Complete genome sequence of fish-pathogenic Aeromonas hydrophila HX-3 and a comparative analysis: insights into virulence factors and quorum sensing.

The gram-negative, aerobic, rod-shaped bacterium Aeromonas hydrophila, the causative agent of motile aeromonad septicaemia, has attracted increasing attention due to its high pathogenicity. Here, we constructed the complete genome sequence of a virulent strain, A. hydrophila HX-3 isolated from Pseudosciaena crocea and performed comparative genomics to investigate its virulence factors and quorum sensing features in comparison with those of other Aeromonas isolates. HX-3 has a circular chromosome of 4,941,513 bp with a 61.0% G + C content encoding 4483 genes, including 4318 protein-coding genes, and 31 rRNA, 127 tRNA and 7 ncRNA operons. Seventy interspersed repeat and 153 tandem repeat sequences, 7 transposons, 8 clustered regularly interspaced short palindromic repeats, and 39 genomic islands were predicted in the A. hydrophila HX-3 genome. Phylogeny and pan-genome were also analyzed herein to confirm the evolutionary relationships on the basis of comparisons with other fully sequenced Aeromonas genomes. In addition, the assembled HX-3 genome was successfully annotated against the Cluster of Orthologous Groups of proteins database (76.03%), Gene Ontology database (18.13%), and Kyoto Encyclopedia of Genes and Genome pathway database (59.68%). Two-component regulatory systems in the HX-3 genome and virulence factors profiles through comparative analysis were predicted, providing insights into pathogenicity. A large number of genes related to the AHL-type 1 (ahyI, ahyR), LuxS-type 2 (luxS, pfs, metEHK, litR, luxOQU) and QseBC-type 3 (qseB, qseC) autoinducer systems were also identified. As a result of the expression of the ahyI gene in Escherichia coli BL21 (DE3), combined UPLC-MS/MS profiling led to the identification of several new N-acyl-homoserine lactone compounds synthesized by AhyI. This genomic analysis determined the comprehensive QS systems of A. hydrophila, which might provide novel information regarding the mechanisms of virulence signatures correlated with QS.

Experimental Induction of Bacterial Resistance to the Antimicrobial Peptide Tachyplesin I and Investigation of the Resistance Mechanisms.

Tachyplesin I is a 17-amino-acid cationic antimicrobial peptide (AMP) with a typical cyclic antiparallel ß-sheet structure that is a promising therapeutic for infections, tumors, and viruses. To date, no bacterial resistance to tachyplesin I has been reported. To explore the safety of tachyplesin I as an antibacterial drug for wide clinical application, we experimentally induced bacterial resistance to tachyplesin I by using two selection procedures and studied the preliminary resistance mechanisms. Aeromonas hydrophila XS91-4-1, Pseudomonas aeruginosa CGMCC1.2620, and Escherichia coli ATCC 25922 and F41 showed resistance to tachyplesin I under long-term selection pressure with continuously increasing concentrations of tachyplesin I. In addition, P. aeruginosa and E. coli exhibited resistance to tachyplesin I under UV mutagenesis selection conditions. Cell growth and colony morphology were slightly different between control strains and strains with induced resistance. Cross-resistance to tachyplesin I and antimicrobial agents (cefoperazone and amikacin) or other AMPs (pexiganan, tachyplesin III, and polyphemusin I) was observed in some resistant mutants. Previous studies showed that extracellular protease-mediated degradation of AMPs induced bacterial resistance to AMPs. Our results indicated that the resistance mechanism of P. aeruginosa was not entirely dependent on extracellular proteolytic degradation of tachyplesin I; however, tachyplesin I could induce increased proteolytic activity in P. aeruginosa Most importantly, our findings raise serious concerns about the long-term risks associated with the development and clinical use of tachyplesin I.

Polar Glycosylated and Lateral Non-Glycosylated Flagella from Aeromonas hydrophila Strain AH-1 (Serotype O11).

