First Report of Kocuria kristinae in the Skin of a Cuban Boa (Epicrates angulifer).

During a routine clinical examination of a four-year-old female Cuban boa (Epicrates angulifer) belonging to a zoological park located in northern Portugal, a skin lesion was observed. A skin swab was taken. Bacteriological analysis conducted using the Vitek® 2 Compact system identified the presence of the bacteria species Kocuria kristinae, a new bacterial pathogen that may be a potential pathogen in wild animals. This K. kristinae strain was resistant to kanamycin, pradofloxacin, erythromycin, clindamycin, tetracycline, nitrofurantoin, and trimethoprim/sulphamethoxazole and was therefore classified as a multidrug-resistant bacterium. To the authors' knowledge, this is the first time that K. kristinae has been described in the skin of a Cuban boa. This report serves as a cautionary warning about the importance of recognising and investigating the potential pathogenicity of this agent, as well as contributing to the development of strategies to prevent the spread of antibiotic-resistant microorganisms.

Mesenchymal Stem Cell Studies in the Goat Model for Biomedical Research-A Review of the Scientific Literature.

Mesenchymal stem cells (MSCs) are multipotent cells, defined by their ability to self-renew, while maintaining the capacity to differentiate into different cellular lineages, presumably from their own germinal layer. MSCs therapy is based on its anti-inflammatory, immunomodulatory, and regenerative potential. Firstly, they can differentiate into the target cell type, allowing them to regenerate the damaged area. Secondly, they have a great immunomodulatory capacity through paracrine effects (by secreting several cytokines and growth factors to adjacent cells) and by cell-to-cell contact, leading to vascularization, cellular proliferation in wounded tissues, and reducing inflammation. Currently, MSCs are being widely investigated for numerous tissue engineering and regenerative medicine applications. Appropriate animal models are crucial for the development and evaluation of regenerative medicine-based treatments and eventual treatments for debilitating diseases with the hope of application in upcoming human clinical trials. Here, we summarize the latest research focused on studying the biological and therapeutic potential of MSCs in the goat model, namely in the fields of orthopedics, dermatology, ophthalmology, dentistry, pneumology, cardiology, and urology fields.

Screening of Natural Molecules as Adjuvants to Topical Antibiotics to Treat Staphylococcus aureus from Diabetic Foot Ulcer Infections.

Diabetic foot ulcers (DFUs) are a common result of a complex secondary complication of diabetes mellitus. More than half of DFUs become infected due to frequent colonization with Staphylococcus aureus. The use of topical antibiotics is proposed, especially in combination with natural adjuvants, to minimize the negative impacts caused by generalized use of systemic antibiotics. In this study, 13 different phytochemicals-namely chalcone, juglone, cinnamic acid, trigonelline, Furvina-and four nitrovinylfuran derivatives-guaiazulene, α-bisabolol, farnesol and nerolidol-were selected to be tested as antibiotic enhancers. After minimum inhibitory and bactericidal concentration (MIC and MBC) determination of each molecule against different strains of S. aureus, including clinical isolates from diabetic foot wounds (CECT 976, Xu212, SA 1199B, RN4220, MJMC102, MJMC109, MJMC110 and MJMC111), their potentiation effects on the antibiotics fusidic acid, mupirocin, gentamicin, oxacillin and methicillin were evaluated through the disc diffusion method. Farnesol at sub-MIC was able to restore the activity of methicillin and oxacillin on the MJMC102 and MJMC111 strains, as well as two MRSA clinical isolates, and potentiated the effect of the remaining antibiotics. The results obtained demonstrate the great potential for the topical application of phytochemicals and derivatives as antibiotic resistance modifier agents to combat multidrug resistance in bacterial wound infections.

In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon bacterial challenge.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection.

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

Black-and-White Ruffed Lemur (Varecia variegata) in Captivity: Analysis of the Oral Microbiota in a One Health Perspective.

This study aimed to characterize the susceptibility profile to antibiotics and biofilm formation of Gram-negative bacterial isolates obtained from the oral cavity of the black-and-white ruffed lemur (Varecia variegata). From eight individuals from a zoo located in Portugal, samples of the oral microbiota were collected with sterile swabs and then placed in closed tubes with a transport medium. Culture was carried out for media of Gram-negative bacteria. Twenty-two isolates were obtained and subjected to susceptibility tests to twenty-five antimicrobial agents belonging to seven different classes. All tested isolates demonstrated resistance to, at least, one antibiotic, and it was possible to observe multidrug resistance in 11 of the 22 isolates (50%). It should be noted that an isolate showed phenotypic resistance to imipenem, an antibiotic for exclusive use in a hospital environment. All the isolates showed an increasing ability of biofilm formation over time. The obtained results show that wild mammals in captivity could be reservoirs and potential sources of multi-resistant pathogens. In view of this fact and considering the One Health concept, it will be advisable to establish local monitoring programs worldwide that benefit and protect human, animal and environmental health.

