pathogens and the impact of the proposed quorum sensing disrupting techniques on the aquaculture system of interest should be studied in more depth.     

具有群体感应抑制作用的地衣芽孢杆菌T-1的动物安全及生态评价

摘要：该研究以具有群体感应抑制作用的地衣芽孢杆菌（Bacillus licheniformis）T-1为试验对象,参照国标的急性毒性实验方法检测其对动物的安全性及对水体生态环境的影响。安全评价试验采用异育银鲫、斑马鱼、小鼠为测试动物,结果显示：异育银鲫腹腔注射浓度为11200mg/L（2.6×1011 CFU/Ml）菌株T–1菌液后,连续观察96h,异育银鲫均健康生长,未出现不良症状或死亡;将斑马鱼浸泡在浓度为2240mg/L（5.2×1010 CFU/mL）的菌株 T–1菌液中,连续观察96h,斑马鱼均健康生长,未出现不良症状或死亡;SPF级小鼠(ICR品系)灌胃16800 mg/kg（3.9×1011 CFU/mL）体重浓度的菌株T-1,观察9d小白鼠健康生长无不良症状。水体生态评价采用小球藻、大型蚤及萼花臂尾轮虫为测试对象,不同浓度的菌株T-1在96h内,对小球藻生长无毒且促进其生长,对大型蚤生长无不良影响,对萼花臂尾轮虫生长无害,且在高浓度下对萼花臂尾轮虫的生长繁殖有促进作用。研究表明,具有群体感应抑制作用的地衣芽孢杆菌T-1对异育银鲫、斑马鱼及小鼠安全无毒害;对水体生态环境中的浮游生物也无不良影响,具有开发成微生态制剂的潜力。     

Disruption of quorum sensing in Vibrio harveyi by the AiiA protein of Bacillus thuringiensis

Quorum sensing is a mechanism in which bacteria coordinate the expression of certain genes in response to their population density by producing, releasing and detecting signal molecules called autoinducers. Quorum sensing is responsible for controlling a plethora of virulence genes in several bacterial pathogens. Disruption of the quorum sensing system of Vibrio harveyi has been proposed as a new anti-infective strategy. AiiA is a protein which can block the bacterial quorum sensing by hydrolyzing AHL-lactone, and could greatly attenuate the disease caused by many bacterial pathogens in which quorum sensing regulate the expression of virulence genes. In this study, primers were designed from the conserved sequences of aiiA gene in the genomes of several Bacillus strains, and the gene was amplified from Bacillus thuringiensis BF1 by PCR. AiiA gene was cloned to a cloning vector pUC and sequenced. The open reading frame of the aiiA gene was 753 bp, and the similarity of aiiA gene from B. thuringiensis BF1 to other sequences in the GenBank was as high as 99%. This gene was subsequently cloned into an expression vector pET-24d(+), and the recombinant AiiA protein was overexpressed in Escherichia coli overexpression strain BL21(DE3). The molecular weight of the expressed AiiA protein was estimated to be 28 kDa by SDS-PAGE. The optimized expression condition for the recombinant AiiA protein was at 25 °C for 6 h with 1 mM IPTG induction. The lysate of the recombinant E. coli could not only repress the pigment synthesize of the quorum sensing system reporter strain Chromobacterium violaceum ATCC 12472, but also attenuate the intensity of bioluminescence of V. harveyi VIB391 by 85%. This study was very important for further research on the disruption of infections caused by V. harveyi in aquaculture.     

