Induction and Genome Analysis of HY01, a Newly Reported Prophage from an Emerging Shrimp Pathogen Vibrio campbellii.

Vibrio campbellii is an emerging aquaculture pathogen that causes luminous vibriosis in farmed shrimp. Although prophages in various aquaculture pathogens have been widely reported, there is still limited knowledge regarding prophages in the genome of pathogenic V. campbellii. Here, we describe the full-genome sequence of a prophage named HY01, induced from the emerging shrimp pathogen V. campbellii HY01. The phage HY01 was induced by mitomycin C and was morphologically characterized as long tailed phage. V. campbellii phage HY01 is composed of 41,772 bp of dsDNA with a G+C content of 47.45%. A total of 60 open reading frames (ORFs) were identified, of which 31 could be predicted for their biological functions. Twenty seven out of 31 predicted protein coding regions were matched with several encoded proteins of various Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, and other phages of Gram-negative bacteria. Interestingly, the comparative genome analysis revealed that the phage HY01 was only distantly related to Vibrio phage Va_PF430-3_p42 of fish pathogen V. anguillarum but differed in genomic size and gene organization. The phylogenetic tree placed the phage together with Siphoviridae family. Additionally, a survey of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers revealed two matching sequences between phage HY01 genome and viral spacer sequence of Vibrio spp. The spacer results combined with the synteny results suggest that the evolution of V. campbellii phage HY01 is driven by the horizontal genetic exchange between bacterial families belonging to the class of Gammaproteobacteria.

Biologically rapid synthesized silver nanoparticles from aqueous Eucalyptus camaldulensis leaf extract: Effects on hyphal growth, hydrolytic enzymes, and biofilm formation in Candida albicans.

Bionanotechnology has increasingly gained attention in biomedical fields as antifungal and antibiofilm agents. In this study, biosynthesized silver nanoparticles (bio-AgNPs) using aqueous Eucalyptus camaldulensis leaf extract were successfully performed by a one-step green approach. Spherical-shaped nanoparticles, approximately 8.65 nm, exhibited noncytotoxicity to erythrocytes, HeLa, and HaCaT cells. The synthesized nanoparticles showed strong fungicidal activity ranging from 0.5 to 1 µg/ml. The nanoparticles affected Candida adhesion and invasion into host cells by reduced germ tube formation and hydrolytic enzyme secretion. Inhibitory effects of bio-AgNPs on Candida biofilms were evaluated by the prevention of yeast-to-hyphal transition. A decrease in cell viability within mature biofilm demonstrated the ability of bio-AgNPs to penetrate into the extracellular matrix and destroy yeast cell morphology, leading to cell death. Molecular biology study on biofilms confirmed downregulation in the expression of genes ALS3, HWP1, ECE1, EFG1, TEC1, ZAP1, encoding hyphal growth and biofilm development and PLB2, LIP9, SAP4, involved in hydrolytic enzymes. In addition to candida treatment, the bio-AgNPs could be applied as an antioxidant to protect against oxidative stress-related human diseases. The findings concluded that bio-AgNPs could be used as an antifungal agent for candida treatment, as well as be incorporated in medical devices to prevent biofilm formation.

Indole and Derivatives Modulate Biofilm Formation and Antibiotic Tolerance of Klebsiella pneumoniae.

Intercellular communication is a crucial process for the multicellular community in both prokaryotes and eukaryotes. Indole has been recognized as a new member of the signal molecules which enables the regulated control of various bacterial phenotypes. To elucidate the inter-species relationship among enteric microorganisms via indole signaling, Klebsiella pneumoniae (KP) culture was treated with indole solution and examined for the pathogenicity by using various phenotypic tests. Both synthetic and naturally-produced indole preparations had no deteriorating effect on growth and autoaggregative capacity of KP. The results showed that biofilm formation of carbapenem-susceptible K. pneumoniae (KP-S) was clearly induced by indole exposure (≈ 2-10 folds), whereas no significant difference was observed in the resistant counterpart. In addition, the tolerance to ß-lactam antibiotics of KP was altered upon exposure to indole and/or derivatives assessed by Kirby-Bauer disk diffusion test. Taken together, our finding indicates the functional role of indole in changing or modulating pathogenic behaviors of other bacteria.

Role of Indole Production on Virulence of Vibrio cholerae Using Galleria mellonella Larvae Model.

Cell to cell communication facilitated by chemical signals plays crucial roles in regulating various cellular functions in bacteria. Indole, one such signaling molecule has been demonstrated to control various bacterial phenotypes such as biofilm formation and virulence in diverse bacteria including Vibrio cholerae. The present study explores some key factors involved in indole production and the subsequent pathogenesis of V. cholerae. Indole production was higher at 37 °C than at 30 °C, although the growth at 37 °C was slightly higher. A positive correlation was observed between indole production and biofilm formation in V. cholerae. Maximum indole production was detected at pH 7. There was no significant difference in indole production between clinical and environmental V. cholerae isolates, although indole production in one environmental isolate was significantly different. Both growth and indole production showed relevant changes with differences in salinity. An indole negative mutant strain was constructed using transposon mutagenesis and the direct effect of indole on the virulence of V. cholerae was evaluated using Galleria mellonella larvae model. Comparison to the wild type strain, the mutant significantly reduced the mortality of G. mellonella larvae which regained its virulence after complementation with exogenous indole. A gene involved in indole production and the virulence of V. cholerae was identified.

Characterization of tryptophanase from Vibrio cholerae.

Tryptophanase (Trpase) is a pyridoxal phosphate (PLP)-dependent enzyme responsible for the production of indole, an important intra- and interspecies signaling molecule in bacteria. In this study, the tnaA gene of Vibrio cholerae coding for VcTrpase was cloned into the pET-20b(+) vector and expressed in Escherichia coli BL21(DE3) tn5:tnaA. Using Ni(2+)-nitrilotriacetic acid (NTA) chromatography, VcTrpase was purified, and it possessed a molecular mass of ∼49 kDa with specific absorption peaks at 330 and 435 nm and a specific activity of 3 U/mg protein. The VcTrpase had an 80 % homology to the Trpase of Haemophilus influenzae and E. coli, but only around 50 % identity to the Trpase of Proteus vulgaris and Porphyromonas gingivalis. The optimum conditions for the enzyme were at pH 9.0 and 45 °C. Recombinant VcTrpase exhibited analogous kinetic reactivity to the EcTrpase with K m and k cat values of 0.612 × 10(-3) M and 5.252 s(-1), respectively. The enzyme catalyzed S-methyl-L-cysteine and S-benzyl-L-cysteine degradation, but not L-phenylalanine and L-serine. Using a site-directed mutagenesis technique, eight residues (Thr52, Tyr74, Arg103, Asp137, Arg230, Lys269, Lys270, and His463) were conserved for maintaining enzyme catalysis. All amino acid substitutions at these sites either eliminated or remarkably diminished Trpase activity. These sites are thus potential targets for the design of drugs to control the V. cholerae Trpase and to further investigate its functions.

Occurrence of potentially pathogenic vibrios and related environmental factors in Songkhla Lake, Thailand.

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of Vibrio spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and Vibrio parahaemolyticus in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and V. cholerae non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, V. alginolyticus was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included V. vulnificus , V. fluvialis , and V. mimicus .