Isolation and Characterization of Fish-Gut Bacillus spp. as Source of Natural Antimicrobial Compounds to Fight Aquaculture Bacterial Diseases.

Aquaculture is responsible for more than 50% of global seafood consumption. Bacterial diseases are a major constraint to this sector and associated with misuse of antibiotics, pose serious threats to public health. Fish-symbionts, co-inhabitants of fish pathogens, might be a promising source of natural antimicrobial compounds (NACs) alternative to antibiotics, limiting bacterial diseases occurrence in aquafarms. In particular, sporeforming Bacillus spp. are known for their probiotic potential and production of NACs antagonistic of bacterial pathogens and are abundant in aquaculture fish guts. Harnessing the fish-gut microbial community potential, 172 sporeforming strains producing NACs were isolated from economically important aquaculture fish species, namely European seabass, gilthead seabream, and white seabream. We demonstrated that they possess anti-growth, anti-biofilm, or anti-quorum-sensing activities, to control bacterial infections and 52% of these isolates effectively antagonized important fish pathogens, including Aeromonas hydrophila, A. salmonicida, A. bivalvium, A. veronii, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei. By in vitro quantification of sporeformers' capacity to suppress growth and biofilm formation of fish pathogens, and by assessing their potential to interfere with pathogens communication, we identified three promising candidates to become probiotics or source of bioactive molecules to be used in aquaculture against bacterial aquaculture diseases.

Antibacterial activity and mode of action of selected glucosinolate hydrolysis products against bacterial pathogens.

Plants contain numerous components that are important sources of new bioactive molecules with antimicrobial properties. Isothiocyanates (ITCs) are plant secondary metabolites found in cruciferous vegetables that are arising as promising antimicrobial agents in food industry. The aim of this study was to assess the antibacterial activity of two isothiocyanates (ITCs), allylisothiocyanate (AITC) and 2-phenylethylisothiocyanate (PEITC) against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. The antibacterial mode of action was also characterized by the assessment of different physiological indices: membrane integrity, intracellular potassium release, physicochemical surface properties and surface charge. The minimum inhibitory concentration (MIC) of AITC and PEITC was 100 µg/mL for all bacteria. The minimum bactericidal concentration (MBC) of the ITCs was at least 10 times higher than the MIC. Both AITC and PEITC changed the membrane properties of the bacteria decreasing their surface charge and compromising the integrity of the cytoplasmatic membrane with consequent potassium leakage and propidium iodide uptake. The surface hydrophobicity was also non-specifically altered (E. coli and L. monocytogenes become less hydrophilic; P. aeruginosa and S. aureus become more hydrophilic). This study shows that AITC and PEITC have strong antimicrobial potential against the bacteria tested, through the disruption of the bacterial cell membranes. Moreover, phytochemicals are highlighted as a valuable sustainable source of new bioactive products.

Insights on antimicrobial resistance, biofilms and the use of phytochemicals as new antimicrobial agents.

Antimicrobial resistance is one of the most serious public health problems. This is of particular concern when bacteria become resistant to various antimicrobial agents simultaneously and when they form biofilms. Consequently, therapeutic options for the treatment of infections have become limited, leading frequently to recurrent infections, treatment failure and increase of morbidity and mortality. Both, persistence and spread of antibiotic resistance, in combination with decreased effectiveness and increased toxicity of current antibiotics have emphasized the urgent need to search alternative sources of antimicrobial substances. Plants are recognized as a source of unexplored chemical structures with high therapeutic potential, including antimicrobial activity against clinically important microorganisms. Additionally, phytochemicals (plant secondary metabolites) present several advantages over synthetic molecules, including green status and different mechanisms of action from antibiotics which could help to overcome the resistance problem. In this study, an overview of the main classes of phytochemicals with antimicrobial properties and their mode of action is presented. A revision about the application of phytochemicals for biofilm prevention and control is also done. Moreover, the use of phytochemicals as scaffolds of new functional molecules to expand the antibiotics pipeline is reviewed.

Evaluation of the effects of selected phytochemicals on quorum sensing inhibition and in vitro cytotoxicity.

