Antibacterial Activity of Long-Chain Polyunsaturated Fatty Acids against Propionibacterium acnes and Staphylococcus aureus

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

Paving the way to acceptance of Galleria mellonella as a new model insect

First paragraph: The larva of the greater wax moth Galleria mellonella is an alternative host used commonly in studies of microbial infection and innate immunity. Indeed, this insect host is often used when quantifying or comparing the virulence of bacterial and fungal pathogens of vertebrates and it has been used successfully to establish the importance of microbial virulence factors and to determine the relative virulence of different isolates of the same species. The recent popularity of G. mellonella as an alternative host system stems from numerous benefits, including the ability to perform experiments at a range of temperatures including human body core temperature; the technical simplicity of establishing infections by various routes such as through feeding, topical application or injection; the convenient size of the insect, which means it is large enough to permit simple injection of inoculums or chemicals but small enough to require little space in the laboratory; the ability to assess the efficacy and toxicity of antimicrobial therapies; and the ease and reliability with which these insects can be sourced in their final instar stage from commercial suppliers. It has also found approval amongst many researchers due to the favourable reproducibility between experiments in the same laboratory. Nevertheless, relatively small variations in susceptibility to infection can occur between batches of larvae from the same supplier and such variation probably arises from factors such as age, size and nutritional status on receipt; conditions encountered during transit to the laboratory; and the presence of any underlying natural infections. These issues are largely uncontrollable when purchasing larvae from a commercial supplier but on reaching the laboratory standardised pre-experimentation storage conditions can improve reproducibility between studies. In recent years the Kavanagh group have raised awareness for the role of a number of variables during storage that require consideration to ensure optimal reproducibility when experimenting with this insect, and factors influencing G. mellonella susceptibility to infections include physical stress, incubation temperature and access to food. In this edition of Virulence, the Kavanagh group report that larvae become increasingly susceptible to infection by pathogens as laboratory storage time increases, highlighting the need to consider this parameter when using the G. mellonella model. Browne et al. elaborate further in the study and relate this observation to a reduction in the total abundance of haemocytes that function in immune defence against pathogens and changes in the relative flux of metabolic pathways. Interestingly, the number of haemocytes after 3 weeks of incubation was approximately half that compared to the population at one week, while qualitative changes in the relative abundance of the various types of haemocytes were also reported. Both these factors probably contribute to reduced immune capacity and thus increased susceptibility to infection.Output Type: Editoria

Wax moth larva (Galleria mellonella): An in vivo model for assessing the efficacy of antistaphylococcal agents

Objectives - To investigate whether the wax moth larva, Galleria mellonella, is a suitable host for assessing the in vivo efficacy of antistaphylococcal agents against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) infections. Methods - Wax moth larvae were infected with increasing doses of S. aureus to investigate the effect of inoculum size on larval survival. In addition, infected wax moth larvae were treated with daptomycin, penicillin or vancomycin to examine whether these agents were effective against S. aureus and MRSA infections in vivo. Results - Increasing inoculum doses of live S. aureus cells resulted in greater larval mortality, but heat-killed bacteria and cell-free culture filtrates had no detrimental effects on survival. Larval mortality rate also depended on the post-inoculation incubation temperature. After larvae were infected with S. aureus, larval survival was enhanced by administering the antistaphylococcal antibiotics daptomycin or vancomycin. Larval survival increased with increasing doses of the antibiotics. Moreover, penicillin improved survival of larvae infected with a penicillin-susceptible methicillin-susceptible S. aureus (MSSA) strain, but it was ineffective at similar doses in larvae infected with MRSA (penicillin resistant). Daptomycin and vancomycin were also effective when administered to the larvae prior to infection with bacteria. Conclusions - This is the first report to demonstrate that antibiotics are effective in the wax moth larva model for the treatment of infections caused by Gram-positive bacteria. The new wax moth larva model is a useful preliminary model for assessing the in vivo efficacy of candidate antistaphylococcal agents before proceeding to mammalian studies, which may reduce animal experimentation and expense

Antibacterial Effect of Eicosapentaenoic Acid against Bacillus cereus and Staphylococcus aureus: Killing Kinetics, Selection for Resistance, and Potential Cellular Target

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

Aeromonas dhakensis: A Zoonotic Bacterium of Increasing Importance in Aquaculture

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

Bacterial Communities of Ballan Wrasse (Labrus bergylta) Eggs at a Commercial Marine Hatchery

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

Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis

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

Prophylactic properties of biofloc- or Nile tilapia-conditioned water against Vibrio parahaemolyticus infection of whiteleg shrimp (Penaeus vannamei)

Isolates of Vibrio parahaemolyticus (VpAHPND) that carry a plasmid encoding two Pir-like toxins cause acute hepatopancreatic necrosis disease (AHPND), a disease that has caused devastating economic losses to the shrimp industry, particularly in Asia. However, lower prevalence of AHPND infection has been associated with farms that operate with biofloc or lower salinity culture water. Therefore, the aim of this present study was to investigate the effects of biofloc and Nile tilapia (Oreochromis niloticus)-conditioned water prepared at different culture water salinities on survival of whiteleg shrimp (Penaeus vannamei) bath-challenged experimentally with VpAHPND. First, groups of shrimp were bath-challenged with VpAHPND in clear 15 ppt seawater (CW) or in the presence of a pre-cultured biofloc at 25%, 50% and 100% (v/v). Survival during 96 h post-challenge was significantly greater in groups cultured in 50% and 100% biofloc (p

Antimicrobial action of chromatin extracellular traps released by neutrophils of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792)

First paragraph: Chromatin extracellular traps (ETs) are released in vitro by certain fish polymorphonuclear leucocytes (PMNs), including neutrophils, in response to various chemical and biological stimuli such as bacterial cells and components like flagellin and lipopolysaccharide [[1], [2], [3], [4]]. For mammals in particular, ETs have been shown to exert antimicrobial properties and function as part of the innate response [5,6]. However, there are few studies on the antimicrobial actions of ETs released by fish, with these properties reported for ETs in immune cell suspensions prepared from common carp (Cyprinus carpio) [7], turbot (Scophthalmus maximus) [8,9] and tongue sole (Cynoglossus semilaevis) [1]. In contrast, some studies failed to provide evidence for antibacterial activities, including those examining tongue sole ETs against Edwardsiella tarda [1] and Atlantic salmon (Salmo salar) ETs against Aeromonas salmonicida [4], indicating complexity in the interactions between bacteria and the actions of the ETs. This present study investigated whether ETs released by neutrophils of rainbow trout (Oncorhynchus mykiss) exert antibacterial activity against the important finfish pathogen, Vibrio anguillarum

Release of chromatin extracellular traps by phagocytes of Atlantic salmon, Salmo salar (LINNAEUS, 1758)

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