Hydroalcoholic extract based-ointment from Punica granatum L. peels with enhanced in vivo healing potential on dermal wounds

The present study reports for the first time, the in vivo wound healing potential of Punica granatum L. peels. A 5% (w/w) methanolic extract based-ointment was formulated and evaluated for its wound healing in guinea pigs. The ointment was applied in vivo on the paravertebral area of twelve excised wounded models once a day for 10 consecutive days. The ointment significantly enhanced the wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, DNA and proteins synthesis) and the histopathological characteristics. Such investigation was encouraged by the efficiency of the methanolic extract as antimicrobial and antioxidant. Indeed, the extract showed antioxidant activity as strong as natural and synthetic compounds (Trolox, BHA, Quercetin). Furthermore, the extract exhibited significant antibacterial and antifungal activity against almost all tested bacteria: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae, Salmonella anatum, Salmonella typhimurium, Streptococcus pneumoniae, and fungi Candida albicans, Candida glabrata, Trichopyton rubrum and Aspergillus niger. The formulated ointment might well find use as skin repair agent without hazard to human health based on these results and on the fact that it has been well established that the extracts of pomegranate used in conditions similar to those applied by traditional medicine, showed no toxic effects.     

Mixture of periodontopathogenic bacteria influences interaction with KB cells

IntroductionThe purpose of this study was to investigate the adhesion and invasion of periodontopathogenic bacteria in varied mixed infections and the release of interleukins from an epithelial cell line (KB cells).MethodsKB cells were co-cultured with Porphyromonas gingivalis ATCC 33277 and M5-1-2, Tannerella forsythia ATCC 43037, Treponema denticola ATCC 35405 and Fusobacterium nucleatum ATCC 25586 in single and mixed infections. The numbers of adherent and internalized bacteria were determined up to 18 h after bacterial exposure. Additionally, the mRNA expression and concentrations of released interleukin (IL)-6 and IL-8 were measured.ResultsAll periodontopathogenic bacteria adhered and internalized in different numbers to KB cells, but individually without any evidence of co-aggregation also to F. nucleatum. High levels of epithelial mRNA of IL-6 and IL-8 were detectable after all bacterial challenges. After the mixed infection of P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 the highest levels of released interleukins were found. No IL-6 and IL-8 were detectable after the mixed infection of P. gingivalis M5-1-2 and F. nucleatum ATCC 25586 and the fourfold infection of P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037 and F. nucleatum ATCC 25586.ConclusionAnaerobic periodontopathogenic bacteria promote the release of IL-6 and IL-8 by epithelial cells. Despite a continuous epithelial expression of IL-8 mRNA by all bacterial infections these effects are temporary because of the time-dependent degradation of cytokines by bacterial proteases. Mixed infections have a stronger virulence potential than single bacteria. Further research is necessary to evaluate the role of mixed infections and biofilms in the pathogenesis of periodontitis.     

Assessing the diversity of bacterial communities from marine sponges and their bioactive compounds

Symbiotic bacteria play vital roles in the survival and health of marine sponges. Sponges harbor rich, diverse and species-specific microbial communities. Symbiotic marine bacteria have increasingly been reported as promising source of bioactive compounds. A culturomics-based study was undertaken to study the diversity of bacteria from marine sponges and their antimicrobial potential. We have collected three sponge samples i.e. Acanthaster carteri, Rhytisma fulvum (soft coral) and Haliclona caerulea from north region (Obhur) of Red Sea, Jeddah Saudi Arabia. Total of 144 bacterial strains were isolated from three marine sponges using culture dependent method. Screening of isolated strains showed only 37 (26%) isolates as antagonists against oomycetes pathogens (P. ultimum and P. capsici). Among 37 antagonistic bacteria, only 19 bacterial strains exhibited antibacterial activity against human pathogens (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 29212). Four major classes of bacteria i.e γ-Proteobacteria, α-Proteobacteria, Firmicutes and Actinobacteria were recorded from three marine sponges where γ-Proteobacteria was dominant class. One potential bacterial strain Halomonas sp. EA423 was selected for identification of bioactive metabolites using GC and LC-MS analyses. Bioactive compounds Sulfamerazine, Metronidazole-OH and Ibuprofen are detected from culture extract of strain Halomonas sp. EA423. Overall, this study gives insight into composition and diversity of antagonistic bacterial community of marine sponges and coral from Red Sea and presence of active metabolites from potential strain. Our results showed that these diverse and potential bacterial communities further need to be studied to exploit their biotechnological significance.     