Quorum sensing (QS) is an important regulatory mechanism in biofilm formation and differentiation. Interference with QS can affect biofilm development and antimicrobial susceptibility. This study evaluates the potential of selected phytochemical products to inhibit QS. Three isothiocyanates (allylisothiocyanate - AITC, benzylisothiocyanate - BITC and 2-phenylethylisothiocyanate - PEITC) and six phenolic products (gallic acid - GA, ferulic acid - FA, caffeic acid - CA, phloridzin - PHL, (-) epicatechin - EPI and oleuropein glucoside - OG) were tested. A disc diffusion assay based on pigment inhibition in Chromobacterium violaceum CV12472 was performed. In addition, the mechanisms of QS inhibition (QSI) based on the modulation of N-acyl homoserine lactone (AHLs) activity and synthesis by the phytochemicals were investigated. The cytotoxicity of each product was tested on a cell line of mouse lung fibroblasts. AITC, BITC and PEITC demonstrated a capacity for QSI by modulation of AHL activity and synthesis, interfering the with QS systems of C. violaceum CviI/CviR homologs of LuxI/LuxR systems. The cytotoxic assays demonstrated low effects on the metabolic viability of the fibroblast cell line only for FA, PHL and EPI.

Evaluation of biological value and appraisal of polyphenols and glucosinolates from organic baby-leaf salads as antioxidants and antimicrobials against important human pathogenic bacteria.

The present investigation has been carried out to investigate the biological role of four different types of baby-leaf salads and to study their potential as natural sources of antioxidants and antimicrobials against several isolates from important human pathogenic bacteria. Four single types of salads (green lettuce, red lettuce, rucola and watercress) and two mixtures [(1) red lettuce+green lettuce; (2) green lettuce + red lettuce + watercress + rucola] were assayed. The HPLC analysis revealed interesting levels of polyphenols and glucosinolates. The results showed a significant variation (p < 0.05) of polyphenols and glucosinolates with plant material. Nine different types of polyphenols grouped in three major classes were found: gallic acid, chlorogenic acid, caffeic acid and dicaffeoyltartaric acid (phenolic acids); quercitin-3-O-rutinoside, quercitin-3-O-rhamnoside, luteolin-7-O-glucoside and isorhamnetin (flavonoids); and cyanidin-3-glucoside (anthocyanins). Only three different glucosinolates were found: glucoraphanin; gluconasturtiin and 4-methoxy-glucobrassicin. A positive correlation was detected between polyphenol contents and antioxidant activity. Red lettuce and mixture 1 were the baby-leaf salads with the highest antioxidant potential. As for the antimicrobial activity, the results showed a selective effect of chemicals against Gram-positive and Gram-negative bacteria and Enterococcus faecalis and Staphylococcus aureus were the bacteria most affected by the phytochemicals. Based on the results achieved baby-leaf salads represent an important source of natural antioxidants and antimicrobial substances.

Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria.

The increased resistance of pathogenic microorganisms is frequently attributed to the extreme and inadequate use of antibiotics and transmission of resistance within and between individuals. To counter the emergence of resistant microorganisms, considerable resources have been invested in the search for new antimicrobials. Plants synthesize a diverse array of secondary metabolites (phytochemicals) known to be involved in defense mechanisms, and in the last few years it is recognized that some of these molecules have health beneficial effects, including antimicrobial properties. In this study, the mechanism of action of gallic (GA) and ferulic (FA) acids, a hydroxybenzoic acid and a hydroxycinnamic acid, was assessed on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes. The targets of antimicrobial action were studied using different bacterial physiological indices: minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), membrane permeabilization, intracellular potassium release, physicochemical surface properties, and surface charge. It was found that FA and GA had antimicrobial activity against the bacteria tested with MIC of 500 µg/mL for P. aeruginosa, 1500 µg/mL for E. coli, 1750 µg/mL for S. aureus, and 2000 µg/mL for L. monocytogenes with GA; 100 µg/mL for E. coli and P. aeruginosa, 1100 µg/mL and 1250 µg/mL for S. aureus and L. monocytogenes, respectively, with FA. The MBC for E. coli was 2500 µg/mL (FA) and 5000 (GA), for S. aureus was 5000 µg/mL (FA) and 5250 µg/mL (GA), for L. monocytogenes was 5300 µg/mL (FA) and 5500 µg/mL (GA), and 500 µg/mL for P. aeruginosa, with both phytochemicals. GA and FA led to irreversible changes in membrane properties (charge, intra and extracellular permeability, and physicochemical properties) through hydrophobicity changes, decrease of negative surface charge, and occurrence of local rupture or pore formation in the cell membranes with consequent leakage of essential intracellular constituents. The overall study emphasizes the potential of plant-derived molecules as a green and sustainable source of new broad spectrum antimicrobial products.

Effects of agriculture production systems on nitrate and nitrite accumulation on baby-leaf salads.

