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Antimicrobial resistance (AMR) evolution and onward transmission of resistance genes is impacted by interrelated biological and social drivers, with evidence and impacts observed across human, animal and environmental One Health domains. Systems-based research examining how food production impacts on AMR in complex agrifood systems is lacking, with little written on management approaches in the UK that might prevent and respond to this challenge. One approach is the creation of a transdisciplinary network to enhance capacity, capability and collaboration between agrifood-focused disciplines and stakeholders. This co-creation platform for network-wide systems-based activities would reduce inefficiencies in AMR-related activities around agrifood, providing a cross-cutting, cohesive community to deliver transformational guidance on relevant, practical agrifood solutions that add value by reducing AMR, antimicrobial usage and associated costs, and decreasing resultant environmental contamination by prioritising challenges, sharing knowledge and best practice, and co-creating practical solutions with key stakeholders. An online survey determined prospective network focus, structure and priorities, with responses analysed using mixed methods. Survey results suggested respondents have interests in synthesising data using systems-approaches and using certain disciplines such as 'social sciences' within network activities. There were disconnects in how and whom to work with on this, with generalised use of 'social science/scientists' but lack of disciplinary understanding (e.g., anthropology, sociology) suggesting disciplinary differences awareness-training is useful. A similar generalisation is seen for mathematics/statistics. There are strong interests in working with food system practitioners (e.g., farmers/vets), providing opportunities for farm/field visits/knowledge exchange, and human health, reflecting the need for farm-to-fork understanding of impacts. There were notable mentions of policy/governance, emphasising translational research desires to create meaningful change. Disciplines/fields did not always align with identified interests e.g., systems and implementation science, suggesting the utility of network activity around introducing these disciplines e.g., methodology-focused rather than subject-focused conferences exploring lateral thinking about subjects. We suggest starting by developing understanding of the most important research questions by working with stakeholders, then working back to how we would achieve desirable project outcomes and who else is needed for this., Competing Interests: None of the authors have competing financial or non-financial interests in the writing of this manuscript., (© 2024 The Authors.)

Aeromonas dhakensis is increasingly recognised to be an important pathogen responsible for disease losses in warm-water aquaculture and, similar to several other Aeromonas species, it can infect humans. Knowledge of A. dhakensis is accumulating, but this species remains relatively under-investigated compared to its close relative, Aeromonas hydrophila. The significance of A. dhakensis may have been overlooked in disease events of aquatic animals due to issues with reliable identification. Critical to appreciating the importance of this pathogen is the application of dependable molecular tools that enable accurate identification and discrimination from A . hydrophila and other motile aeromonads. This review aims to synthesise the key literature on A. dhakensis , particularly with relevance to aquaculture, including knowledge of the bacterium derived from disease case studies in aquatic hosts. Identification methods and strain phylogeny are discussed, with accurate detection important for prompt diagnosis and for distinguishing strains with heightened virulence. Increasing evidence suggests that A. dhakensis may be more virulent than A. hydrophila and correct identification is required to determine the zoonotic risks posed, which includes concerns for antibiotic-resistant strains. This review provides an impetus to improve species identification in the future and screen strain collections of presumptive Aeromonas spp. retrospectively to reveal the true prevalence and impact of A. dhakensis in aquaculture, the environment, and healthcare settings.

Fish erythrocytes remain nucleated, unlike mammalian erythrocytes that undergo enucleation during maturation. Besides oxygen transport, fish erythrocytes are capable of several immune defence processes and thus these cells are candidates for carrying out ETotic responses. ETosis is an evolutionarily conserved innate immune defence process found in both vertebrates and invertebrates, which involves the extrusion of DNA studded with antimicrobial effector proteins into the extracellular space that traps and kills microorganisms. In this present report, we demonstrate that erythrocytes from Danio rerio (zebrafish) produce ETotic-like responses when exposed to both chemical and physiological inducers of ETosis. Furthermore, erythrocytes from Salmo salar (Atlantic salmon) behaved in a similar way. We have termed these ET-like formations, as Fish Erythrocyte Extracellular Traps (FEETs). Several inducers of mammalian ETosis, such as the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, induced FEETs. Moreover, we found that FEETs depend on the activation of PKC and generation of mitochondrial reactive oxygen species (mROS). This present report is the first demonstration that fish erythrocytes can exhibit ETotic-like responses, unveiling a previously unknown function, which sheds new light on the innate immune arsenal of these cells., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Competing Interests: Declaration of competing interest The authors confirm they have no known conflicts of interest.

Introduction: Aeromonads are ubiquitous in aquatic environments and several species are opportunistic pathogens of fish. Disease losses caused by motile Aeromonas species, particularly Aeromonas hydrophila , can be challenging in intensive aquaculture, such as at striped catfish ( Pangasianodon hypophthalmus ) farms in Vietnam. Outbreaks require antibiotic treatments, but their application is undesirable due to risks posed by resistance. Vaccines are an attractive prophylactic and they must protect against the prevalent strains responsible for ongoing outbreaks., Methods: This present study aimed to characterize A. hydrophila strains associated with mortalities in striped catfish culture in the Mekong Delta by a polyphasic genotyping approach, with a view to developing more effective vaccines., Results: During 2013-2019, 345 presumptive Aeromonas spp. isolates were collected at farms in eight provinces. Repetitive element sequence-based PCR, multi-locus sequence typing and whole-genome sequencing revealed most of the suspected 202 A. hydrophila isolates to belong to ST656 ( n = 151), which corresponds to the closely-related species Aeromonas dhakensis , with a lesser proportion belonging to ST251 ( n = 51), a hypervirulent lineage (vAh) of A. hydrophila already causing concern in global aquaculture. The A. dhakensis ST656 and vAh ST251 isolates from outbreaks possessed unique gene sets compared to published A. dhakensis and vAh ST251 genomes, including antibiotic-resistance genes. The sharing of resistance determinants to sulphonamides ( sul1 ) and trimethoprim ( dfrA1 ) suggests similar selection pressures acting on A. dhakensis ST656 and vAh ST251 lineages. The earliest isolate (a vAh ST251 from 2013) lacked most resistance genes, suggesting relatively recent acquisition and selection, and this underscores the need to reduce antibiotics use where possible to prolong their effectiveness. A novel PCR assay was designed and validated to distinguish A. dhakensis and vAh ST251 strains., Discussion: This present study highlights for the first time A. dhakensis , a zoonotic species that can cause fatal human infection, to be an emerging pathogen in aquaculture in Vietnam, with widespread distribution in recent outbreaks of motile Aeromonas septicaemia in striped catfish. It also confirms vAh ST251 to have been present in the Mekong Delta since at least 2013. Appropriate isolates of A. dhakensis and vAh should be included in vaccines to prevent outbreaks and reduce the threat posed by antibiotic resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bartie, Ngô, Bekaert, Hoang Oanh, Hoare, Adams and Desbois.)

