Explore the vast range of titles available.

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 µM, respectively. Violacein showed a MIC of 15 µM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Violacein is a natural indole-derived purple pigment of microbial origin that has attracted attention for its remarkable biological properties. Due to its poor solubility in aqueous media, most studies of this pigment use extracts of the compound obtained with common solvents. Violacein is also transported in bacterial extracellular vesicles (EVs) and transferred via this type of carrier remains stable in an aqueous environment. This paper is the first to present an in-depth study of Janthinobacterium lividum EVs as violacein carriers. J. lividum EVs were studied for their contribution to violacein translocation, size, morphology and protein composition. The production of violacein encapsulated in EVs was more efficient than the intracellular production of this compound. The average size of the violacein-containing EVs was 124.07 ± 3.74 nm. Liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) revealed 932 proteins common to three independent EVs isolations. The high proportion of proteins with intracellular localisation, which are involved in many fundamental cellular processes, suggests that J. lividum EVs could be generated in a cell lysis model, additionally stimulated by violacein production. Using human keratinocytes and melanoma cell lines, it was confirmed that J. lividum EVs are able to react with and deliver their cargo to mammalian cells. The EVs-delivered violacein was shown to retain its activity against melanoma cells, and the dose and timing of treatment can be selected to target only cancer cells. The characterisation of J. lividum EVs, described in the following paper, represents a milestone for their future potential anticancer application. Key points • This report focuses on the investigation of Janthinobacterium lividum EVs as a new delivery vehicle for violacein, a compound with a previously demonstrated broad spectrum of activity. • EVs were characterised for size, morphology and protein composition. • Studies on human keratinocytes and a melanoma cell model confirmed that the activity of violacein applied in the encapsulated form of EVs is similar to that of its organic solvent extract, but their production is much more environmentally friendly.

The aim of the current research was to improve violacein production with Janthinobacterium lividum using abiotic stresses and bacterial adaptation against stress. Initially, the effect of carbon sources and the medium volume: air ratio on violacein production was assessed. Then, the production of violacein under hydrogen peroxide (H 2 O 2 ) and ampicillin (Amp) stresses and acyl homoserine lactone (AHL) was evaluated. In the next step, J. lividum was adapted against increased concentrations of Amp. Finally, the production of violacein was analyzed in adapted bacterium cultivated in the presence of optimal amounts of H 2 O 2 , Amp, and AHL. The alterations in the expression of some of genes involved in violacein production was evaluated using Real-time PCR (RT-PCR). The highest amount of violacein was achieved using medium volume: air ratio of 10% v/v (in 100 ml flasks) and glycerol as carbon source. Also, H 2 O 2 (103 mg/l) and Amp (130 mg/l) stresses increased the production of violacein significantly compared to normal conditions (57 mg/l) and violacein production in the presence of crude AHL increased from 56 mg/l to 210 mg/l. The production of violacein with adapted bacterium under the above-mentioned stresses and AHL was about 1.3 g/l. RT-PCR results showed that the expression of the AHL encoding gene ( lux I) was repressed in the presence of stresses and glycerol. Also, the expression of vio A increased in the presence of Amp but H 2 O 2 had no significant effect on vio A expression. Totally, we showed that microbial adaptation and abiotic stresses are cost-effective methods to generate significant improvement in violacein production.

Regional and global trade of live animals can contribute to the spread and emergence of novel pathogens, including several important pathogens of amphibians. However, understanding the spread or even frequency of infections in large, complex amphibian trade networks has been difficult, in part because businesses tend to be reluctant to participate in surveillance programs. Thus, we developed a novel approach to surveillance in which anonymous participating businesses were sent surveillance kits through a trusted trade advocacy partner, samples were returned to researchers via anonymous prepaid envelopes, and results were provided via a secure website with access regulated by a unique personal identification number (PIN) created by the business. We tested samples for the amphibian pathogens, Batrachochytrium salamandrivorans (Bsal), Batrachochytrium dendrobatidis (Bd), and Ranavirus spp. (Rv), as well as the beneficial microbe, Janthinobacterium lividum (Jliv), using quantitative real‐time polymerase chain reaction (qPCR). Out of 120 businesses invited to complete an anonymous socioeconomic survey, 24 volunteered to participate in pathogen surveillance, of which 14 were sent surveillance kits. Eight of these businesses returned samples consisting of swabs collected from amphibians in 78 terrestrial habitats and water filters from 49 aquatic habitats. Copies of a highly conserved vertebrate gene (EBF3N), quantified using qPCR, were consistently low (<100 copies) in returned samples, but similar to those collected by researchers, indicating comparable sample quality. Three samples (from two facilities) had detectable levels of Bd DNA; Bsal, Rv, and Jliv were not detected. This pilot study provides evidence that information about pathogens in pet trade networks can be acquired by developing partnerships with industry, and business participation might be enhanced by ensuring anonymity and inclusion of a trade advocacy partner.

