In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.

Acute nitrite exposure causes gut microbiota dysbacteriosis and proliferation of pathogenic Photobacterium in shrimp.

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1ß and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Effects of aqueous and ethanolic leaf extracts from drumstick tree (Moringa oleifera) on gilthead seabream (Sparus aurata L.) leucocytes, and their cytotoxic, antitumor, bactericidal and antioxidant activities.

Aqueous and ethanolic extracts of drumstick, Moringa oleifera, leaves were evaluated in vitro to ascertain their principal active components and determine their immunostimulant, cytotoxic, antitumoral, bactericidal and antioxidant activities. Phytochemical screening of M. oleifera leaf extracts showed a greater abundance of phenolic and cyanogenic glycosides in aqueous than in ethanolic extracts, characterized by several flavonoids, condensed tannins and saponins. No significant effects on gilthead seabream (Sparus aurata) head-kidney leucocyte activities (phagocytic ability and capacity, respiratory burst and peroxidase) were detected after incubation for 24 h with different concentrations (0.001/1 mg mL-1) of either extract. In addition, the aqueous extract showed a marked cytotoxic effect on both SAF-1 (at doses above 0.01 mg mL-1) and PLHC-1 (at doses above 0.25 mg mL-1) cell lines. The ethanolic extract improved the viability of SAF-1 cells and decreased the viability of PLHC-1 cells when used at higher concentrations. Both the ethanolic and, particularly, the aqueous extracts showed significant bactericidal activity on pathogenic Vibrio anguillarum and Photobacterium damselae strains. The antiradical activity of M. oleifera, as determined by the ABTS assay, increased in a linear dose-response with increasing extract concentrations. The results as a whole for the cytotoxic, bactericidal and antioxidant activities of M. oleifera leaf extracts point to their possible use as additives in functional diets for farmed fish.

Use of bacteriophage vB_Pd_PDCC-1 as biological control agent of Photobacterium damselae subsp. damselae during hatching of longfin yellowtail (Seriola rivoliana) eggs.

AIMS: This study describes the effect of phage therapy on hatching of longfin yellowtail (Seriola rivoliana) eggs challenged with Photobacterium damselae subsp. damselae. METHODS AND RESULTS: A lytic phage (vB_Pd_PDCC-1) against P. damselae subsp. damselae was isolated and characterized. The use of phage vB_Pd_PDCC-1 increased the hatching rate of eggs, and reduced presumptive Vibrio species to non-detectable numbers, even in non-disinfected eggs. High-throughput 16S rRNA gene sequencing analysis revealed that phage vB_Pd_PDCC-1 caused significant changes in the composition and structure of the associated microbiota, allowing that members (e.g. those belonging to the family Vibrionaceae) of the class Gammaproteobacteria to be displaced by members of the class Alphaproteobacteria. CONCLUSIONS: To the best of our knowledge, this represents the first study evaluating phage therapy to control potential negative effects of P. damselae subsp. damselae during hatching of longfin yellowtail eggs. SIGNIFICANCE AND IMPACT OF THE STUDY: The Seriola genus includes several important commercial fish species due to its rapid growth and easy adaptability to confinement conditions. However, bacterial infections (especially those caused by Vibrio and Photobacterium species) are among the main limiting factors for the intensification of marine fish aquaculture, particularly during early development stages. Therefore, the use of phages, which are natural killers of bacteria, represents a promising strategy to reduce the mortality of farmed organisms caused by pathogenic bacteria.

Molecular and histopathological characterization of Photobacterium damselae in naturally and experimentally infected Nile tilapia (Oreochromis niloticus).