24-Branched Δ5 sterols from Laurencia papillosa red seaweed with antibacterial activity against human pathogenic bacteria

Methanol extract of thirty-eight seaweeds samples were first screened against Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6051) and -negative (Escherichia coli ATCC 8739 and Pseudomonas aerugenosa ATCC 9027) bacteria. Laurencia papillosa (Ceramiales, Rhodomelaceae, Rhodophyta) gave maximum antimicrobial activity against these bacteria. It was finally tested against four clinical Gram-negative isolates (E. coli, P. aerugenosa, Klebsiella pneumoniae and Shigella flexineri) and exhibited antibacterial activity. The extract was fractionated by column chromatography and the active fraction was identified as a cholesterol derivative, 24-propylidene cholest-5-en-3β-ol using gas chromatography mass spectrometry (GC–MS). The electrospray ionization mass spectrometry (ESI-MS) and FT-IR spectroscopic analysis also supported the structure of the compound. The minimum inhibitory concentration ranged from 1.2 to 1.7 μg/mL (IC₅₀) against clinical isolates. This is the first report of antibacterial activity of this cholesterol derivative. This compound could be exploited as potential lead molecule against broad spectrum drug development. The results also affirm the potential of seaweeds as an important natural source of antimicrobial compounds for pharmaceutical industries.     

Indigenous oil-degrading bacteria in crude oil-contaminated seawater of the Yellow sea,China

Indigenous oil-degrading bacteria play an important role in efficient remediation of polluted marine environments. In this study, we investigated the diversity and abundance of indigenous oil-degrading bacteria and functional genes in crude oil-contaminated seawater of the Dalian coast. The gene copy number bacterial 16S rRNA in total were determined to be about 10¹⁰ copies L^?1 in contaminated seawater and 10⁹ copies L^?1 in uncontaminated seawater. Bacteria of Alcanivorax, Marinobacter, Novosphingobium, Rhodococcus, and Pseudoalteromonas were found to be predominant oil-degrading bacteria in the polluted seawater in situ. In addition, bacteria belonging to Algoriphagus, Aestuariibacter, Celeribacter, Fabibacter, Zobellia, Tenacibaculum, Citreicella, Roseivirga, Winogradskyella, Thioclava, Polaribacter, and Pelagibaca were confirmed to be the first time as an oil-degrading bacterium. The indigenous functional enzymes, including AlkB or polycyclic aromatic hydrocarbons ring-hydroxylating dioxygenases α (PAH-RHDα) coding genes from Gram-positive (GP) and Gram-negative bacteria (GN), were revealed and quite diverse. About 10¹⁰ to 10¹¹ copies L^?1 for the expression of alkB genes were recovered and showed that the two-thirds of all the AlkB sequences were closely related to widely distributed Alcanivorax and Marinobacter isolates. About 10⁹ copies L^?1 seawater for the expression of RHDαGN genes in contaminated seawater and showed that almost all RHDαGN sequences were closely related to an uncultured bacterium; however, RHDαGP genes represented only about 10⁵ copies L^?1 seawater for the expression of genes in contaminated seawater, and the naphthalene dioxygenase sequences from Rhodococcus and Mycobacterium species were most abundant. Together, their data provide evidence that there exists an active aerobic microbial community indigenous to the coastal area of the Yellow sea that is capable of degrading petroleum hydrocarbons.     