Polar and but not lateral flagellin proteins from Aeromonas hydrophila strain AH-1 (serotype O11) were found to be glycosylated. Top-down mass spectrometry studies of purified polar flagellins suggested the presence of a 403 Da glycan of mass. Bottom-up mass spectrometry studies showed the polar flagellin peptides to be modified with 403 Da glycans in O-linkage. The MS fragmentation pattern of this putative glycan was similar to that of pseudaminic acid derivative. Mutants lacking the biosynthesis of pseudaminic acid (pseB and pseI homologues) were unable to produce polar flagella but no changes were observed in lateral flagella by post-transcriptional regulation of the flagellin. Complementation was achieved by reintroduction of the wild-type pseB and pseI. We compared two pathogenic features (adhesion to eukaryotic cells and biofilm production) between the wild-type strain and two kinds of mutants: mutants lacking polar flagella glycosylation and lacking the O11-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. Results suggest that polar flagella glycosylation is extremely important for A. hydrophila AH-1 adhesion to Hep-2 cells and biofilm formation. In addition, we show the importance of the polar flagella glycosylation for immune stimulation of IL-8 production via toll-"like" receptor 5 (TLR5).

Knockout of the alanine racemase gene in Aeromonas hydrophila HBNUAh01 results in cell wall damage and enhanced membrane permeability.

This study focused on the alanine racemase gene (alr-2), which is involved in the synthesis of d-alanine that forms the backbone of the cell wall. A stable alr-2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-alanine, growth was unaffected; deprivation of d-alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No alanine racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-alanine for growth indicated that inactivation of alr-2 does not impose an auxotrophic requirement for d-alanine.

Influence of carvacrol and 1,8-cineole on cell viability, membrane integrity, and morphology of Aeromonas hydrophila cultivated in a vegetable-based broth.

This study investigated the effects of carvacrol (CAR) and 1,8-cineole (CIN) alone (at the MIC) or in combination at subinhibitory amounts (both at 1/8 MIC) on the cell viability, membrane permeability, and morphology of Aeromonas hydrophila INCQS 7966 (A. hydrophila) cultivated in a vegetable-based broth. CAR and CIN alone or in combination severely affected the viability of the bacteria and caused dramatic changes in the cell membrane permeability, leading to cell death, as observed by confocal laser microscopy. Scanning and transmission electron microscopy images of bacterial cells exposed to CAR or CIN or the mixture of both compounds revealed severe changes in cell wall structure, rupture of the plasma membrane, shrinking of cells, condensation of cytoplasmic content, leakage of intracellular material, and cell collapse. These findings suggest that CAR and CIN alone or in combination at subinhibitory amounts could be applied to inhibit the growth of A. hydrophila in foods, particularly as sanitizing agents in vegetables.

Role of Aeromonas hydrophila flagella glycosylation in adhesion to Hep-2 cells, biofilm formation and immune stimulation.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).

Aeromonas hydrophila produces conductive nanowires.

Aeromonas hydrophila is a facultative anaerobe which, under conditions of oxygen depletion, uses Fe(III) as electron acceptor. A. hydrophila produces pili during growth with Fe(III). The study was focused on the characterization of the morphology, the electrical properties and the nature of the bacterial pili. Scanning electron microscopy and conductive-probe atomic force microscopy revealed the presence of filaments between cells and substrate and their conductive nature. Our results indicate that pili of A. hydrophila strain A might serve as biological nanowires, transferring electrons from the cell surface to the surface of Fe(III) oxides and, in addition, the possibility of playing a role in inter/intra species signaling. Quorum sensing (QS) is recognized as one of the main regulatory ways for extracellular polymeric substances (EPS) production and biofilm formation. We present evidence that nanowire formation can be regulated by addition of synthetic acyl-homoserine lactones (AHL). These conductive pili may be involved in various interactions, and their protein components might be usable in the future for biotechnological approaches in materials science.

A multi-approach study of influence of growth temperature and nutrient deprivation in a strain of Aeromonas hydrophila.

