Fish-derived antimicrobial peptides: Activity of a chionodracine mutant against bacterial models and human bacterial pathogens.

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.

Psychrobacter sanguinis Wound Infection Associated with Marine Environment Exposure, Washington, USA.

We report a 26-year-old man with Psychrobacter sanguinis cellulitis of a wound sustained during ocean fishing in Washington, USA, in 2017. Psychrobacter spp. are opportunistic pathogens found in a wide range of environments. Clinicians should be aware of Psychrobacter spp. and perform 16S rRNA sequencing if this pathogen is suspected.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus.

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

Antibiotic resistance of bacteria isolated from the internal organs of edible snow crabs.

Antibiotic resistance and microbiota within edible snow crabs are important for the Chionoecetes (snow crab) fishing industry. We investigated these parameters using culture methods and antibiotic susceptibility tests with six internal organs from three species of Chionoecetes. Each sample revealed many unexpected microbial species within Chionoecetes internal organs. On the basis of 16S rRNA sequence analysis of 381 isolates, the most abundant genera identified in Chionoecetes opilio were Acinetobacter spp. (24%), Bacillus spp. (4%), Pseudomonas spp. (34%), Stenotrophomonas spp. (28%), and Agreia spp. (11%). In Chionoecetes sp. crabs, Acinetobacter spp. (23%), Bacillus spp. (12%), and Psychrobacter spp. (20%) were most prevalent, while Agreia spp. (11%), Bacillus spp. (31%), Microbacterium spp. (10%), Rhodococcus spp. (12%), and Agrococcus spp. (6%) were most abundant in C. japonicus. Our antibiotic resistance test found resistance to all nine antibiotics tested in 19, 14, and two of the isolates from C. opilio, Chionoecetes sp., and, C. japonicus respectively. Our results are the first to show that microbes with antibiotic resistance are widely distributed throughout the internal organs of natural snow crabs.

Psychrobacter arenosus bacteremia after blood transfusion, France.

We report a case of transfusion-associated bacteremia caused by Psychrobacter arenosus. This psychrotolerant bacterium was previously isolated in 2004 from coastal sea ice and sediments in the Sea of Japan, but not from humans. P. arenosus should be considered a psychrotolerant bacterial species that can cause transfusion-transmitted bacterial infections.

p-Benzoquinone-mediated amperometric biosensor developed with Psychrobacter sp. for toxicity testing of heavy metals.

A rapid and reliable p-benzoquinone-mediated amperometric biosensor (ToxTell) incorporated with Psychrobacter sp. to detect toxicities of heavy metal ions has been developed. This ToxTell biosensor relied on the real-time monitoring of inhibition effect for metabolism by toxicant to provide early detection and assessment of the degree of toxicity to living cells. The effect of growth phase on the sensitivity of Psychrobacter sp. biosensor was studied. The results showed that at the middle of the logarithmic phase or transition from logarithmic to stationary phase, the Psychrobacter sp. ToxTell biosensor had a higher sensitivity to toxicants. The effects of pH, salinity in respiratory substrates and incubation time on the performance of Psychrobacter sp. biosensor were also investigated. EC(50) values of Cu(2+), Cd(2+), Zn(2+), Cr(6+), Hg(2+) and Pb(2+) to Psychrobacter sp. determined at incubation time 30 min were 2.6 mg/L, 47.3 mg/L, 10.9 mg/L, 14.0 mg/L, 0.8 mg/L and 110.1 mg/L, respectively. The ToxTell microbial biosensor developed in this work demonstrated excellent storage stability for more than 60 days. The biosensor could incorporate different microbial species as biocomponent to reflect the comprehensive values for toxicants in real samples and the results therefore had high degree of validity.

Inoculation of Ni-resistant plant growth promoting bacterium Psychrobacter sp. strain SRS8 for the improvement of nickel phytoextraction by energy crops.

This study was conducted to elucidate effects of inoculating plant growth-promoting bacterium Psychrobacter sp. SRS8 on the growth and phytoextraction potential of energy crops Ricinus communis and Helianthus annuus in artificially Ni contaminated soils. The toxicity symptom in plants under Ni stress expressed as chlorophyll, protein content, growth inhibition, and Fe, P concentrations were studied, and the possible relationship among them were also discussed. The PGPB SRS8 was found capable of stimulating plant growth and Ni accumulation in both plant species. Further, the stimulation effect on plant biomass, chlorophyll, and protein content was concomitant with increased Fe and P assimilation from soil to plants. Further, the induction of catalase and peroxidase activities was also involved in the ability of SRS8 to increase the tolerance in both plant species under Ni stress. The findings suggest that strain SRS8 play an important role in promoting the growth and phytoextraction efficiency of R. communis and H. annuus, which may be used for remediation of metal contaminated sites.

Treatment of fresh poultry carcases with emulsions of glycerol monocaprate (monocaprin) to reduce contamination with Campylobacter and psychrotrophic bacteria.

1. A previous study has shown that emulsions of monocaprin in citrate lactate buffer at pH 4·1-4·3 are highly active in killing Campylobacter in water, where they reduce viable bacterial counts by more than 6 log(10) colony forming units (cfu) in 1 min at a concentration of 1·25 mM (0·03%). 2. The present study was carried out to evaluate whether monocaprin emulsions could be used to kill Campylobacter on raw poultry. 3. It was shown that immersion of naturally contaminated chicken legs in 20 mM (0·5%) monocaprin emulsion at pH 4·1 for 1 min at 20°C reduced the number of Campylobacter by 2·0 to 2·7 log(10) cfu. Pre-chill dipping of whole carcases into 20 mM monocaprin emulsion in the slaughterhouse also caused a significant reduction in Campylobacter contamination. 4. Immersion in monocaprin emulsions at pH 4·1 was also assessed as a means to reduce the number of psychrotrophic spoilage bacteria. There were lower psychrotrophic bacteria counts on treated chicken parts than on untreated controls after storage at 3°C for up to 14 d. 5. Immersion in emulsions of monocaprin, which is a natural lipid classified as GRAS, may be a feasible method to reduce the number of Campylobacter and spoilage bacteria on raw poultry. This method could reduce the risk of human exposure to Campylobacter, and at the same time increase the shelf-life of poultry products.

Short peptide induces an "uncultivable" microorganism to grow in vitro.

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.