Culture-Dependent and Independent Studies of Microbial Diversity in Highly Copper-Contaminated Chilean Marine Sediments

Cultivation and molecular-based approaches were used to study microbial diversity in two Chilean marine sediments contaminated with high (835 ppm) and very high concentrations of copper (1,533 ppm). The diversity of cultivable bacteria resistant to copper was studied at oxic and anoxic conditions, focusing on sulfate-, thiosulfate-, and iron-reducing bacteria. For both sediments, the cultivable bacteria isolated at oxic conditions were mostly affiliated to the genus Bacillus, while at anoxic conditions the majority of the cultivable bacteria found were closely related to members of the genera Desulfovibrio, Sphingomonas, and Virgibacillus. Copper resistance was between 100 and 400 ppm, with the exception of a strain affiliated to members of the genus Desulfuromonas, which was resistant up to 1,000 ppm of copper. In parallel, cloning and sequencing of 16S rRNA was performed to study the total bacterial diversity in the sediments. A weak correlation was observed between the isolated strains and the 16S rRNA operational taxonomic units detected. The presence of copper resistance genes (copA, cusA, and pcoA) was tested for all the strains isolated; only copA was detected in a few isolates, suggesting that other copper resistance mechanisms could be used by the bacteria in those highly copper-contaminated sediments.     

Genomic and Phylogenetic Characterization of Luminous Bacteria Symbiotic with the Deep-Sea Fish Chlorophthalmus albatrossis (Aulopiformes: Chlorophthalmidae)

Bacteria forming light-organ symbiosis with deep-sea chlorophthalmid fishes (Aulopiformes: Chlorophthalmidae) are considered to belong to the species Photobacterium phosphoreum. The identification of these bacteria as P. phosphoreum, however, was based exclusively on phenotypic traits, which may not discriminate between phenetically similar but evolutionarily distinct luminous bacteria. Therefore, to test the species identification of chlorophthalmid symbionts, we carried out a genomotypic (repetitive element palindromic PCR genomic profiling) and phylogenetic analysis on strains isolated from the perirectal light organ of Chlorophthalmus albatrossis. Sequence analysis of the 16S rRNA gene of 10 strains from 5 fish specimens placed these bacteria in a cluster related to but phylogenetically distinct from the type strain of P. phosphoreum, ATCC 11040^T, and the type strain of Photobacterium iliopiscarium, ATCC 51760^T. Analysis of gyrB resolved the C. albatrossis strains as a strongly supported clade distinct from P. phosphoreum and P. iliopiscarium. Genomic profiling of 109 strains from the 5 C. albatrossis specimens revealed a high level of similarity among strains but allowed identification of genomotypically different types from each fish. Representatives of each type were then analyzed phylogenetically, using sequence of the luxABFE genes. As with gyrB, analysis of luxABFE resolved the C. albatrossis strains as a robustly supported clade distinct from P. phosphoreum. Furthermore, other strains of luminous bacteria reported as P. phosphoreum, i.e., NCIMB 844, from the skin of Merluccius capensis (Merlucciidae), NZ-11D, from the light organ of Nezumia aequalis (Macrouridae), and pjapo.1.1, from the light organ of Physiculus japonicus (Moridae), grouped phylogenetically by gyrB and luxABFE with the C. albatrossis strains, not with ATCC 11040^T. These results demonstrate that luminous bacteria symbiotic with C. albatrossis, together with certain other strains of luminous bacteria, form a clade, designated the kishitanii clade, that is related to but evolutionarily distinct from P. phosphoreum. Members of the kishitanii clade may constitute the major or sole bioluminescent symbiont of several families of deep-sea luminous fishes.     