In the present study we investigated the behavior of an Aeromonas hydrophila strain in prolonged nutrient deprivation condition analyzing the possible link among survival, cell morphology and adhesive characteristics and correlating them with the expression of the 43kDa outer membrane protein (OMP). The strain was inoculated in mineral and drinking chlorinated water, and in Nutrient Broth as a control with incubation at 4 and 24°C for 176days. Specimens were analyzed at different times during starvation stress. Viability was assessed by flow cytometry and growth by plate count technique; morphology and adhesivity were detected by optical and electron microscopy. The 43kDa OMP expression at different times was determined after immunoblotting assay using a polyclonal antibody produced in rabbit. The results showed a long-term viability as evidenced by cytofluorimetric analysis; however, the prolonged starvation led to the shift from the normal rod shaped cells to spherical forms in the last phases of incubation especially at 24°C. Concomitantly with the appearance of spherical cells we noted a reduction of the 43kDa OMP content and adhesive ability. Therefore, our results suggest a role of the 43kDa OMP as adhesin in A. hydrophila. In conclusion, we demonstrated that the bacterium can long survive under stress conditions, however adopting strategies which can lead to a loss of some cell surface components involved in the interactions with eukaryotic cells, therefore modifying its virulence properties.

Aeromonas hydrophila flagella glycosylation: involvement of a lipid carrier.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.

Effects of material morphology on the phototoxicity of nano-TiO2 to bacteria.

Nanostructured titania (nano-TiO2) is produced in diverse shapes, but it remains largely unknown how tuning the morphology of nano-TiO2 may alter its toxicity. Herein, we show that material morphology plays a critical role in regulating the phototoxicity of nano-TiO2 to bacteria. Low-dimensional nano-TiO2, including nanotubes, nanorods, and nanosheets, were synthesized hydrothermally, and their effects on the bacterial viability of Escherichia coli and Aeromonas hydrophila were compared to spherical nanostructures (anatase nanospheres and P25). Results reveal that TiO2 nanotubes and nanosheets are less phototoxic than their rod- and sphere-shape counterparts under simulated solar irradiation. None of the tested nano-TiO2 shows toxicity in the dark. In contrast to their diminished phototoxicity, however, TiO2 nanotubes and nanosheets exhibit comparable or even higher photoactivity than other nanostructures. Observations by scanning transmission electron microscopy suggest that material morphology influences nano-TiO2 phototoxicity by governing how nano-TiO2 particles align at the bacterial cell surface. Overall, when comparing materials with different morphologies and dimensionality, nano-TiO2 phototoxicity is not a simple function of photocatalytic reactivity or ROS production. Instead, we propose that the evaluation of nano-TiO2 phototoxicity encompasses a three-pronged approach, involving the intrinsic photoactivity, aggregation of nano-TiO2, and the nano-TiO2/bacteria surface interactions.

The cytotoxic effect of essential oils from Origanum vulgare L. and/or Rosmarinus officinalis L. on Aeromonas hydrophila.

This study aimed to evaluate the antibacterial activities of the essential oils from Origanum vulgare L. (OV) and Rosmarinus officinalis L. (RO), both singly and in combination at sub-inhibitory concentrations (» MIC + » MIC), against Aeromonas hydrophila and to investigate the possible mechanisms underlying these activities. Used singly (OV: 2.5 µL/mL; RO: 20 µL/mL) or in a mixture (OV: 0.625 µL/mL + RO: 5 µL/L), these essential oils led to a significant decrease (p<0.01) in bacterial viability after 24 h of exposure. A decrease in glucose consumption by A. hydrophila and release of cellular material were observed immediately after the addition of the essential oils, both singly and as a mixture, and continued for up to 6 h. Electron microscopy of cells exposed to the essential oils revealed severe changes in the plasma membrane, cytoplasmic appearance, and cell shape during the 6-h exposure period. OV and RO essential oils combined at sub-inhibitory concentrations could be rationally applied to inhibit the growth of A. hydrophila in food products, particularly minimally processed vegetables.

Transcriptional hierarchy of Aeromonas hydrophila polar-flagellum genes.

Aeromonas hydrophila polar-flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that the A. hydrophila polar flagellum is constitutively expressed. In contrast to other bacteria with dual flagellar systems such as Vibrio parahaemolyticus, the A. hydrophila LafK protein does not compensate for the lack of the polar-flagellum regulator FlrA (V. parahaemolyticus FlaK homologue). This is consistent with the fact that the A. hydrophila FlrA mutation abolishes polar-flagellum formation in liquid and on solid surfaces but does not affect inducible lateral-flagellum formation. The results highlight that the polar- and lateral-flagellum interconnections and control networks are specific and that there are differences between the dual flagellar systems in A. hydrophila and V. parahaemolyticus. Furthermore, our results indicate that the A. hydrophila polar-flagellum transcriptional hierarchy (also in class II, III, and IV genes) shares some similarities with but has many important differences from the transcriptional hierarchies of Vibrio cholerae and Pseudomonas aeruginosa. The A. hydrophila flhF and flhG genes are essential for the assembly of a functional polar flagellum because in-frame mutants fail to swim in liquid medium and lack the polar flagellum. In Vibrio and Pseudomonas flhG disruption increases the number of polar flagella per cell, and Pseudomonas flhF disruption gives an aberrant placement of flagellum. Here, we propose the gene transcriptional hierarchy for the A. hydrophila polar flagellum.