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWBD) and rainbow trout fry syndrome (RTFS), which affect salmonids. To better understand this pathogen and its interaction with the host during infection, including to support the development of resistant breeds and new vaccines and treatments, there is a pressing need for reliable and reproducible immersion challenge models that more closely mimic natural routes of infection. The aim of this present study was to evaluate a challenge model developed previously for rainbow trout for use in Atlantic salmon. First, preliminary challenges were conducted in Atlantic salmon (n = 120) and rainbow trout (n = 80) fry using two F. psychrophilum isolates collected from each fish species, respectively; fish had been pretreated with 200 mg/L hydrogen peroxide for 1 h. Thereafter, the main challenge was performed for just one F. psychrophilum isolate for each species (at 2 × 10 7  CFU/mL) but using larger cohorts (Atlantic salmon: n = 1187; rainbow trout: n = 2701). Survival in the main challenge was 81.2% in Atlantic salmon (21 days post-challenge) and 45.3% in rainbow trout (31 days post-challenge). Mortalities progressed similarly during the preliminary and main challenges for both species, demonstrating the reproducibility of this model. This is the first immersion challenge model of F. psychrophilum to be developed successfully for Atlantic salmon., (© 2022 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Background: Antimicrobial resistance (AMR) is a growing global crisis with long-term and unpredictable health, social and economic impacts, with which climate change is likely to interact. Understanding how to govern AMR amidst evolving climatic changes is critical. Scenario planning offers a suitable approach. By envisioning alternative futures, stakeholders more effectively can identify consequences, anticipate problems, and better determine how to intervene. This study explored future worlds and actions that may successfully address AMR in a changing climate in a high-income country, using Sweden as the case., Methods: We conducted online scenario-building workshops and interviews with eight experts who explored: (1) how promising interventions ( taxation of antimicrobials at point of sale , and infection prevention measures ) could each combat AMR in 2050 in Sweden given our changing climate; and (2) actions to take starting in 2030 to ensure success in 2050. Transcripts were thematically analyzed to produce a narrative of participant validated alternative futures., Results: Recognizing AMR to be a global problem requiring global solutions, participants looked beyond Sweden to construct three alternative futures: (1) "Tax Burn Out" revealed taxation of antimicrobials as a low-impact intervention that creates inequities and thus would fail to address AMR without other interventions, such as infection prevention measures. (2) "Addressing the Basics" identified infection prevention measures as highly impactful at containing AMR in 2050 because they would contribute to achieving the Sustainable Development Goals (SDGs), which would be essential to tackling inequities underpinning AMR and climate change, and help to stabilize climate-induced mass migration and conflicts; and (3) "Siloed Nations" described a movement toward nationalism and protectionism that would derail the "Addressing the Basics" scenario, threatening health and wellbeing of all. Several urgent actions were identified to combat AMR long-term regardless which future un-folds, such as global collaboration, and a holistic approach where AMR and climate change are addressed as interlinked issues., Conclusion: Our participatory scenario planning approach enabled participants from different sectors to create shared future visions and identify urgent actions to take that hinge on global collaboration, addressing AMR and climate change together, and achieving the SDGs to combat AMR under a changing climate., Competing Interests: BM is an employee of the Swedish Pharmaceutical Industry Association, and was previously employed by Pfizer AB. He is also a shareholder of several pharmaceutical companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lambraki, Cousins, Graells, Léger, Abdelrahman, Desbois, Gallagher, Staaf Larsson, Mattson, Henriksson, Troell, Søgaard Jørgensen, Wernli, Carson, Parmley and Majowicz.)