Mass mortality has occurred among cultured Nile tilapia, Oreochromis niloticus, on fish farms in Manzala, Dakahlia province, Egypt, in the summer season, 2019. Moribund fish were reported with deep ulcers, septicaemic lesions and sampled for bacterial isolation. In this study, most isolates were subjected to bacteriological examination, antibiotic sensitivity test, 16S rRNA gene sequencing and histopathological examination. Following isolate identification, intraperitoneal challenge of Nile tilapia with a bacterial suspension 2 × 106  CFU/ml was performed. Samples from liver, spleen and kidney were collected for histological and biochemical analysis. The results showed a high similarity (99%) to Photobacterium damselae strains using phylogenetic analysis of 16S rRNA. P. damselae exhibited resistance to amoxicillin and erythromycin, as well it was highly sensitive to chloramphenicol and doxycycline. Moreover, haemorrhage, oedema, hemosiderosis and melanomacrophage activation in the liver and head kidney of infected fish were detected by light and electron microscopy. Also, significant higher levels of CAT and SOD in the spleen and head kidney, as well as the serum levels of NO were observed in experimentally challenged O. niloticus, compared to the control fish. Our data identified P. damselae for the first time from infected Nile tilapia, describing its sensitivity to a variety of antibiotics, histopathological alterations and oxidative stress impact, and it could be useful indicators for understanding P. damselae pathogenesis, which might provide a preventive efficacy for P. damselae.

Bioluminescent method for the rapid screening of toxic heayy metals in environmental samples using Photobacterium leiognathi strain AK-MIE.

Assessment of eco-toxicant using bioluminescent bacterial assay is a widely used and globally accepted method. In this work, a new luminescent bacterium was isolated from squid (Loligo duvauceli) and identified as Photobacterium leiognathi strain AK-MIE using 16S rRNA, phylogeny analysis. The predicted optimum conditions by RSM were 2.76% (w/v) NaCl, 2.28% (w/v) peptone, 0.34% (w/v) yeast extract, and pH 6.83 with 541,211.80 RLU of luminescent production whereas the predicted optimum conditions by ANN were 2.21% (w/v) NaCl, 2.27% (w/v) peptone, 0.39% (w/v) yeast extract, and pH 6.94 which produced 541,986.20 RLU. The validation analysis of both RSM and ANN show 0.60% and 0.69% deviation from the predicted results indicating that both models provided good quality predictions with ANN showing a superior data fitting capability for non-linear regression analysis. Toxicity tests show strain AK-MIE was sensitive to mercury (concentration causing 50% inhibition or IC50 of 0.00978 mgL-1), followed by cadmium (IC50 of 0.5288 mgL-1), copper IC50 of (0.8117 mgL-1), silver (IC50 of 1.109 mgL-1), and lead (IC50 of 10.71 mgL-1) which are more sensitive than previously isolated luminescent bacteria, suggesting that strain AK-MIE has the potential to be used in toxicity assessment of heavy metals in the environment. Based on the field trial results, several sediment samples from industrial areas in Bangi, Selangor managed to inhibit the bioluminescence of strain AK-MIE. Validation method carried out using ICP-MS proved the presence of several toxic heavy metal elements.

Mechanism study of cyfluthrin biodegradation by Photobacterium ganghwense with comparative metabolomics.

A high-efficiency pyrethroid-degrading bacterium, Photobacterium ganghwense strain 6046 (PGS6046), was first isolated from an offshore seawater environment. Metabolomics method was used to investigate the biotransformation pathway of PGS6046 to cyfluthrin wherein 156 metabolites were identified. The growth rates of the PGS6046 cultivated in nourishing media were much higher than those cultivated in seawater, regardless of the presence of cyfluthrin. Statistical analyses revealed that the metabolic profile of PGS6046 was associated with the culture medium, the presence of cyfluthrin, and culture time. The PGS6046 cultivated in a nourishing medium was characterized by higher levels of amino acids, a lower abundance of intermediates in the tricarboxylic acid cycle, and the presence of some fatty acids than those cultivated in seawater. The effects of cyfluthrin on PGS6046 metabolism varied based on the culture medium, whereas the cyanoalanine levels increased under both culture conditions. Culture time significantly affected the metabolism of amino acids and carbohydrates in PGS6046. The present study revealed the metabolic characteristics of PGS6046 under different culture conditions and will further facilitate the exploration of the fundamental questions regarding PGS6046 and its potential applications in environmental bioremediation.

Degradation of atrazine by catalytic ozonation in the presence of iron scraps: performance, transformation pathway, and acute toxicity.