PCR for Detection of Shigella spp. in Mayonnaise

The use of PCR to amplify a specific virA gene fragment serves as a highly specific and sensitive method to detect virulent bacteria of the genus Shigella and enteroinvasive Escherichia coli. Amplification of a 215-bp DNA band was obtained by using isolated genomic DNA of Shigella, individual cells of Shigella dysenteriae, and mayonnaise contaminated with S. dysenteriae. Moreover, a multiplex PCR with specific (virA) and bacterium-restricted (16S ribosomal DNA) primers generated an amplification product of approximately 755 bp for all bacteria tested and an additional 215-bp product for Shigella and enteroinvasive E. coli.     

Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

The Antibacterial Properties of 4, 8, 4′, 8′-Tetramethoxy (1,1′-biphenanthrene) -2,7,2′,7′-Tetrol from Fibrous Roots of Bletilla striata

Biphenanthrene compound, 4, 8, 4′, 8′-tetramethoxy (1, 1′-biphenanthrene)—2, 7, 2′, 7′-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.     

Antimicrobial and wound healing activities of certain Sudanese medicinal plants

The emergence of drug-resistant organisms have been increasing globally; therefore, it is a burning need to find an alternative drug to get rid of the diseases caused by resistant strains. This study aims to evaluate the antimicrobial and wound healing activities of Loranthus acacia, Cassia obtusifolia and Cymbopogon proximus plants. All the plants were collected and extracted — by maceration method. Antimicrobial activities determined using standard ATCC strain for Gram-positive bacteria (Bacillus subtilis, Bacillus crew, Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus) and Gram-negative bacteria (Shigella sonnnei, Salmonella Typhimurium, Salmonella typhi, Klebsiella pnuemoniae, Escherichia coli and Pseudomonas aeruginosa) following agar well diffusion method. Plants extracts were prepared as gel and investigated for in vivo wound healing activities in rats. Histological studies were performed on animals’ skin. The results showed that all tested plants have various antimicrobial and wound healing activities. Out of these plants, L. acacia exhibited the best result; it revealed a significant result for antimicrobial activities counter to all Gram-positive, Gram-negative bacteria and wound healing activities in comparing with the reference drug. Thus, it is essential to consider L. acacia as a prospective source in progress in the synthesis of a new antimicrobial drug for the treatment of infectious diseases.     

Differential Role of the T6SS in Acinetobacter baumannii Virulence

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.     

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.     

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.     

Evaluation of a Modified Cefsulodin-Irgasan-Novobiocin Agar for Isolation of Yersinia spp

Y. enterocolitica and Y. pseudotuberculosis are important food borne pathogens. However, the presence of competitive microbiota makes the isolation of Y. enterocolitica and Y. pseudotuberculosis from naturally contaminated foods difficult. We attempted to evaluate the performance of a modified Cefsulodin-Irgasan-Novobiocin (CIN) agar in the differentiation of Y. enterocolitica from non-Yersinia species, particularly the natural intestinal microbiota. The modified CIN enabled the growth of Y. enterocolitica colonies with the same efficiency as CIN and Luria-Bertani agar. The detection limits of the modified CIN for Y. enterocolitica in culture medium (10 cfu/ml) and in artificially contaminated pork (10⁴ cfu/ml) were also comparable to those of CIN. However, the modified CIN provided a better discrimination of Yersinia colonies from other bacteria exhibiting Yersinia-like colonies on CIN (H₂S-producing Citrobacter freundii, C. braakii, Enterobacter cloacae, Aeromonas hydrophila, Providencia rettgeri, and Morganella morganii). The modified CIN exhibited a higher recovery rate of Y. enterocolitica from artificially prepared bacterial cultures and naturally contaminated samples compared with CIN. Our results thus demonstrated that the use of modified CIN may be a valuable means to increase the recovery rate of food borne Yersinia from natural samples, which are usually contaminated by multiple types of bacteria.     