Introduction: Antimicrobial resistance (AMR) is a global crisis that evolves from a complex system of factors. Understanding what factors interact is key to finding solutions. Our objective was to identify the factors influencing AMR in the European food system and places to intervene., Materials and Methods: We conducted two workshops involving participants with diverse perspectives to identify the factors influencing AMR and leverage points (places) to target interventions. Transcripts were open coded for factors and connections, then transcribed into Vensim 8.0.4 to develop a causal loop diagram (CLD) and compute the number of feedback loops. Thematic analysis followed to describe AMR dynamics in Europe's food system and places for intervention. The CLD and themes were confirmed via participant feedback., Results: Seventeen participants representing human, animal and agricultural sectors identified 91 CLD factors and 331 connections. Seven themes (e.g., social and economic conditions) describing AMR dynamics in Europe's food system, five 'overarching factors' that impact the entire CLD system (e.g., leadership) and fourteen places for intervention (e.g., consumer demand) emerged from workshop discussions. Most leverage points fell on highly networked feedback loops suggesting that intervening at these places may create unpredictable consequences., Conclusions: Our study produced a CLD of factors influencing AMR in Europe's food system that implicates sectors across the One Health spectrum. The high connectivity between the CLD factors described by participants and our finding that factors are connected with many feedback mechanisms underscores the complexity of the AMR problem and the challenge with finding long-term solutions. Identifying factors and feedbacks helped identify relevant leverage points in the system. Some actions, such as government's setting AMU standards may be easier to implement. These actions in turn can support multi-pronged actions that can help redefine the vision, values and goals of the system to sustainably tackle AMR., Competing Interests: I.A.L., M.C., T.G., A.L., S.H., D.W., P.S.J., and E.J.P. have no competing interests to declare. P.H. is a member of the Technical Committee for the BAP Vanguard Climate Action Standard and P.H. and M.T. act as scientific advisors to the Seafood Business for Ocean Stewardship (SeaBOS) initiative. P.H. was supported by the CGIAR Research Program on Fish Agri-Food Systems (FISH) led by WorldFish and on Climate Change, Agriculture and Food Security (CCAFS) supported by contributors to the CGIAR Trust Fund. P.H. and M.T. acknowledge the Kjell and Märta Beijer Foundation for supporting this work through the Beijer Institute’s Aquaculture and Sustainable Seafood programme, and the SEAWIN project funded by FORMAS (2016-00227). P.H. is partially funded by FORMAS Inequality and the Biosphere project (2020-00454). A.P.D. is (or has been in the last 5 years) engaged in research grants funded by the Sustainable Aquaculture Innovation Centre, UK Research and Innovation, International Veterinary Vaccinology Network, Scottish Funding Council, Great British Prawns Ltd and Benchmark Animal Health. C.A.C. works for the Government of Canada at the Public Health Agency of Canada. C.A.C. is/has been a subject matter expert for the World Organisation for Animal Health (OIE) and the World Health Organization (WHO) and has been a co-topic editor for two volumes of a Frontiers in Veterinary Science, Research Topic: Antimicrobial Usage in Companion and Food Animals: Methods, Surveys and Relationships with Antimicrobial Resistance in Animals and Humans. S.E.M. is (or has been in the last 5 years) engaged in research grants/contracts funded by the Canadian Institutes for Health Research, the Bill and Melinda Gates Foundation/UK Dept., International Development, the Natural Sciences and Engineering Research Council of Canada, Institut de recherche Robert-Sauvé en santé et en sécurité du travail, Dairy Farmers of Canada Research Funding Program, the World Health Organization, British Columbia Centre for Disease Control, Indian and Northern Affairs Canada, and Ontario Ministry of Agriculture, Food and Rural Affairs. She currently sits on the Editorial Boards of Foodborne Pathogens and Disease, and Epidemiology and Infection, and is a member of the World Health Organization’s Foodborne Disease Burden Epidemiology Reference Group, and she has served as a paid expert on behalf of the Attorney General of Canada in legal proceedings, providing evidence on the public health risks and benefits of unpasteurized milk. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5 μg/ml calcium ionophore (CaI) for 1 h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3 h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (p < 0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5 μg/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Essential genes in bacterial pathogens are potential drug targets and vaccine candidates because disrupting their function is lethal. The development of new antibiotics, in addition to effective prevention measures such as vaccination, contributes to addressing the global problem of bacterial antibiotic resistance. The aim of this present study was to determine the essential genes of Vibrio anguillarum , a bacterial pathogen of aquatic animals, as a means to identify putative targets for novel drugs and to assist the prioritisation of potential vaccine candidates. Essential genes were characterised by a Tn-seq approach using the TnSC189 mariner transposon to construct a library of 52,662 insertion mutants. In total, 329 essential genes were identified, with 34.7% found within the core genome of this species; each of these genes represents a strong potential drug target. Seven essential gene products were predicted to reside in the cell membrane or be released extracellularly, thus serving as putative vaccine candidates. Comparison to essential gene data from five other studies of Vibrio species revealed 13 proteins to be conserved across the studies, while 25 genes were specific to V. anguillarum and not found to be essential in the other Vibrio spp. This study provides new information on the essential genes of Vibrio species and the methodology may be applied to other pathogens to guide the development of new drugs and vaccines, which will assist efforts to counter antibiotic resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bekaert, Goffin, McMillan and Desbois.)