Degradation of atrazine by catalytic ozonation in the presence of iron scraps (ZVI/O3) was carried out. The key operational parameters (i.e., initial pH, ZVI dosage, and ozone dosage) were optimized by the batch experiments, respectively. This ZVI/O3 system exhibited much higher degradation efficiency of atrazine than the single ozonation, ZVI, and traditional ZVI/O2 systems. The result shows that the pseudo-first-order constant (0.0927 min-1) and TOC removal rate (86.6%) obtained by the ZVI/O3 process were much higher than those of the three control experiments. In addition, X-ray diffraction (XRD) analysis indicates that slight of γ-FeOOH and Fe2O3 were formed on the surface of iron scrap after ZVI/O3 treatment. These corrosion products exhibit high catalytic ability for ozone decomposition, which could generate more hydroxyl radical (HO•) to degrade atrazine. Six transformation intermediates were identified by liquid chromatography-mass spectrometry (LC-MS) analysis in ZVI/O3 system, and the degradation pathway of atrazine was proposed. Toxicity tests based on the inhibition of the luminescence emitted by Photobacterium phosphoreum and Vibrio fischeri indicate the detoxification of atrazine by ZVI/O3 system. Finally, reused experiments indicate the approving recyclability of iron scraps. Consequently, the ZVI/O3 system could be as an effective and promising technology for pesticide wastewater treatment.

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities.

Origanum vulgare is a well-known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF-1 cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF-1 cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC-1 cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish.

Evaluation of a novel automated water analyzer for continuous monitoring of toxicity and chemical parameters in municipal water supply.

A novel tool, the DAMTA analyzer (Device for Analytical Monitoring and Toxicity Assessment), designed for fully automated toxicity measurements based on luminescent bacteria as well as for concomitant determination of chemical parameters, was developed and field-tested. The instrument is a robotic water analyzer equipped with a luminometer and a spectrophotometer, integrated on a thermostated reaction plate which contains a movable carousel with 80 cuvettes. Acute toxicity is measured on-line using a wild type Photobacterium phosphoreum strain with measurable bioluminescence and unaltered sensitivity to toxicants lasting up to ten days. The EC50 values of reference compounds tested were consistent with A. fischeri and P. phosphoreum international standards and comparable to previously published data. Concurrently, a laboratory trial demonstrated the feasibility of use of the analyzer for the determination of nutrients and metals in parallel to the toxicity measurements. In a prolonged test, the system was installed only in toxicity mode at the premises of the World Fair "Expo Milano-2015″, a high security site to ensure the quality of the supplied drinking water. The monitoring program lasted for six months during which ca. 2400 toxicity tests were carried out; the results indicated a mean non-toxic outcome of -5.5 ±â€¯6.2%. In order to warrant the system's robustness in detecting toxic substances, Zn was measured daily with highly reproducible inhibition results, 70.8 ±â€¯13.6%. These results assure that this novel toxicity monitor can be used as an early warning system for protection of drinking water sources from emergencies involving low probability/high impact contamination events in source water or treated water.

Algerian propolis extracts: Chemical composition, bactericidal activity and in vitro effects on gilthead seabream innate immune responses.

Propolis has been used as a medicinal agent for centuries. The chemical composition of four propolis samples collected from four locations of the Sétif region, Algeria, using gas chromatography-mass spectrometry was determined. More than 20 compounds and from 30 to 35 compounds were identified in the aqueous and ethanolic extracts, respectively. Furthermore, the antimicrobial activity of the propolis extracts against two marine pathogenic bacteria was evaluated. Finally, the in vitro effects of propolis on gilthead seabream (Sparus aurata L.) leucocyte activities were measured. The bactericidal activity of ethanolic extracts was very high against Shewanella putrefaciens, average against Photobacterium damselae and very low against Vibrio harveyi. The lowest bactericidal activity was always that found for the aqueous extracts. When the viability of gilthead seabream head-kidney leucocytes was measured after 30 min' incubation with the different extracts, both the ethanolic and aqueous extracts of one of the propolis samples (from Babor) and the aqueous extract of another (from Ain-Abbassa) provoked a significant decrease in cell viability when used at concentrations of 100 and 200 µg ml-1. Furthermore, significant inhibitory effects were recorded on leucocyte respiratory burst activity when isolated leucocytes where preincubated with the extracts. This effect was dose-dependent in all cases except when extracts from a third propolis sample (from Boutaleb) were used. Our findings suggest that some of Algerian propolis extracts have bactericidal activity against important bacterial pathogens in seabream and significantly modulate in vitro leucocyte activities, confirming their potential as a source of new natural biocides and/or immunomodulators in aquaculture practice.