The aim of this paper is to describe a new variant of Janthinobacterium lividum - ROICE173, isolated from Antarctic snow, and to investigate the antimicrobial effect of the crude bacterial extract against 200 multi-drug resistant (MDR) bacteria of both clinical and environmental origin, displaying various antibiotic resistance patterns. ROICE173 is extremotolerant, grows at high pH (5.5–9.5), in high salinity (3%) and in the presence of different xenobiotic compounds and various antibiotics. The best violacein yield (4.59 ± 0.78 mg·g −1 wet biomass) was obtained at 22 °C, on R2 broth supplemented with 1% glycerol. When the crude extract was tested for antimicrobial activity, a clear bactericidal effect was observed on 79 strains (40%), a bacteriostatic effect on 25 strains (12%) and no effect in the case of 96 strains (48%). A very good inhibitory effect was noticed against numerous MRSA, MSSA, Enterococci , and Enterobacteriaceae isolates. For several environmental E. coli strains, the bactericidal effect was encountered at a violacein concentration below of what was previously reported. A different effect (bacteriostatic vs. bactericidal) was observed in the case of Enterobacteriaceae isolated from raw vs. treated wastewater, suggesting that the wastewater treatment process may influence the susceptibility of MDR bacteria to violacein containing bacterial extracts.

In the course of assessing the sensitivity of the Janthinobacterium lividum VKM B-3515 strain to low concentrations of hydrogen peroxide, it was found that at a content of 0.003% H 2 O 2 , the growth properties of the bacterium during submerged cultivation without pigmentation differed statistically insignificantly relative to the control variant at 16 hours of incubation and beyond. Whereas in the presence of peroxide at 12 hours the optical density was lower than in the control by 97%. When cultivating by the surface method, the respiration intensity did not significantly differ between the control and experimental variants. However, during the extraction of the pigment, it was found that in the presence of hydrogen peroxide, the optical density of the acetone extract significantly exceeds the control variant by 28%. It can be assumed that, at the same growth parameters of the culture, the biosynthesis of violacein is stimulated and the population can adapt to the peroxide content, and the peroxide concentration itself decreases due to the cost of catalytic reactions. Further studies of the sensitivity of J. lividum VKM B-3515 to various oxidizing agents will allow us to consider the effect of weak oxidative stress on the biosynthesis of violacein.

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides—the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin—from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

Violacein and its biosynthesis by-product deoxyviolacein are valuable natural pigments with different biological activities. Various efforts have been made to enhance violacein and deoxyviolacein production in microbes. However, the effect of different culture media, agitation, and fungal elicitation on biosynthesis in Janthinobacterium has not been evaluated. In this study, the effect of eight different culture media, agitation, and fungal elicitation by Agaricus bisporus on violacein and deoxviolacein production in Janthinobacterium agaricidamnosum DSM 9628 and Janthinobacterium lividum DSM 1552 were examined. The results showed that violacein and deoxviolacein are produced at high-levels when Janthinobacterium is cultivated in minimal media such as Davis minimal broth with glycerol (DMBgly), shipworm basal medium (SBM), and MM9 media. A 50-fold increase was observed in violacein production when Janthinobacterium was cultivated in these media compared to cultivation in Luria–Bertani (LB), nutrient broth (NB), and King’s B (KB). Agitation reduces violacein and deoxyviolacein production, while fungal elicitation decreases violacein but increases deoxyviolacein when Janthinobacterium is cultured in KB media, SBM, and modified SBM (MSBM). An antibacterial assay using various pathogenic bacteria showed that violacein and deoxyviolacein extracted from Janthinobacterium are effective against both Gram-positive and Gram-negative pathogens, confirming their functionality as antibacterial agents. The findings suggest that cultivation in minimal media and fungal elicitation might invoke a stress response, enhancing the production of violacein and deoxviolacein in Janthinobacterium.

Violacein is a natural violet pigment produced by several Gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH2 at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006-2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.[PUBLICATION ABSTRACT]