Antibiotics are used in aquaculture to maintain the health and welfare of stocks; however, the emergence and selection of antibiotic resistance in bacteria poses threats to humans, animals and the environment. Mitigation of antibiotic resistance relies on understanding the flow of antibiotics, residues, resistant bacteria and resistance genes through interconnecting systems, so that potential solutions can be identified and issues around their implementation evaluated. Participatory systems-thinking can capture the deep complexity of a system while integrating stakeholder perspectives. In this present study, such an approach was applied to Nile tilapia ( Oreochromis niloticus ) production in the Nile Delta of Egypt, where disease events caused by antibiotic-resistant pathogens have been reported. A system map was co-produced with aquaculture stakeholders at a workshop in May 2018 and used to identify hotspots of antibiotic use, exposure and fate and to describe approaches that would promote fish health and thus reduce antibiotic use. Antibiotics are introduced into the aquaculture system via direct application for example in medicated feed, but residues may also be introduced into the system through agricultural drainage water, which is the primary source of water for most fish farms in Egypt. A follow-up survey of stakeholders assessed the perceived feasibility, advantages and disadvantages of potential interventions. Interventions that respondents felt could be implemented in the short-term to reduce antibiotic usage effectively included: more frequent water exchanges , regular monitoring of culture water quality parameters , improved storage conditions for feed , use of probiotics and greater access to farmer and service providers training programmes . Other potential interventions included greater access to suitable and rapid diagnostics , high quality feeds , improved biosecurity measures and genetically-improved fish , but these solutions were expected to be achieved as long-term goals, with cost being of one of the noted barriers to implementation. Identifying feasible and sustainable interventions that can be taken to reduce antibiotic use, and understanding implementation barriers, are important for addressing antibiotic resistance and ensuring the continued efficacy of antibiotics. This is vital to ensuring the productivity of the tilapia sector in Egypt. The approach taken in the present study provides a means to identify points in the system where the effectiveness of interventions can be evaluated and thus it may be applied to other food production systems to combat the problem of antibiotic resistance., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Ballan wrasse (Labrus bergylta, Ascanius 1767) are cleaner fish cultured in northern Europe to remove sea lice from farmed Atlantic salmon (Salmo salar, Linnaeus 1758). Despite increasing appreciation for the importance of the microbiota on the phenotypes of vertebrates including teleosts, the microbiota of wrasse eggs has yet to be described. Therefore, the aim of this present study was to describe the bacterial component of the microbiota of ballan wrasse eggs shortly after spawning and at 5 days, once the eggs had undergone a routine incubation protocol that included surface disinfection steps in a common holding tank. Triplicate egg samples were collected from each of three spawning tanks and analysis of 16S rRNA gene sequences revealed that 88.6% of reads could be identified to 186 taxonomic families. At Day 0, reads corresponding to members of the Vibrionaceae, Colwelliaceae and Rubritaleaceae families were detected at greatest relative abundances. Bacterial communities of eggs varied more greatly between tanks than between samples deriving from the same tank. At Day 5, there was a consistent reduction in 16S rRNA gene sequence richness across the tanks. Even though the eggs from the different tanks were incubated in a common holding tank, the bacterial communities of the eggs from the different tanks had diverged to become increasingly dissimilar. This suggests that the disinfection and incubation exerted differential effects of the microbiota of the eggs from each tank and that the influence of the tank water on the composition of the egg microbiota was lower than expected. This first comprehensive description of the ballan wrasse egg bacterial community is an initial step to understand the role and function of the microbiota on the phenotype of this fish. In future, mass DNA sequencing methods may be applied in hatcheries to screen for pathogens and as a tool to assess the health status of eggs.

Atypical Aeromonas salmonicida causes furunculosis infections of non-salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one-quarter of that preventing visible bacterial growth, whereas for florfenicol it was one-half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re-infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic-resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida., (© 2020 The Authors. Journal of Fish Diseases published by John Wiley & Sons Ltd.)

Mass production and use of antibiotics has led to the rise of resistant bacteria, a problem possibly exacerbated by inappropriate and non-optimal application. Antibiotic treatment often follows fixed-dose regimens, with a standard dose of antibiotic administered equally spaced in time. But are such fixed-dose regimens optimal or can alternative regimens be designed to increase efficacy? Yet, few mathematical models have aimed to identify optimal treatments based on biological data of infections inside a living host. In addition, assumptions to make the mathematical models analytically tractable limit the search space of possible treatment regimens (e.g. to fixed-dose treatments). Here, we aimed to address these limitations by using experiments in a Galleria mellonella (insect) model of bacterial infection to create a fully parametrised mathematical model of a systemic Vibrio infection. We successfully validated this model with biological experiments, including treatments unseen by the mathematical model. Then, by applying artificial intelligence, this model was used to determine optimal antibiotic dosage regimens to treat the host to maximise survival while minimising total antibiotic used. As expected, host survival increased as total quantity of antibiotic applied during the course of treatment increased. However, many of the optimal regimens tended to follow a large initial 'loading' dose followed by doses of incremental reductions in antibiotic quantity (dose 'tapering'). Moreover, application of the entire antibiotic in a single dose at the start of treatment was never optimal, except when the total quantity of antibiotic was very low. Importantly, the range of optimal regimens identified was broad enough to allow the antibiotic prescriber to choose a regimen based on additional criteria or preferences. Our findings demonstrate the utility of an insect host to model antibiotic therapies in vivo and the approach lays a foundation for future regimen optimisation for patient and societal benefits., Competing Interests: The authors have declared that no competing interests exist.

Vibrio anguillarum is the causative agent of vibriosis in many species important to aquaculture. We generated whole genome sequence (WGS) data on a diverse collection of 64 V. anguillarum strains, which we supplemented with 41 publicly available genomes to produce a combined dataset of 105 strains. These WGS data resolved six major lineages (L1-L6), and the additional use of multilocus sequence analysis (MLSA) clarified the association of L1 with serotype O1 and Salmonidae hosts (salmon/trout), and L2 with serotypes O2a/O2b/O2c and Gadidae hosts (cod). Our analysis also revealed a large-scale homologous replacement of 526-kb of core genome in an L2 strain from a con-specific donor. Although the strains affected by this recombination event are exclusively associated with Gadidae , we find no clear genetic evidence that it has played a causal role in host specialism. Whilst it is established that Vibrio species freely recombine, to our knowledge this is the first report of a contiguous recombinational replacement of this magnitude in any Vibrio genome. We also note a smaller accessory region of high single nucleotide polymorphism (SNP) density and gene content variation that contains lipopolysaccharide biosynthesis genes which may play a role in determining serotype., (Copyright © 2020 Coyle, Bartie, Bayliss, Bekaert, Adams, McMillan, Verner-Jeffreys, Desbois and Feil.)