Nitrate and nitrite are widespread contaminants of vegetables, fruits, and waters. The levels of these compounds are increased as a result of using organic wastes from chemical industries, domestic wastes, effluents, nitrogenous fertilizers, and herbicides in agriculture. Therefore, determining the nitrate and nitrite levels in biological, food, and environmental samples is important to protect human health and the environment. In this context, we set this study, in which we report the effect of production system (conventional and organic) on the accumulation of nitrates and nitrites in fresh baby-leaf samples. The average levels of the nitrate ([Formula: see text]) and nitrite ([Formula: see text]) contents in six different baby-leaf salads of a single species (green lettuce, red lettuce, watercress, rucola, chard, and corn salad) produced in organic and conventional agriculture system were evaluated. Spectrophotometric analytical method recently published was validated and used. Nitrates and nitrites were detected in all samples. The nitrates levels from organic production varied between 1.45 and 6.40 mg/kg fresh weight (FW), whereas those from conventional production ranged from 10.5 to 45.19 mg/kg FW. The nitrites content was lower than nitrates and ranged from 0.32 to 1.89 mg/kg FW in organic production system and between 0.14 and 1.41 mg/kg FW in conventional production system. Our results showed that the nitrate content was dependent on the agricultural production system, while for nitrites, this dependency was less pronounced.

The activity of ferulic and gallic acids in biofilm prevention and control of pathogenic bacteria.

The activity of two phenolic acids, gallic acid (GA) and ferulic acid (FA) at 1000 µg ml(-1), was evaluated on the prevention and control of biofilms formed by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. In addition, the effect of the two phenolic acids was tested on planktonic cell susceptibility, bacterial motility and adhesion. Biofilm prevention and control were tested using a microtiter plate assay and the effect of the phenolic acids was assessed on biofilm mass (crystal violet staining) and on the quantification of metabolic activity (alamar blue assay). The minimum bactericidal concentration for P. aeruginosa was 500 µg ml(-1) (for both phenolic acids), whilst for E. coli it was 2500 µg ml(-1) (FA) and 5000 µg ml(-1) (GA), for L. monocytogenes it was >5000 µg ml(-1) (for both phenolic acids), and for S. aureus it was 5000 µg ml(-1) (FA) and >5000 µg ml(-1) (GA). GA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. FA caused total inhibition of swimming (L. monocytogenes) and swarming (L. monocytogenes and E. coli) motilities. Colony spreading of S. aureus was completely inhibited by FA. The interference of GA and FA with bacterial adhesion was evaluated by the determination of the free energy of adhesion. Adhesion was less favorable when the bacteria were exposed to GA (P. aeruginosa, S. aureus and L. monocytogenes) and FA (P. aeruginosa and S. aureus). Both phenolics had preventive action on biofilm formation and showed a higher potential to reduce the mass of biofilms formed by the Gram-negative bacteria. GA and FA promoted reductions in biofilm activity >70% for all the biofilms tested. The two phenolic acids demonstrated the potential to inhibit bacterial motility and to prevent and control biofilms of four important human pathogenic bacteria. This study also emphasizes the potential of phytochemicals as an emergent source of biofilm control products.

First study on antimicriobial activity and synergy between isothiocyanates and antibiotics against selected Gram-negative and Gram-positive pathogenic bacteria from clinical and animal source.

The emergence of new diseases and the resurgence of several infections that were controlled in the past, associated with recent increase of bacterial resistance have created the necessity for more studies towards to the development of new antimicrobials and new treatment strategies. The aim of the present study was to evaluate the in vitro synergy between different classes of important glucosinolates hydrolysis products-isothiocyanates with antibiotics (gentamycin and vancomycin), against important pathogenic bacteria: Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus. A disc diffusion method was used to evaluate the antibacterial activity. The antimicrobial activity of phytochemicals and combinations between gentamycin, vancomycin and phytochemicals were quantitatively assessed by measuring the inhibitory halos. The results showed a selective antimicrobial effect of isothiocyanates, and this effect was strictly related with their chemical structure. In general the benzylisothiocyanate was the most effective compound against both Gram-positive and Gram-negative bacteria. The Listeria monocytogenes and Staphylococcus aureus were the bacteria most affected either by the phytochemicals alone or by the combination phytochemical-antibiotic. The bacteria Pseudomonas aeruginosa was the less affected pathogen. The most important synergism detected occurred between the commercial antibiotics with benzylisothiocyanate and 2-phenylethylisothiocyanate. In conclusion, some isothiocyanates are effective inhibitors of in vitro bacterial growth, and they can act synergistically with antibiotics.