[Excretion of extracellular membrane nanovesicles by aeromonas].

AIM: Study of extracellular membrane nanovesicles production by Aeromonas hydrophila and Aeromonas salmonicida bacteria on a subcellular level. MATERIALS AND METHODS: 4 strains of A. hydrophila: 342-1, E 8-8, H 336 and H 1-6-05 and 1 strain of A. salmonicida A-450 as well as intact Wistar line rats were used. Methods of transmission electron microscopy: ultrathin sectioning and negative contrasting were used. RESULTS. A. hydrophila and A. salmonicida bacteria produced in pure cultures excrete into the environment extracellular membrane nanovesicles. The size of these vesicles varies from 20 to 200 nm in diameter. The process of gemmation from bacterial cell and possibility of obtaining isolated membrane nanovesicles preparations is shown. These vesicles are detected in ultrathin sections of apical surface of intact rat intestine among accumulations ofparietal microorganisms that colonize mucous membrane. Extracellular membrane nanovesicles excreted by aeromonas are analogous by size and ultrastructure to vesicles of other species of gram-negative bacteria described in the literature. CONCLUSION: During production of A. hydrophila and A. salmonicida bacteria in vitro nanovesicles are formed from the outer membranes of the cells and excreted into the environment, the nanovesicles are similar to those detected in ultrathin sections of the surface of intestine of rats among accumulations of parietal microorganisms and in glycocalix between epitheliocyte microvilli.

Effect of acute and chronic arsenic exposure on growth, structure and virulence of Aeromonas hydrophila isolated from fish.

Aeromonas hydrophila being a ubiquitous bacterium is prone to arsenic exposure. The present study was designed to determine the role of arsenic on growth and virulence of A. hydrophila. Exposure to arsenic (1 mg L(-1) and 2 mg L(-1)) had no effect on growth but significantly inhibited the hemolytic and cytotoxic potential of exposed bacteria. Transmission electron microscopy revealed loss of membrane integrity and presence of condensed cytoplasm suggestive of acute stress in bacteria exposed to arsenic. Arsenic-adapted bacteria were developed by repeated sub-culturing in presence of arsenic. Arsenic-adaptation led to significant recovery in hemolytic and cytotoxic potential. The arsenic-adapted bacteria exhibited normal membrane integrity, decreased cytoplasmic condensation and possessed scattered polysome like structures in the cytoplasm. A positive correlation was observed between arsenic tolerance and resistance to several antimicrobials. Arsenic-adaptation failed to confer cross-protection to mercury and cadmium stress. SDS-PAGE analysis revealed the expression of two new proteins of approximately 85 kDa and 79 kDa respectively in arsenic-adapted A. hydrophila. Plasmid-curing and transformation studies clearly indicate plasmid has no role on arsenic resistance trait of the bacteria. Our study, for the first time, reports a structure and function relationship of xenobiotics on bacteria.

Vanillin, a potential agent to prevent biofouling of reverse osmosis membrane.

Reverse osmosis (RO) membrane systems are widely used in water purification plants. Reduction in plant performance due to biofilm formation over the membrane is an inherent problem. As quorum sensing (QS) mechanisms of microorganisms have been reported to be involved in the formation of biofilm, ways are sought for quorum quenching (QQ) and thereby prevention of biofilm formation. In this study using a chemostat culture run for seven days in a CDC reactor it was found that a natural QQ compound, vanillin considerably suppressed bacterial biofilm formation on RO membrane. There was 97% reduction in biofilm surface coverage, when grown in the presence of vanillin. Similarly, the average thickness, total biomass and the total protein content of the biofilm that formed in the presence of vanillin were significantly less than that of the control. However vanillin had no effect on 1-day old pre-formed biofilm.