Neutrophils release nuclear chromatin decorated with antimicrobial proteins into the extracellular milieu as an innate immune defence mechanism to counter invading microbes. These chromatin structures, called extracellular traps (ETs) and released by a process called NETosis, have been detected in mammals, certain invertebrates and some fish species, including fathead minnow, zebrafish, common carp, turbot, sole and barramundi. However, there have been no previous studies of ETs in the Salmonidae. ETs are released in response to chemical and biological stimuli, but observations from different fish species are inconsistent, particularly regarding the potency of various inducers and inhibitors. Thus, this present study aimed to describe ET release in a salmonid (rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)) and uncover the inducers and inhibitors that can control this response. Highly enriched suspensions of polymorphonuclear cells (PMNs; mainly neutrophils) were prepared from head kidney tissues by a triple-layer Percoll gradient technique. ET structures were visualised in PMN-enriched suspensions through staining of the chromatin with nucleic acid-specific dyes and immunocytochemical probing of characteristic proteins expected to decorate the structure. ET release was quantified after incubation with inducers and inhibitors known to affect this response in other organisms. Structures resembling ETs stained positively with SYTOX Green (a stain specific for nucleic acid) while immunocytochemistry was used to detect neutrophil elastase, myeloperoxidase and H2A histone in the structures, which are diagnostic proteinaceous markers of ETs. Consistent with other studies on mammals and some fish species, calcium ionophore and flagellin were potent inducers of ETs, while cytochalasin D inhibited NETosis. Phorbol 12-myristate 13-acetate (PMA), used commonly to induce ETs, exerted only weak stimulatory activity, while heat-killed bacteria and lipopolysaccharide did not induce ET release. Unexpectedly, the ET-inhibitor diphenyleneiodonium chloride acted as an inducer of ET release, an observation not reported elsewhere. Taken together, these data confirm for the first time that ETs are released by salmonid PMNs and compounds useful for manipulating NETosis were identified, thus providing a platform for further studies to explore the role of this mechanism in fish immunity. This new knowledge provides a foundation for translation to farm settings, since manipulation of the innate immune response offers a potential alternative to the use of antibiotics to mitigate against microbial infections, particularly for pathogens where protection by vaccination has yet to be realised., Competing Interests: Declaration of competing interest The authors confirm they have no known conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence., Results: Larvae were killed by Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology (haematoxylin and eosin staining, Gram Twort and immunohistochemistry) and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts., Conclusions: This study shows that an alternative host, G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.

Aquaculture systems are highly complex, dynamic and interconnected systems influenced by environmental, biological, cultural, socio-economic and human behavioural factors. Intensification of aquaculture production is likely to drive indiscriminate use of antibiotics to treat or prevent disease and increase productivity, often to compensate for management and husbandry deficiencies. Surveillance or monitoring of antibiotic usage (ABU) and antibiotic resistance (ABR) is often lacking or absent. Consequently, there are knowledge gaps for the risk of ABR emergence and human exposure to ABR in these systems and the wider environment. The aim of this study was to use a systems-thinking approach to map two aquaculture systems in Vietnam - striped catfish and white-leg shrimp - to identify hotspots for emergence and selection of resistance, and human exposure to antibiotics and antibiotic-resistant bacteria. System mapping was conducted by stakeholders at an interdisciplinary workshop in Hanoi, Vietnam during January 2018, and the maps generated were refined until consensus. Thereafter, literature was reviewed to complement and cross-reference information and to validate the final maps. The maps and component interactions with the environment revealed the grow-out phase, where juveniles are cultured to harvest size, to be a key hotspot for emergence of ABR in both systems due to direct and indirect ABU, exposure to water contaminated with antibiotics and antibiotic-resistant bacteria, and duration of this stage. The pathways for human exposure to antibiotics and ABR were characterised as: occupational (on-farm and at different handling points along the value chain), through consumption (bacterial contamination and residues) and by environmental routes. By using systems thinking and mapping by stakeholders to identify hotspots we demonstrate the applicability of an integrated, interdisciplinary approach to characterising ABU in aquaculture. This work provides a foundation to quantify risks at different points, understand interactions between components, and identify stakeholders who can lead and implement change., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Numerous United Kingdom and European Union expert panels recommend that the general adult population consumes ~250 mg of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day through the consumption of one portion of oily fish per week. The long-chain omega-3 fatty acids EPA and DHA are only found in appreciable amounts in marine organisms. Increasing oily fish consumption conflicts with sustaining fisheries, so alternative dietary sources of EPA and DHA must be explored. Mussels are high in omega-3 polyunsaturated fatty acids (PUFAs) and a good source of essential amino acids. Therefore, we aimed to investigate the impact of introducing mussels as a protein source in the lunchtime meal three times per week for two weeks on the omega-3 status of free-living participants. Following an initial two-week monitoring period, 12 participants (eight male and four female) attended the nutrition laboratory three times per week for two weeks. Each participant received a personalised lunch constituting one-third of their typical daily calorie consumption with ~20% of the calories supplied as cooked mussels. A portion of cooked mussels from each feeding occasion was tested for total omega-3 content. The mean ± SD mussel EPA + DHA content was 518.9 ± 155.7 mg/100 g cooked weight, meaning that each participant received on average 709.2 ± 252.6 mg of EPA + DHA per meal or 304.0 ± 108.2 mg of EPA + DHA per day. Blood spot analysis revealed a significant increase in the omega-3 index (week 1 = 4.27 ± 0.81; week 4 = 5.07 ± 1.00) and whole blood EPA content during the study (%EPA week 1 = 0.70 ± 0.0.35; %EPA week 4 = 0.98 ± 0.35). Consuming mussels three times per week for two weeks as the protein source in a personalised lunchtime meal is sufficient to moderately improve the omega-3 index and whole blood DHA + EPA content in young healthy adults.

Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA; C20:5n-3), are attracting interest as possible new topical antibacterial agents, particularly due to their potency and perceived safety. However, relatively little is known of the underlying mechanism of antibacterial action of EPA or whether bacteria can develop resistance quickly against this or similar compounds. Therefore, the aim of this present study was to determine the mechanism of antibacterial action of EPA and investigate whether bacteria could develop reduced susceptibility to this fatty acid upon repeated exposure. Against two common Gram-positive human pathogens, Bacillus cereus and Staphylococcus aureus , EPA inhibited bacterial growth with a minimum inhibitory concentration of 64 mg/L, while minimum bactericidal concentrations were 64 mg/L and 128 mg/L for B. cereus and S. aureus , respectively. Both species were killed completely in EPA at 128 mg/L within 15 min at 37 °C, while reduced bacterial viability was associated with increased release of 260-nm-absorbing material from the bacterial cells. Taken together, these observations suggest that EPA likely kills B. cereus and S. aureus by disrupting the cell membrane, ultimately leading to cell lysis. Serial passage of the strains in the presence of sub-inhibitory concentrations of EPA did not lead to the emergence or selection of strains with reduced susceptibility to EPA during 13 passages. This present study provides data that may support the development of EPA and other fatty acids as antibacterial agents for cosmetic and pharmaceutical applications., Competing Interests: The authors declare no conflict of interest.