Pathogenesis of Photobacterium damselae subspecies infections in sea bass and sea bream.

Photobacterium damselae is a Gram negative bacterium causes photobacteriosis, a worldwide septicemic disease in aquaculture including sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogenicity of bacterial subspecies and the disease pathological changes in natural and experimental infections have thus far yielded inconsistency of effective preventive measures. This study aimed to represent a comprehensive analysis of the potential pathogenic capacities of the two subspecies of P. damselae in cultured sea bass and sea bream in the Northwestern region of Egypt. Diseased 321 sea bass and 257 sea bream, in addition to 99 healthy sea bass fingerlings were sampled from three farms located along the Mediterranean Sea. P. damselae subspecies were isolated from diseased fish and characterized using bacteriological, molecular, and antimicrobial susceptibility methods. Healthy fish were challenged by a virulent P. damselae subsp. piscicida, monitored for disease signs and mortality, and the histopathological abnormalities and hematological disorders were carried out. Clinical signs and gross lesions in naturally infected sea bass and sea bream showed great similarities with absence of a subspecies-specific characteristic sign or lesion. The two subspecies were recovered through the entire year from individual fish sample, suggests a coexistence of two subspecies endemic infection. In diseased sea bass, 38.32% and 16.20% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. However in diseased sea bream, 44.47% and 26.46% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. High mortalities and devastating clinicopathologic abnormalities represented by sever clinical signs, hematological disorders and histological abnormalities strengthen the pathogenicity of P. damselae subspecies in the two fish species and therefore, a vaccination strategy against both subspecies should be taken into account.

Tetrodecamycin: An unusual and interesting tetronate antibiotic.

The tetrodecamycins are a group of secondary metabolites that are characterized by the presence of a tetronate ring in their structure. Originally discovered for their antibiotic activity against Photobacterium damselae ssp. piscicida, the causative agent of pseudotuberculosis in fish, this family of molecules has also been shown to have potent antibiotic activity against methicillin-resistant Staphylococcus aureus. Due to their small size and highly cyclized nature, they represent an unusual member of the much larger group of bioactive molecules called the tetronates. Herein, we review what is known about the mechanism of action of these molecules and also present a hypothesis for their biosynthesis. A deeper understanding of the tetrodecamycins will provide a more holistic view of the tetronate-family, provide new chemical probes of bacterial biology, and may provide therapeutic lead molecules.

Eco-toxicological bioassay of atmospheric fine particulate matter (PM2.5) with Photobacterium Phosphoreum T3.

A bioluminescent bacterium, Photobacterium phosphoreum T3 (PPT3), was used as a bio-indicator for the atmospheric fine particulate matter (PM2.5) to determine the eco-toxicity of PM2.5. The PM2.5 contains toxic chemicals, which reduce light output. The PM2.5 samples were collected in the period from March 2014 to January 2015 in Nanjing and analyzed for the chemical composition versus their eco-toxicity. The eco-toxicological responses of each toxicant were detected in PM2.5 samples with PPT3. The dose-response curves obtained were verified using the Weibull fitting function. According to the measured EC50 values (EC50, the concentration of a toxicant that inhibits 50% of the bioluminescence), the toxicity sequence was: B[a]P>hexa-PCB>tetra-PCB>tri-PCB>Pb(2+)>DEHP>Cu(2+)>DBP>BDE209>Zn(2+)>DMP>DEP, where B[a]P is benzo(a)pyrene, PCB is polychlorinated biphenyl, DEHP is diethylhexyl phthalate, DBP is dibutyl phthalate, BDE209 is decabromodiphenyl ether, DMP is dimethyl phthalate, and DEP is diethyl phthalate. All the PM2.5 samples analyzed proved to be weak toxic for PPT3. The toxicity of PM2.5 was assessed by the dose-addition of organic species and heavy metallic elements existing in PM2.5 with PPT3. The bioluminescence test showed that the metals and organics detected in PM2.5 promoted PM2.5 toxicity. The total detectable organics (denoted by ΣOrs) exhibited slightly higher toxicity than the total metals (denoted by ΣMs). In contrast, the sum of water-soluble ions (denoted by ΣIons) was beneficial to PPT3. The PM2.5 toxicity increased as the PM2.5 trapped more organics or metallic elements from the industrial or densely populated urban areas, where the PM2.5 had a high inhibition rate of bioluminescence for PPT3 in contrast to the residential PM2.5 samples, where the minimum inhibition rate was observed. The toxicity of PM2.5 samples varied with the mass concentrations, chemical constituents, and sampling locations. The chemicals in PM2.5, especially organic species and metallic elements, affected its eco-toxicity. These data provided good understanding of the atmospheric PM2.5 pollution in the large portion of the East China.