Seasonal effects on bioactive compounds and antioxidant capacity of six economically important brassica vegetables.

Research on natural and bioactive compounds is increasingly focused on their effects on human health, but there are unexpectedly few studies evaluating the relationship between climate and natural antioxidants. The aim of this study was analyze the biological role of six different Brassica vegetables (Brassica oleracea L. and Brassica rapa L.) as a natural source of antioxidant compounds. The antioxidant activity may be assigned to high levels of L-ascorbic acid, total phenolics and total flavonoids of each sample. The climate seasons affected directly the concentration of bioactive components and the antioxidant activity. Broccoli inflorescences and Portuguese kale showed high antioxidant activity in Spring-Summer whilst turnip leaves did so in Summer-Winter. The Brassica vegetables can provide considerable amounts of bioactive compounds and thus may constitute an important natural source of dietary antioxidants.

Persister cells in a biofilm treated with a biocide.

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.

Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria.

The purpose of the present study was to evaluate the in vitro antibacterial effects of different classes of important and common dietary phytochemicals (5 simple phenolics - tyrosol, gallic acid, caffeic acid, ferulic acid, and chlorogenic acid; chalcone - phloridzin; flavan-3-ol - (-) epicatechin; seco-iridoid - oleuropein glucoside; 3 glucosinolate hydrolysis products - allylisothiocyanate, benzylisothiocyanate and 2-phenylethylisothiocyanate) against Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Staphylococcus aureus. Another objective of this study was to evaluate the effects of dual combinations of streptomycin with the different phytochemicals on antibacterial activity. A disc diffusion assay was used to evaluate the antibacterial activity of the phytochemicals and 3 standard antibiotics (ciprofloxacin, gentamicin and streptomycin) against the four bacteria. The antimicrobial activity of single compounds and dual combinations (streptomycin-phytochemicals) were quantitatively assessed by measuring the inhibitory halos. The results showed that all of the isothiocyanates had significant antimicrobial activities, while the phenolics were much less efficient. No antimicrobial activity was observed with phloridzin. In general P. aeruginosa was the most sensitive microorganism and L. monocytogenes the most resistant. The application of dual combinations demonstrated synergy between streptomycin and gallic acid, ferulic acid, chlorogenic acid, allylisothiocyanate and 2-phenylethylisothiocyanate against the Gram-negative bacteria. In conclusion, phytochemical products and more specifically the isothiocyanates were effective inhibitors of the in vitro growth of the Gram-negative and Gram-positive pathogenic bacteria. Moreover, they can act synergistically with less efficient antibiotics to control bacterial growth.

Aeromonas fluvialis sp. nov., isolated from a Spanish river.

A Gram-stain-negative, facultatively anaerobic bacterial strain, designated 717(T), was isolated from a water sample collected from the Muga river, Girona, north-east Spain. Preliminary analysis of the 16S rRNA gene sequence showed that this strain belonged to the genus Aeromonas, the nearest species being Aeromonas veronii (99.5 % similarity, with seven different nucleotides). A polyphasic study based on a multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) showed isolate 717(T) to be an independent phylogenetic line, with Aeromonas sobria, Aeromonas veronii and Aeromonas allosaccharophila as the closest neighbour species. DNA-DNA reassociation experiments and phenotypic analysis identified that strain 717(T) represents a novel species, for which the name Aeromonas fluvialis sp. nov. is proposed, with type strain 717(T) (=CECT 7401(T) =LMG 24681(T)).

Aeromonas taiwanensis sp. nov. and Aeromonas sanarellii sp. nov., clinical species from Taiwan.

Two clinical Aeromonas strains (A2-50(T) and A2-67(T)) recovered from the wounds of two patients in Taiwan could not be assigned to any known species of this genus based on their 16S rRNA gene sequences, which showed similarities of 99.6-99.8 % to those of the type strains of Aeromonas caviae, A. trota and A. aquariorum. The rpoD phylogenetic tree allocated these strains to two novel and independent phylogenetic lines, the neighbouring species being A. caviae, the type strain of which showed 93.2 % similarity (56 bp differences) to strain A2-50(T) and 92.2 % (63 bp differences) to strain A2-67(T). A multilocus phylogenetic analysis of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) confirmed that the two strains formed independent phylogenetic lineages within the genus. These data, together with phenotypic characterization and DNA-DNA reassociation results, revealed that these strains represent novel Aeromonas species, for which the names Aeromonas taiwanensis sp. nov. (type strain A2-50(T) =CECT 7403(T) =LMG 24683(T)) and Aeromonas sanarellii sp. nov. (type strain A2-67(T) =CECT 7402(T) =LMG 24682(T)) are proposed.