Background: Microbial diseases cause considerable economic losses in aquaculture and new infection control measures often rely on a better understanding of pathogenicity. However, disease studies performed in fish hosts often require specialist infrastructure (e.g., aquaria), adherence to strict legislation and do not permit high-throughput approaches; these reasons justify the development of alternative hosts. This study aimed to validate the use of larvae of the greater wax moth (Galleria mellonella) to investigate virulence of the important fish pathogen, Vibrio anguillarum., Results: Using 11 wild-type isolates of V. anguillarum, these bacteria killed larvae in a dose-dependent manner and replicated inside the haemolymph, but infected larvae were rescued by antibiotic therapy. Crucially, virulence correlated significantly and positively in larva and Atlantic salmon (Salmo salar) infection models. Challenge studies with mutants knocked out for single virulence determinants confirmed conserved roles in larva and fish infections in some cases (pJM1 plasmid, rtxA), but not all (empA, flaA, flaE)., Conclusions: The G. mellonella model is simple, more ethically acceptable than experiments on vertebrates and, crucially, does not necessitate liquid systems, which reduces infrastructure requirements and biohazard risks associated with contaminated water. The G. mellonella model may aid our understanding of microbial pathogens in aquaculture and lead to the timely introduction of new effective remedies for infectious diseases, while adhering to the principles of replacement, reduction and refinement (3Rs) and considerably reducing the number of vertebrates used in such studies.

Marine algae are a relatively untapped source of bioactive natural products, including those with antimicrobial activities. The ability to assess the antimicrobial activity of cell extracts derived from algal cultures is vital to identifying species that may produce useful novel antibiotics. One assay that is used widely for this purpose is the disk diffusion assay due to its simplicity, rapidity, and low cost. Moreover, this assay gives output data that are easy to interpret and can be used to screen many samples at once irrespective of the solvent used during preparation. In this chapter, a step-by-step protocol for performing a disk diffusion assay is described. The assay is particularly well suited to testing algal cell extracts and fractions resulting from separation through bioassay-guided approaches.

New compounds are needed to treat acne and superficial infections caused by Propionibacterium acnes and Staphylococcus aureus due to the reduced effectiveness of agents used at present. Long-chain polyunsaturated fatty acids (LC-PUFAs) are attracting attention as potential new topical treatments for Gram-positive infections due to their antimicrobial potency and anti-inflammatory properties. This present study aimed to investigate the antimicrobial effects of six LC-PUFAs against P. acnes and S. aureus to evaluate their potential to treat infections caused by these pathogens. Minimum inhibitory concentrations were determined against P. acnes and S. aureus, and the LC-PUFAs were found to inhibit bacterial growth at 32-1024 mg/L. Generally, P. acnes was more susceptible to the growth inhibitory actions of LC-PUFAs, but these compounds were bactericidal only for S. aureus. This is the first report of antibacterial activity attributed to 15-hydroxyeicosapentaenoic acid (15-OHEPA) and 15-hydroxyeicosatrienoic acid (HETrE), while the anti-P. acnes effects of the six LC-PUFAs used herein are novel observations. During exposure to the LC-PUFAs, S. aureus cells were killed within 15-30 min. Checkerboard assays demonstrated that the LC-PUFAs did not antagonise the antimicrobial potency of clinical agents used presently against P. acnes and S. aureus. However, importantly, synergistic interactions against S. aureus were detected for combinations of benzoyl peroxide with 15-OHEPA, dihomo-γ-linolenic acid (DGLA) and HETrE; and neomycin with 15-OHEPA, DGLA, eicosapentaenoic acid, γ-linolenic acid and HETrE. In conclusion, LC-PUFAs warrant further evaluation as possible new agents to treat skin infections caused by P. acnes and S. aureus, especially in synergistic combinations with antimicrobial agents already used clinically.

Objectives: To evaluate the in vivo effectiveness of a combination treatment containing ranalexin (a natural antimicrobial peptide) and lysostaphin (an antistaphylococcal endopeptidase) for reducing nasal burden of methicillin-resistant Staphylococcus aureus (MRSA)., Methods: The community-acquired MRSA strain S. aureus NRS384 (USA300-0114) was used in the present study because it is commonly isolated from human nares and it established consistent and reproducible colonization of cotton rat nares. This model was used to evaluate the efficacy of ranalexin/lysostaphin gels (0.1%-1% w/v; administered intranasally once or once per day for 3 consecutive days) for reducing nasal MRSA burden. Control animals were administered vehicle gel only (0.5% hydroxypropyl methylcellulose) or 2% mupirocin, which is used clinically for nasal decolonization of MRSA. Nasal MRSA burden was assessed at 192 h post-inoculation, which was at least 72 h after the final treatment had been administered. An additional study assessed the efficacy of 0.1% ranalexin/lysostaphin against a mupirocin-resistant MRSA strain (MUP20), which had been selected by serial passage of S. aureus NRS384 through subinhibitory concentrations of mupirocin., Results: Gels containing 0.1% ranalexin/lysostaphin consistently reduced median nasal burden of MRSA to an extent similar to or greater than 2% mupirocin. Treatment with 0.1% ranalexin/lysostaphin was also effective against the MUP20 strain. There was evidence for only minimal irritancy in cotton rat nares administered three doses of 0.1% ranalexin/lysostaphin, suggesting that this agent is suitable for short-course therapy such as is employed currently for nasal decolonization with mupirocin., Conclusions: Ranalexin/lysostaphin could serve as an alternative to mupirocin for nasal decolonization of MRSA.