细菌群体感应淬灭的几种调控机制及其潜在应用

群体感应（quorum sensing,QS）作为一种调控机制,细菌通过感知细胞外环境中的信号分子浓度变化调控调节着生物发光、质粒转移、抗生素合成、生物膜形成及毒力因子表达等多种重要的生物学功能,协调细菌群体行为以适应环境变化。文章简要综述细菌细胞之间的群体感应功能及其几种主要淬灭调控机制,为促进基于Qs为靶标的生态防控病害新技术构建提供参考,为Qs相关研究及具有潜在应用价值的Qs抑制剂开发提供思路。     

Characterization of virulence,cell surface characteristics and biofilm‐forming ability of Aeromonas spp. isolates from fish and sea water

Members of the genus Aeromonas are emerging human pathogens, causing a variety of extra‐intestinal, systemic and gastrointestinal infections in both immunocompetent and immunocompromised persons. Aeromonas virulence is multifaceted and involves surface‐associated molecules, motility, biologically active extracellular products and biofilm formation. Aeromonads, isolated from diverse freshwater fish species as well as sea water, were screened for biofilm formation, with varying physicochemical parameters including temperature, agitation and nutrient availability. Motility, cell surface characteristics (auto‐aggregation, hydrophobicity and S layer), and extracellular virulence factor production (haemolysis, proteolysis, DNase production) were also assessed to identify potential associations with the biofilm phenotype. Biofilm formation was influenced by environmental conditions, with isolates preferentially forming biofilms in nutrient‐rich media at 30 °C, although strong biofilm formation also occurred at 37 °C. Strong biofilm formation was observed for Aeromonas culicicola isolates following exposure to nutrient‐rich conditions, while Aeromonas allosaccharophila isolates preferred nutrient‐poor conditions for biofilm formation. Source‐/species‐specific correlations, ranging from weak to strong, were observed between biofilm formation and motility, cell surface characteristics and/or extracellular virulence factor production. Understanding the specific mechanisms by which Aeromonas species adhere to abiotic surfaces may aid in preventing and/or treating disease outbreaks in aquaculture systems and could lead to effective eradication of these fish pathogens.     

Renibacterium salmoninarum iron‐acquisition mechanisms and ASK cell line infection: Virulence and immune response

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron‐acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT‐qPCR to assess the effects of normal and iron‐limited conditions on iron‐uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H‐2 and the type strain DSM20767^T. Further evaluated was the in vitro immune‐related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron‐limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H‐2 than DSM20767^T. These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF‐α, IL‐1β, TLR1 and INF‐γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron‐limited conditions, especially H‐2. These observations indicate that iron‐acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron‐uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.     

The interaction of infectious salmon anaemia virus (ISAV) with the blue mussel,Mytilus edulis

Integrated multi‐trophic aquaculture (IMTA) is an alternative approach to mono‐culture aquaculture that reduces environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter‐feeding particulate‐bound organic nutrients. They may also increase or decrease disease risk on farms by serving as reservoirs or barriers for important finfish pathogens such as infectious salmon anaemia virus (ISAV). This study aimed to optimize culture and molecular assays in shellfish tissues and to determine the fate of ISAV in mussels, Mytilus edulis. To determine detection limits, qRT‐PCR and culture assays in both CHSE‐ and ASK cells were optimized in ISAV‐inoculated mussel tissue homogenates. Both qRT‐PCR and culture assays performed in ASK cells had comparable detection limits of 10^2.8 TCID₅₀ mL^?1. The ISAV RNA genome was consistently detected in digestive gland tissue of ISAV‐exposed mussels. Viable ISAV was not detected in mussel tissues by culture analysis in CHSE‐ and ASK cells. The fact that qRT‐PCR analysis resulted in positive cycle threshold (CT) values that corresponded to the detectable range of ISAV in ASK culture assays suggests that little to no viable ISAV particles are present in the mussel tissues.     