Joint Toxicity of Two Phthalates with Waterborne Copper to Daphnia magna and Photobacterium phosphoreum.

Di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) are two widely used phthalates, while Cu(II) is a common valence state of copper. They have been ubiquitously detected in the aquatic environment, but information on their joint toxicity to aquatic organisms is scarce. In this study, we evaluated the combined effects of copper and these two phthalates to Daphnia magna and Photobacterium phosphoreum by quantifying the acute toxicity expressed by the EC50 (the concentration causing 50 % of maximal effect) value. The toxicity order was DEHP + Cu(II) > DBP + Cu(II) > Cu(II) > DEHP > DBP for both test species. Antagonism effects were found in the joint toxicity of Cu(II) combined with DBP or DEHP using the toxic unit method. These findings have important implications in environmental risk assessment for phthalates in the aquatic environment in the presence of heavy metals.

Photobacterium damselae subsp. piscicida responds to antimicrobial peptides through phage-shock-protein A (PspA)-related extracytoplasmic stress response system.

AIMS: To investigate whether Photobacterium damselae subsp. piscicida (Phdp) can sense and directly respond to the presence of cationic antimicrobial peptides (AMPs). METHODS AND RESULTS: We performed proteomic methodologies to investigate the responsive proteins of Phdp on exposure to AMP Q6. Proteins significantly altered were analysed by two-dimensional gel electrophoresis (2-DE) and LC-ESI-Q-TOF MS/MS, thus resulting in five outer membrane proteins (OMPs), seven inner membrane proteins (IMPs) and 17 cytoplasmic proteins (CPs) identified. Quantitative real-time PCR was also applied to monitor the mRNA expression level of these target proteins. CONCLUSIONS: COG analysis revealed that upon exposure to AMP Q6, the majority of the upregulated proteins were involved in signal transduction mechanism, carbohydrate transport and metabolism, post-translational modification, protein turnover and chaperones, while the downregulated proteins were mainly related to energy production and conversion. Among them, phage-shock-protein A (PspA)-related stress response system was considered to play a crucial role. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report elucidating Phdp AMP-response mechanism using proteomics approach. AMP-responsive proteins identified in this study could serve as attractive targets for developing more effective antimicrobial agents against Phdp and other marine bacterial pathogens.

A zeta potential value determines the aggregate's size of penta-substituted [60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells.

BACKGROUND: The cause-effect relationships between physicochemical properties of amphiphilic [60]fullerene derivatives and their toxicity against bacterial cells have not yet been clarified. In this study, we report how the differences in the chemical structure of organic addends in 10 originally synthesized penta-substituted [60]fullerene derivatives modulate their zeta potential and aggregate's size in salt-free and salt-added aqueous suspensions as well as how these physicochemical characteristics affect the bioenergetics of freshwater Escherichia coli and marine Photobacterium phosphoreum bacteria. Dynamic light scattering, laser Doppler micro-electrophoresis, agarose gel electrophoresis, atomic force microscopy, and bioluminescence inhibition assay were used to characterize the fullerene aggregation behavior in aqueous solution and their interaction with the bacterial cell surface, following zeta potential changes and toxic effects. RESULTS: Dynamic light scattering results indicated the formation of self-assembled [60]fullerene aggregates in aqueous suspensions. The measurement of the zeta potential of the particles revealed that they have different surface charges. The relationship between these physicochemical characteristics was presented as an exponential regression that correctly described the dependence of the aggregate's size of penta-substituted [60]fullerene derivatives in salt-free aqueous suspension from zeta potential value. The prevalence of DLVO-related effects was shown in salt-added aqueous suspension that decreased zeta potential values and affected the aggregation of [60]fullerene derivatives expressed differently for individual compounds. A bioluminescence inhibition assay demonstrated that the toxic effect of [60]fullerene derivatives against E. coli cells was strictly determined by their positive zeta potential charge value being weakened against P. phosphoreum cells in an aquatic system of high salinity. Atomic force microscopy data suggested that the activity of positively charged [60]fullerene derivatives against bacterial cells required their direct interaction. The following zeta potential inversion on the bacterial cells surface was observed as an early stage of toxicity mechanism that violates the membrane-associated energetic functions. CONCLUSIONS: The novel data about interrelations between physicochemical parameters and toxic properties of amphiphilic [60]fullerene derivatives make possible predicting their behavior in aquatic environment and their activity against bacterial cells.