The objective of this study was to determine whether combinations of antimicrobial peptides (AMPs) with caspofungin display enhanced antifungal activity versus Candida albicans in vitro and in vivo. Three conventional AMPs that satisfied criteria favouring their potential development as novel antifungals were selected for investigation. Colistin sulphate was also included as a cyclic peptide antibiotic used in the clinic. Minimum inhibitory concentrations (MICs) were determined for each antifungal agent and checkerboard assays were used to determine fractional inhibitory concentration index (FICI) values for dual combinations of AMPs or colistin with caspofungin. Viability assays were performed for the same combinations in order to investigate fungicidal interactions. Synergistic antifungal combinations were then tested for efficacy in vivo and compared to monotherapies in wax moth larva and murine models of systemic C. albicans infection. In combination with caspofungin, each of the AMPs [hMUC7-12, DsS3(1-16), hLF(1-11)] and colistin were synergistic and candidacidal in vitro. The treatment of infected wax moth larvae with combinations of caspofungin with hMUC7-12, DsS3(1-16) or colistin resulted in significant enhancements in survival compared to treatment with monotherapies. Notably, the treatment of C. albicans-infected mice with a combination of caspofungin and DsS3(1-16) resulted in the enhancement of survival compared to groups treated with just the individual agents. This study demonstrates that combination therapies containing caspofungin and AMPs or colistin merit further development as potential novel treatments for C. albicans infections.

The antimicrobial effects of free fatty acids are well recognised and these compounds can prevent the growth of or directly kill bacteria, fungi and other microbes by affecting multiple cellular targets, including the cell membrane and components found therein. Moreover, fatty acids exert detrimental effects on microbial pathogens by interfering with mechanisms of virulence, such as preventing biofilm formation and inhibiting the production of toxins and enzymes. The antimicrobial properties of free fatty acids can be exploited for the preservation of perishable products, such as food and cosmetics, and for the prevention and treatment of infections. These safe natural products are particularly useful in circumstances where antimicrobial activity is required but where the use of conventional antibiotics is undesirable or forbidden. This review focuses on the most promising prospects for exploiting the antimicrobial properties of free fatty acids for applications in various industries. The benefits of using fatty acids as antimicrobial agents are discussed and relevant recent patents are highlighted.

There is an urgent need for new antimicrobial agents to combat infections caused by drug-resistant pathogens. Once a compound is shown to be effective in vitro, it is necessary to evaluate its efficacy in an animal infection model. Typically, this is achieved using a mammalian model, but such experiments are costly, time consuming, and require full ethical consideration. Hence, cheaper and ethically more acceptable invertebrate models of infection have been introduced, including the larvae of the greater wax moth Galleria mellonella. Invertebrates have an immune system that is functionally similar to the innate immune system of mammals, and often identical virulence and pathogenicity factors are used by human pathogenic microbes to infect wax moth larvae and mammals. Moreover, the virulence of many human pathogens is comparable in wax moth larvae and mammals. Using key examples from the literature, this chapter highlights the benefits of using the wax moth larva model to provide a rapid, inexpensive, and reliable evaluation of the toxicity and efficacy of new antimicrobial agents in vivo and prior to the use of more expensive mammalian models. This simple insect model can bridge the gap between in vitro studies and mammalian experimentation by screening out compounds with a low likelihood of success, while providing greater justification for further studies in mammalian systems. Thus, broader implementation of the wax moth larva model into anti-infective drug discovery and development programs could reduce the use of mammals during preclinical assessments and the overall cost of drug development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many cationic peptides with antimicrobial properties have been isolated from bacteria, fungi, plants, and animals. These peptides vary in molecular size, potency and spectra of activities. This report surveyed the literature to highlight the peptides that have antifungal activity and greatest potential for development as new therapeutic agents. Thus, to be included in the evaluation, each peptide had to fulfil the following criteria: (i) potent antifungal activity, (ii) no, or minimal, mammalian cell toxicity, (iii) of ≤25 amino acids in length, which minimises the costs of synthesis, reduces immunogenicity and enhances bioavailability and stability in vivo, (iv) minimal post-translational modifications (also reduces the production costs). The ~80 peptides that satisfied these criteria are discussed with respect to their structures, mechanisms of antimicrobial action and in vitro and in vivo toxicities. Certainly, some of these small peptides warrant further study and have potential for future exploitation as new antifungal agents.

Drug-resistant staphylococci constitute a serious problem that urgently requires the discovery of new therapeutic agents. There has been a resurgence in interest in using lysostaphin (a specific anti-staphylococcal enzyme) as a treatment for infections caused by these important pathogens. However, bacterial resistance to lysostaphin is a problem, but the use of a combination treatment may surmount this issue. In this present study, using viable counts from suspension incubations, lysostaphin is shown to be synergistically bactericidal in combination with various conventional antimicrobial peptides, the antimicrobial protein bovine lactoferrin, a lantibiotic (nisin), and certain lipopeptides used clinically (colistin, daptomycin and polymyxin B). Combinations that act in synergy are of clinical importance as these reduce the doses of the compounds needed for effective treatments and decrease the chances of resistance being selected. The use of lysostaphin in combination with a peptide may represent a new avenue in tackling drug-resistant staphylococci.