Antibiofilm potential of a tropical marine Bacillus licheniformis isolate: role in disruption of aquaculture associated biofilms

Microbial biofilms are important in aquaculture industries as they resist antibiotic treatments. In this study, we have investigated the antibiofilm potential of a tropical marine culture Bacillus licheniformis D1 (containing an antimicrobial protein BLDZ1) against two aquaculture associated pathogens namely, Vibrio harveyi and Pseudomonas aeruginosa. Both the test cultures formed biofilms on polystyrene and glass surfaces. The cell free supernatant (CFS) of B. licheniformis inhibited V. harveyi and P. aeruginosa biofilms on polystyrene surfaces up to around 80% and 78% respectively. In addition, the CFS disrupted pre‐formed biofilms of test cultures by about 73%. Fluorescence and scanning electron microscope analysis confirmed the antibiofilm potential of the CFS. The cell free supernatant displayed antiadhesive activity that inhibited the initial attachment of the bacteria during the process of biofilm formation. In addition, the CFS exhibited antimicrobial activity and mediated cell death via cytoplasmic membrane disruption.     

Asian carp farming systems: towards a typology and increased resource use efficiency

Resource use efficiency in Asian carp farming systems is analysed based on a survey of 2493 farms of nine countries. Multivariate classification of farms by intensity and diversity identified six farm types: four types of specialized aquaculture farms at different levels of intensity, and two types of integrated agriculture–aquaculture systems. Pond‐based, specialized semi‐extensive systems (using mainly inorganic fertilizers and feeds of off‐farm origin), and integrated semi‐intensive systems (using feeds and fertilizer of both on and off‐farm origin) are by far the most common types, accounting for 59% and 27% of all farms respectively. Specialized semi‐extensive systems also show the highest protein and nutrient (N and P) use efficiencies, and among the highest labour use efficiency. Super‐intensive cage farms are less efficient in nutrient and labour use, but provide very high returns to land and capital investment. On average, the aquaculture components of integrated agriculture– aquaculture systems are less nutrient, land, and labour efficient than specialized semi‐extensive systems. Integrated semi‐extensive systems (using organic fertilizers of on‐farm origin) are particularly inefficient across all indicators. Hence in practice, gains in overall resource use efficiency through on‐farm integration with agricultural production are constrained by the relative inefficiency of the aquaculture subsystems on integrated farms. Although such systems can likely be improved, integration as such is not a panacea to increasing resource use efficiency. Wide variation in resource use efficiency within all systems indicates potential for substantial efficiency gains through improved management regardless of the fundamental choice of system.     

Review of climate change impacts on marine aquaculture in the UK and Ireland

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Characterization of phenotype variations of luminescent and non‐luminescent variants of Vibrio harveyi wild type and quorum sensing mutants

Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.     

Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention

Vibrio anguillarum, also known as Listonella anguillarum, is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. In both aquaculture and larviculture, this disease is responsible for severe economic losses worldwide. Because of its high morbidity and mortality rates, substantial research has been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease‐prevention strategies. This review summarizes the current state of knowledge pertaining to V. anguillarum, focusing on pathogenesis, known virulence factors, diagnosis, prevention and treatment.     

Green fluorescent protein visualization of Vibrio parahaemolyticus infections in Indian white shrimp Fenneropenaeus indicus (H Milne Edwards)

The pathways by which pathogens invade Fenneropenaeus indicus, the potential colonization in various tissues and the disease transmission mechanisms are unclear. The aims of the present study were to visualize the colonization and pathogenesis of GFP‐tagged Vibrio parahaemolyticus, in various tissues of F. indicus to evaluate the pathogen interaction. Among the three strains isolated, a virulent strain VpDAHV2 was tagged with green fluorescent protein (GFP‐VpDAHV2) and validated for both its growth characteristics and its virulence as a genuine model for F. indicus infection. VpDAHV2 was positive for toxR and tlh genes and negative for tdh genes. CLSM images revealed that maximum colonization was observed in the haemolymph of the F. indicus challenged with GFP‐VpDAHV2. The haemolymph was the primary site for the colonization of GFP‐VpDAHV2 in F. indicus. The enteric localization occurred independently of the flagellum or motility of GFP‐VpDAHV2 through the intestinal route. The F. indicus infection model suggests that the haemolymph and the intestine represent the sites of infection by GFP‐VpDAHV2, and hence are the active sites of pathogen interactions. GFP tagging of V. parahaemolyticus is a new and systemic approach to determine the presence of bacteria in vivo for the confirmation of host pathogen interactions in aquaculture studies.     

