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Tachyplesin I is a 17-amino-acid cationic antimicrobial peptide (AMP) with a typical cyclic antiparallel β-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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aeromonas hydrophila is an opportunistic pathogenic bacterium causing diseases in human and fish. The emergence of multidrug-resistant A. hydrophila isolates has been increasing in recent years. In this study, we have isolated a novel virulent podophage of A. hydrophila, designated as Ahp1, from waste water. Ahp1 has a rapid adsorption (96% adsorbed in 2 min), a latent period of 15 min, and a burst size of 112 PFU per infected cell. At least eighteen Ahp1 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 36-kDa protein being the predicted major capsid protein. Genome analysis of Ahp1 revealed a linear doubled-stranded DNA genome of 42,167 bp with a G + C content of 58.8%. The genome encodes 46 putative open reading frames, 5 putative phage promoters, and 3 transcriptional terminators. Based on high degrees of similarity in overall genome organization and among most of the corresponding ORFs, as well as phylogenetic relatedness among their DNAP, RNAP and major capsid proteins, we propose a new subgroup, designated Ahp1-like subgroup. This subgroup contains Ahp1 and members previously belonging to phiKMV-like subgroup, phiAS7, phi80-18, GAP227, phiR8-01, and ISAO8. Since Ahp1 has a narrow host range, for effective phage therapy, different phages are needed for preparation of cocktails that are capable of killing the heterogeneous A. hydrophila strains., Competing Interests: The authors have declared that no competing interests exist.

Co-infection with infectious spleen and kidney necrosis virus (ISKNV) and Aeromonas hydrophila is becoming ever more widespread in Chinese perch (Siniperca chuatsi) aquaculture industry, so that it's necessary to develop the combined vaccine against ISKNV and A. hydrophila disease. The surface display of heterologous on bacteria using anchoring motifs from outer membranes proteins has already been explored as an effective delivery system of viral antigens. In present study, the ISKNV orf086 gene, which is verified as a protective antigen, was inserted into ompA gene cassette of A. hydrophila GYK1 strain by homologous recombination. And an ompA-orf086 fusion A. hydrophila mutant strain K28 was constructed. Then the ISKNV orf086 was verified to express on the surface of A. hydrophila K28 by RT-PCR, western blot and indirect immunofluorescence assay. Next, Chinese perch were intraperitoneally inoculated with formalin inactivated A. hydrophila k28 emulsified with ISA763 adjuvant with a dose of 9 × 10(8) CFU per fish. Transcriptional analysis of non-specific and specific immune related genes revealed that the expression levels of IRF-7, IRAK1, Mx, Viperin, Lysozyme and IgM were strongly up-regulated in Chinese perch post-inoculation. In addition, specific antibodies were detected by ELISA, and the results showed that antibody titer against ISKNV or A. hydrophila reached the highest with 1:800 or 1:1200 on 14dpv, respectively. Lymphocyte proliferation were detected by MTT methods, and the results showed that the SI values of AH-K28 vaccinated group to three different stimulators were significantly higher than those of control group. At last, protective efficacy were determined by challenge trials. The cumulative mortality rates of vaccinated groups were significantly lower than the control one (P < 0.05) after ISKNV or A. hydrophila challenge, and the relative percentage survival (RPS) value was 73.3% and 60%, respectively. This system provides a novel approach to the surface display of heterologous antigenic proteins on A. hydrophila and suggests the possibility to use the recombinant K28 strain as a combined vaccine against ISKNV and A. hydrophila infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium., (Copyright © 2016. Published by Elsevier Ltd.)