New treatments are urgently required for infections caused by meticillin-resistant Staphylococcus aureus (MRSA) as these strains are often resistant to multiple conventional antibiotics. Earlier studies showed that ranalexin, an antimicrobial peptide (AMP), in combination with lysostaphin, an antistaphylococcal endopeptidase, synergistically inhibits the growth of MRSA, meaning that it deserved consideration as a new anti-S. aureus therapy. Using haemolysis and Vero cell viability assays, ranalexin with lysostaphin is proven to be non-toxic at antibacterial concentrations. In human serum, ranalexin with lysostaphin is significantly more effective against MRSA than treatment with either component alone. In a rabbit model of wound infection, ranalexin with lysostaphin reduced MRSA in the wound by ca. 3.5log(10) colony-forming units (CFU) compared with the untreated control. The combination is significantly more effective than treatment with ranalexin or lysostaphin alone. In a mouse model of systemic infection, ranalexin with lysostaphin reduced MRSA kidney burden by ca. 1log(10)CFU/g compared with untreated controls or treatment with ranalexin or lysostaphin alone. Importantly, the combination is synergistically bactericidal against various S. aureus isolates in vitro, including those with reduced susceptibility to lysostaphin or vancomycin. Ranalexin and lysostaphin could be incorporated in wound dressings for the prevention and treatment of topical S. aureus infections. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci., (2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

All eukaryotic organisms, single-celled or multi-cellular, produce a diverse array of natural anti-infective agents that, in addition to conventional antimicrobial peptides, also include proteins and other molecules often not regarded as part of the innate defences. Examples range from histones, fatty acids, and other structural components of cells to pigments and regulatory proteins. These probably represent very ancient defence factors that have been re-used in new ways during evolution. This review discusses the nature, biological role in host protection and potential biotechnological uses of some of these compounds, focusing on those from fish, marine invertebrates and marine micro-algae.

Aims: To characterize the antibacterial synergy of the antimicrobial peptide, ranalexin, used in combination with the anti-staphylococcal endopeptidase, lysostaphin, against methicillin-resistant Staphylococcus aureus (MRSA), and to assess the combination's potential as a topical disinfectant or decolonizing agent for MRSA. MRSA causes potentially lethal infections, and pre-operative patients colonized with MRSA are often treated with chlorhexidine digluconate and mupirocin cream to eradicate carriage. However, chlorhexidine is unsuitable for some patients, and mupirocin resistance is increasingly encountered, indicating new agents are required., Methods and Results: Using an ex vivo assay, ranalexin and lysostaphin tested in combination reduced viable MRSA on human skin to a greater extent than either compound individually. The combination killed bacteria within 5 min and remained effective and synergistic even in high salt and low pH conditions., Conclusions: The combination is active against MRSA on human skin and under conditions that may be encountered in sweat., Significance and Impact of the Study: Although the exact mechanism of activity remains unresolved, considering its specific spectrum of activity, fast killing kinetics and low likelihood of resistance arising, the combination of ranalexin with lysostaphin warrants consideration as a new agent to eradicate nasal and skin carriage of Staph. aureus, including MRSA.

Amongst the diverse and potent biological activities of free fatty acids (FFAs) is the ability to kill or inhibit the growth of bacteria. The antibacterial properties of FFAs are used by many organisms to defend against parasitic or pathogenic bacteria. Whilst their antibacterial mode of action is still poorly understood, the prime target of FFA action is the cell membrane, where FFAs disrupt the electron transport chain and oxidative phosphorylation. Besides interfering with cellular energy production, FFA action may also result from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and auto-oxidation degradation products or direct lysis of bacterial cells. Their broad spectrum of activity, non-specific mode of action and safety makes them attractive as antibacterial agents for various applications in medicine, agriculture and food preservation, especially where the use of conventional antibiotics is undesirable or prohibited. Moreover, the evolution of inducible FFA-resistant phenotypes is less problematic than with conventional antibiotics. The potential for commercial or biomedical exploitation of antibacterial FFAs, especially for those from natural sources, is discussed.

Pathogenic bacteria, such as multidrug-resistant Staphylococcus aureus (MRSA), which are not susceptible to most conventional antibiotics, are causing increased concern in healthcare institutions worldwide. The discovery of novel antibacterial compounds for biomedical exploitation is one avenue that is being pursued to combat these problematic bacteria. Marine eukaryotic microalgae are known to produce numerous useful products but have attracted little attention in the search for novel antibiotic compounds. Cell lysates of the marine diatom, Phaeodactylum tricornutum Bohlin, have been reported to display antibacterial activity in vitro, but the compounds responsible have not been fully identified. In this paper, using column chromatography and reversed-phase high-performance liquid chromatography, we report the isolation of an antibacterial fatty acid. Mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy revealed it to be the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). We show that EPA is active against a range of both Gram-positive and Gram-negative bacteria, including MRSA, at micromolar concentrations. These data indicate that it could find application in the topical and systemic treatment of drug-resistant bacterial infections.

One solution to the global crisis of antibiotic resistance is the discovery of novel antimicrobial compounds for clinical application. Marine organisms are an attractive and, as yet, relatively untapped resource of new natural products. Cell extracts from the marine diatom, Phaeodactylum tricornutum, have antibacterial activity and the fatty acid, eicosapentaenoic acid (EPA), has been identified as one compound responsible for this activity. During the isolation of EPA, it became apparent that the extracts contained further antibacterial compounds. The present study was undertaken to isolate these additional antibacterial factors using silica column chromatography and reverse-phase high-performance liquid chromatography. Two antibacterial fractions, each containing a pure compound, were isolated and their chemical structures were investigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The antibacterial compounds were identified as the monounsaturated fatty acid (9Z)-hexadecenoic acid (palmitoleic acid; C16:1 n-7) and the relatively unusual polyunsaturated fatty acid (6Z, 9Z, 12Z)-hexadecatrienoic acid (HTA; C16:3 n-4). Both are active against Gram-positive bacteria with HTA further inhibitory to the growth of the Gram-negative marine pathogen, Listonella anguillarum. Palmitoleic acid is active at micro-molar concentrations, kills bacteria rapidly, and is highly active against multidrug-resistant Staphylococcus aureus. These free fatty acids warrant further investigation as a new potential therapy for drug-resistant infections.