Effect of quorum quenching bacteria on growth,virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout

Five N‐acyl homoserine lactone‐degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo‐C8‐HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo‐C8‐HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 10⁸ CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.     

Virulence genes,serobiotypes and antibiotic resistance profile of Escherichia coli strains isolated from aquaculture and other sources

In order to determine the prevalence of pathogenic Escherichia coli, a total number of 155 E. coli isolates from aquaculture, clinical and veterinary sources were screened for seven pathogenic virulence markers and a house‐keeping gene by a polymerase chain reaction. The targeted virulence genes included eaeA of enteropathogenic E. coli, elt and est of enterotoxigenic E. coli (ETEC), ipaH of enteroinvasive E. coli, pCVD432 of enteroaggregative E. coli, stx, hlyA and eaeA of shigatoxigenic E. coli (STEC) and Enterohaemorrhagic E. coli. All the isolates were positive for phoA, the house‐keeping gene for E. coli. Among the 155 isolates, seven numbers (4.5%) harboured the virulence markers belonging to the pathogenic group ETEC and STEC. The virulent genes detected in these groups were elt, est, hlyA and stx. The sources of these virulence genes were fish (hlyA), shrimp (elt), feeder canal water (hlyA and elt) of aquaculture origin and from diarrhoea affected cow (hlyA, est and stx). The isolates with pathogenic traits belonged to the serogroups O6 or O29 and the remaining could not be typed. They showed resistance to two to four antibiotics out of the 12 antibiotics tested. Biotyping revealed that three isolates belonged to a single biotype (7333) and the remaining isolates were of diverse types. In conclusion, a molecular tool such as PCR proves as more effective tool for detection of this pathogen than the conventional methods. Detection of these emerging pathogens in aquaculture samples warrants for strict adherence to hygienic handling at retail outlets and proper cooking by the consumer before consumption.     

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north‐western Mexico

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10‐fold serial dilution of tissue infected with strain WSSV Mx‐H, and the virulence of four WSSV strains from north‐western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD₅₀ of the Mx‐H strain was a dilution dose of 10^?7.5; the mortality titre was 10^9.2 LD₅₀ per gram. In shrimp injected with 10^2.5 to 10^6.5 LD₅₀, no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx‐F strain (intermediate virulence) and the Mx‐C strain (high virulence) showed more genetic differences than those observed between the Mx‐G (low‐virulence) and Mx‐H (high‐virulence) strains, in ORF94 and ORF125. The application of high‐viral‐load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.     

Impact of ozonation on water quality in marine recirculation systems

Ozone (O₃) is a powerful oxidant and is becoming popular in various aquaculture systems for disinfection and improving water quality by oxidation of inorganic and/or organic compounds. However, the use of ozone in marine-based aquaculture systems has been limited because of the potential to form bromate, which is formed during the oxidation of naturally occurring bromide by ozone. Because bromate is a human carcinogen, there are concerns with its chronic impact on fish health. In addition, the use of O₃ is hindered by lack of quantitative as well as qualitative design and performance information on O₃ for recirculating systems. This study investigated the application of ozonation to control pathogens and enhance the process water quality in a recirculating aquaculture system while minimizing bromate formation. A field scale monitoring program was conducted on process water quality from Atlantic halibut (Hippoglossus hippoglossus) recirculating systems. Ozonated modules showed reduction of 15% total organic carbon (TOC) and less than 25 μg/l bromate concentration was formed. In addition, ozonated modules showed reduction in nitrate, color and suspended solids, as compared to those that did not use ozone. The results of this study elucidates the formation of bromate in marine water recirculation systems.