Integration of microfiltration and anion-exchange nanoparticles-based magnetic separation with MALDI mass spectrometry for bacterial analysis.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is powerful in characterizing and identifying bacterial isolates. However, sufficient quantities of bacterial cells are required for generating MALDI mass spectra and a procedure to isolate and enrich target bacteria from sample matrix prior to MALDI-MS analysis is often necessary. In this paper, anion-exchange superparamagnetic nanoparticles (NPs), i.e., fluidMAG-DEAE and fluidMAG-Q, were employed to capture Aeromonas, Salmonella, Pseudomonas, Enterococcus, Bacillus, Staphylococcus and Escherichia coli from aqueous solutions and fresh water. The magnetically isolated bacteria were then characterized by whole cell MALDI-MS. The capture efficiency was found to be dependent on bacterial species, medium pH, the functional group and concentration of the NPs. The experimental results demonstrated that fluidMAG-DEAE and fluidMAG-Q were broad spectrum probes for bacteria. Furthermore, both dead and live bacteria could be captured by the NPs, and the live bacteria captured remained viable. Membrane filtration prior to the magnetic isolation could increase enrichment factor and eliminate potential matrix interference. A detection limit of 1 x 10(3)cfu/ml was achieved for the bacteria spiked in tap water and reservoir water, and the analytical time was around 2h.

Morphological changes of Aeromonas hydrophila in response to osmotic stress.

The adaptive response of bacteria to stressful environmental situations may lead to a modification of physiological and phenotypical characteristics, including morphology. The aim of this study was the analysis of the ultrastructural changes in Aeromonas hydrophila exposed to different NaCl concentrations (1.7%, 3.4%, 6%) at 4 and 24 degrees C for 188 days. Bacterial cultures were processed for scanning and transmission electron microscopy, and specimens were analysed at different times during osmotic stress. SEM reveals the presence of three predominant morphotypes: rod, filamentous and spherical forms, depending on the time and culture conditions. Normal rod cells prevail in 1.7% NaCl growth conditions, maintaining high rates until the end of the trial at 4 degrees C. The most favourable conditions for the elongated morphotype are 3.4% NaCl at 4 degrees C. Spherical forms appear later, increase with time and are the prevalent population at the end of the trial at 24 degrees C, in all culture conditions. TEM reveals the presence of normal, necrotic-like and apoptotic-like forms; these latter forms increase with time according to salt concentration and temperature. Initially, a detachment of the external membrane appears, with cytoplasmic clumping into small, dense masses; as the process continues, both these features become more evident with increasing salt concentrations. This behaviour has been compared to that of eukaryotic cells undergoing growth factor deprivation-induced apoptosis. Occasionally, surface blebs are observed. In conclusion, the study suggests that the exposure of A. hydrophila to stressful conditions (osmolarity, temperature and nutrients) leads to the generation of varying morphotypes, which promote cell survival in adverse conditions and a rapid repopulation in post-stress environments.

Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3.

Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.

Effect of over-expression of phasin gene from Aeromonas hydrophila on biosynthesis of copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate.

The gene phaPAh, encoding the protein phasin that is associated with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) granule of Aeromonas hydrophila 4AK4, was cloned and characterized. Recombinant strains harboring additional copies of the phasin gene (phaPAh) and the polyhydroxyalkanoate (PHA) synthase gene (phaCAh) accumulated PHBHHx copolyesters consisting of 21 mol% 3-hydroxyhexanoate (3HHx) as compared to 14 mol% 3HHx produced by wild type strain. The molecular weight of PHBHHx produced by the above recombinants was lower than that obtained from the wild type strain grown under similar conditions. Over-expression of phaPAh led to the production of more PHA granules but with reduced sizes. SDS-PAGE showed that PhaPAh was the predominant protein present in the PHBHHx granules. The RT-PCR results suggested that phasin PhaPAh, regulated phaCAh gene at the transcription level. Gene PhaPWe from Wautersia eutropha (formerly Ralstonia eutropha; encoding a 20 kDa protein with low amino acid homology to the A. hydrophila 13 kDa protein) cloned into A. hydrophila 4AK4 exhibited similar effects on PHBHHx production and PHBHHx composition. These data suggest that the phasins could represent a protein family possessing similar functions but different structures.