Novel macrolide-resistance genes, mef(C) and mph(G), carried by plasmids from Vibrio and Photobacterium isolated from sediment and seawater of a coastal aquaculture site.

UNLABELLED: The aim of this study was to determine whether mef(C) and mph(G), originally found on the transferable multi-drug plasmid pAQU1 from Photobacterium damselae subsp. damselae isolated from seawater of a fish farm, are responsible for conferring macrolide resistance. Since these genes are localized head-to-tail on pAQU1 and only four nucleotides exist between them, the single- and combination-effect of these genes was examined. When mph(G) alone was introduced to Escherichia coli, the minimum inhibitory concentrations (MICs) against erythromycin, clarithromycin and azithromycin increased, whereas introduction of mef(C) alone did not influence macrolide susceptibility. Introduction of both mef(C) and mph(G) dramatically increased the MICs to the same three macrolides, i.e. >512 µg ml(-1) , >512 µg ml(-1) and 128 µg ml(-1) respectively. These results suggest that the macrolide phosphotransferase encoded by mph(G) is essential for macrolide resistance, while the efflux pump encoded by mef(C) is required for high-level macrolide resistance. The tandem-pair arrangements of the mef(C) and mph(G) genes were conserved on plasmids ranging in size from 240 to 350 kb of the 22 erythromycin-resistant strains belonging to Vibrio and Photobacterium obtained from the fish farm. Sixteen of 22 plasmids ranged in size from 300 to 350 kb. This is the first report of novel macrolide resistance genes originating from a marine bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, mef(C) and mph(G) were found to be novel macrolide-resistance genes, and this is the first report of macrolide-resistance genes originating from a marine bacterium. These genes may be responsible for previously reported cases of the emergence of erythromycin-resistant bacteria in aquaculture sites by an unknown mechanism. The introduction of the tandem arrangement of the mef(C) and mph(G) genes in Escherichia coli increased the MICs to erythromycin, clarithromycin and azithromycin, suggesting a novel mechanism conferring high-level macrolide resistance via combined expression of the efflux pump and macrolide phosphotransferase.

Reduction in toxicity of coking wastewater to aquatic organisms by vertical tubular biological reactor.

We conducted a battery of toxicity tests using photo bacterium, algae, crustacean and fish to evaluate acute toxicity profile of coking wastewater, and to evaluate the performance of a novel wastewater treatment process, vertical tubular biological reactor (VTBR), in the removal of toxicity and certain chemical pollutants. A laboratory scale VTBR system was set up to treat industrial coking wastewater, and investigated both chemicals removal efficiency and acute bio-toxicity to aquatic organisms. The results showed that chemical oxygen demand (COD) and phenol reductions by VTBR were approximately 93% and 100%, respectively. VTBR also reduced the acute toxicity of coking wastewater significantly: Toxicity Unit (TU) decreased from 21.2 to 0.4 for Photobacterium phosphoreum, from 9.5 to 0.6 for Isochrysis galbana, from 31.9 to 1.3 for Daphnia magna, and from 30.0 to nearly 0 for Danio rerio. VTBR is an efficient treatment method for the removal of chemical pollutants and acute bio-toxicity from coking wastewater.