Identification of Novel Benzoylformate Decarboxylases by Growth Selection

A growth selection system was established using Pseudomonas putida, which can grow on benzaldehyde as the sole carbon source. These bacteria presumably metabolize benzaldehyde via the β-ketoadipate pathway and were unable to grow in benzoylformate-containing selective medium, but the growth deficiency could be restored by expression in trans of genes encoding benzoylformate decarboxylases. The selection system was used to identify three novel benzoylformate decarboxylases, two of them originating from a chromosomal library of P. putida ATCC 12633 and the third from an environmental-DNA library. The novel P. putida enzymes BfdB and BfdC exhibited 83% homology to the benzoylformate decarboxylase from P. aeruginosa and 63% to the enzyme MdlC from P. putida ATCC 12633, whereas the metagenomic BfdM exhibited 72% homology to a putative benzoylformate decarboxylase from Polaromonas naphthalenivorans. BfdC was overexpressed in Escherichia coli, and the enzymatic activity was determined to be 22 U/ml using benzoylformate as the substrate. Our results clearly demonstrate that P. putida KT2440 is an appropriate selection host strain suitable to identify novel benzoylformate decarboxylase-encoding genes. In principle, this system is also applicable to identify a broad range of different industrially important enzymes, such as benzaldehyde lyases, benzoylformate decarboxylases, and hydroxynitrile lyases, which all catalyze the formation of benzaldehyde.     

Investigation of Reference Genes in Vibrio parahaemolyticus for Gene Expression Analysis Using Quantitative RT-PCR

Vibrio parahaemolyticus is a significant human pathogen capable of causing foodborne gastroenteritis associated with the consumption of contaminated raw or undercooked seafood. Quantitative RT-PCR (qRT-PCR) is a useful tool for studying gene expression in V. parahaemolyticus to characterize its virulence factors and understand the effect of environmental conditions on its pathogenicity. However, there is not a stable gene in V. parahaemolyticus that has been identified for use as a reference gene for qRT-PCR. This study evaluated the stability of 6 reference genes (16S rRNA, recA, rpoS, pvsA, pvuA, and gapdh) in 5 V. parahaemolyticus strains (O3:K6-clinical strain-tdh ⁺, ATCC33846-tdh ⁺, ATCC33847-tdh ⁺, ATCC17802-trh ⁺, and F13-environmental strain-tdh ⁺) cultured at 4 different temperatures (15, 25, 37 and 42°C). Stability values were calculated using GeNorm, NormFinder, BestKeeper, and Delta CT algorithms. The results indicated that recA was the most stably expressed gene in the V. parahaemolyticus strains cultured at different temperatures. This study examined multiple V. parahaemolyticus strains and growth temperatures, hence the finding provided stronger evidence that recA can be used as a reference gene for gene expression studies in V. parahaemolyticus.     

Antimicrobial Resistance of the Coral Pathogen Vibrio coralliilyticus and Caribbean Sister Phylotypes Isolated from a Diseased Octocoral

Vibrio coralliilyticus is a global marine pathogen that has been found to cause disease in several marine organisms, including corals. This study is the first report of the isolation of V. coralliilyticus from a diseased Caribbean octocoral, Pseudopterogorgia americana. Five sister phylotypes were positively identified using 16S rRNA gene sequencing, recA probes specific for V. coralliilyticus, and rep-PCR fingerprinting. The antimicrobial resistance was compared between pathogenic strains of V. coralliilyticus and the Caribbean strains. First, the antimicrobial resistance of V. coralliilyticus-type strain ATCC BAA-450 was determined using an agar-overlay antimicrobial bioassay at 24°C and 27°C, temperatures which are relevant to its known temperature-dependent virulence. From 108 distinct bacteria isolated from P. americana, 12 inhibited the V. coralliilyticus-type strain at 24°C and five at 27°C. Next, the phenotypic comparison of two Caribbean phylotypes and three V. coralliilyticus reference strains against a subset of 30 bacteria demonstrated a similar resistance trend. At both temperatures, the reference strains were inhibited by three bacteria isolates, while the Caribbean strains were inhibited by four to nine bacteria. Additionally, V. coralliilyticus-type strain ATCC BAA-450 and one of the Caribbean strains were inhibited by a higher number of bacteria at 24°C compared with 27°C. Together, these results highlight that V. coralliilyticus strains have antimicrobial resistance to the majority of coral-associated bacteria tested, which may be temperature-dependent in some strains. Furthermore, all V. coralliilyticus strains tested showed multi-drug resistance to a range of 11–16 (out of 26) commercial antibiotics. This study establishes V. coralliilyticus in association with a Caribbean octocoral and demonstrates its resistance to the antimicrobial activity of coral-associated bacteria and to commercial antibiotics.     

Identification and characterization of the channel-forming protein in the cell wall of Corynebacterium amycolatum

The mycolic-acid layer of certain gram-positive bacteria, the mycolata, represents an additional permeability barrier for the permeation of small water-soluble solutes. Consequently, it was shown in recent years that the mycolic acid layer of individual bacteria of the group mycolata contains pores, called porins, for the passage of hydrophilic solutes. Corynebacterium amycolatum, a pathogenic Corynebacterium species, belongs to the Corynebacteriaceae family but it lacks corynomycolic acids in its cell wall. Despite the absence of corynomycolic acids the cell wall of C. amycolatum contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of C. amycolatum. Based on partial sequencing of the protein responsible for channel formation derived from C. amycolatum ATCC 49368 we were able to identify the gene coram0001_1986 within the known genome sequence of C. amycolatum SK46 that codes for the cell wall channel. The corresponding gene of C. amycolatum ATCC 49368 was cloned into the plasmid pXHis for its expression in Corynebacterium glutamicum ?porA?porH. Biophysical characterization of the purified protein (PorA_coram) suggested that coram0001_1986 is indeed the gene coding for the pore-forming protein PorA_coram in C. amycolatum ATCC 49368. The protein belongs to the DUF (Domains of Unknown Function) 3068 superfamily of proteins, mainly found in bacteria from the family Corynebacteriaceae. The nearest relative to PorA_coram within this family is an ORF which codes for PorA_cres, which was also recognized in reconstitution experiments as a channel-forming protein in Corynebacterium resistens.     

Antibacterial substances from marine algae isolated from Jeddah coast of Red sea,Saudi Arabia

Marine algae are known to produce a wide variety of bioactive secondary metabolites and several compounds have been derived from them for prospective development of novel drugs by the pharmaceutical industries. However algae of the Red sea have not been adequately explored for their potential as a source of bioactive substances. In this context Ulva reticulata, Caulerpa occidentalis, Cladophora socialis, Dictyota ciliolata, and Gracilaria dendroides isolated from Red sea coastal waters of Jeddah, Saudi Arabia, were evaluated for their potential for bioactivity. Extracts of the algae selected for the study were prepared using ethanol, chloroform, petroleum ether and water, and assayed for antibacterial activity against Escherichia coli ATCC 25322, Pseudomonas aeruginosa ATCC 27853, Stapylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212. It was found that chloroform was most effective followed by ethanol, petroleum ether and water for the preparation of algal extract with significant antibacterial activities, respectively. Results also indicated that the extracts of red alga G. dendroides were more efficient against the tested bacterial strains followed by green alga U. reticulata, and brown algae D. ciliolata. Chemical analyses showed that G. dendroides recorded the highest percentages of the total fats and total proteins, followed by U. reticulata, and D. ciliolate. Among the bioflavonoids determined Rutin, Quercetin and Kaempherol were present in high percentages in G. dendroides, U. reticulata, and D. ciliolate. Estimation of saturated and unsaturated fatty acids revealed that palmitic acid was present in highest percentage in all the algal species analyzed. Amino acid analyses indicated the presence of free amino acids in moderate contents in all the species of algae. The results indicated scope for utilizing these algae as a source of antibacterial substances.