Aeromonas hydrophila is a gram-negative fish pathogenic bacterium, also responsible for causing opportunistic pathological conditions in humans. It causes a number of diseases in fish due to which the fish industry incurs huge economic losses annually. Due to problems of antibiotic resistance, and the rapidity with which the infection spreads among fishes, vaccination remains the most effective strategy to combat this infection in fish populations. Among various virulence factors associated with bacterial virulence, outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity. In the present study, we have investigated the immunogenic potential of a non-specific porin, outer membrane protein C (OmpC) whose expression is regulated by the two-component regulatory system and plays a major role in the survival of A. hydrophila under different osmolaric conditions. The full-length gene (~1 kb) encoding OmpC of A. hydrophila was cloned, characterized and expressed in E. coli. High yield (~112 mg/L at shake flask level) of the recombinant OmpC (rOmpC) (~40 kDa) of A. hydrophila was obtained upon purification from inclusion bodies using Ni(2+)-NTA affinity chromatography. Immunization with purified rOmpC in murine model generated high endpoint (>1:40,000) titers. IgG isotyping, ELISA and ELISPOT assay indicated mixed immune response with a TH2 bias. Also, the anti-rOmpC antibodies were able to agglutinate A. hydrophila in vitro and exhibited specific cross-reactivity with different Aeromonas strains, which will facilitate easy detection of different Aeromonas isolates in infected samples. Taken together, these data clearly indicate that rOmpC could serve as an effective vaccine against different strains of Aeromonas, a highly heterogenous group of bacteria.

Background: Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation., Results: A total of 542 cell envelope proteins were identified by LC-MS/MS, with 66 down-regulated and 104 up-regulated proteins. Bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane ABC transporters and H(+)-ATP synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. Further q-PCR validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and ATP synthase elevated the susceptibility of A. hydrophila to iron starvation., Conclusions: Our study demonstrates that the redox and energy generation process, and ATP synthase in A. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. An understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating A. hydrophila infection.

Objective: The aim of this study is to explore the changes of sensitivity to tetracyclines and the resistance mechanism of Aeromonas hydrophila (Ah) after in vitro induction by exposure Ah to the increasing concentration of Doxycycline (DO)., Methods: The sensitive Ah isolates were first screened from clinical isolates and then incubated on TSA solid medium containing DO of 1/4×MIC value. The resistant strains were obtained through continuous subculture by increasing the concentration of DO in the medium for geometric series. The minimal inhibitory concentrations (MICs) of induced strains to DO and 16 other non-selected antimicrobials were determined. Meanwhile, MICs were also determined after adding efflux pump inhibitor 1-methyl-2-pyrrolidinone (NMP) to the medium. The relationship between sensitivity changes and efflux function were analyzed. Then five genes of tet from induced strains were amplified and sequenced., Results: The MICs of induced strains to DO increased significantly after induction, whereas the MICs of strains to those non-selected tetracyclines also increased. The MICs of induced strains to fluoroquinolone increased much more than that of control. The induced strains exhibited a little higher sensitivity to aminoglycoside and rifampicin. However, the MICs of all induced strains to DO decreased after adding NMP to the medium. The detection of tet genes indicated that tetA and tetE were positive in No. 7 after induction and the tetC gene was positive in No. 2 before and after induction. The tetE gene was detected in strains No. 1, 3, 4, 5, 6 and 7 no matter whether it was induced or not., Conclusion: This study suggested that tetE gene may be the predominant gene mediating tetracyclines-resistance of Ah, which would provide theoretical basis to clarify the resistance mechanism of Ah to tetracyclines.

Aeromonas species are important pathogens of fishes and aquatic animals capable of infecting humans and other animals via food. Due to the paucity of pan-genomic studies on aeromonads, the present study was undertaken to analyse the pan-genome of three clinically important Aeromonas species (A. hydrophila, A. veronii, A. caviae). Results of pan-genome analysis revealed an open pan-genome for all three species with pan-genome sizes of 9181, 7214 and 6884 genes for A. hydrophila, A. veronii and A. caviae, respectively. Core-genome: pan-genome ratio (RCP) indicated greater genomic diversity for A. hydrophila and interestingly RCP emerged as an effective indicator to gauge genomic diversity which could possibly be extended to other organisms too. Phylogenomic network analysis highlighted the influence of homologous recombination and lateral gene transfer in the evolution of Aeromonas spp. Prediction of virulence factors indicated no significant difference among the three species though analysis of pathogenic potential and acquired antimicrobial resistance genes revealed greater hazards from A. hydrophila. In conclusion, the present study highlighted the usefulness of whole genome analyses to infer evolutionary cues for Aeromonas species which indicated considerable phylogenomic diversity for A. hydrophila and hitherto unknown genomic evidence for pathogenic potential of A. hydrophila compared to A. veronii and A. caviae.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by an emergent, high virulent Aeromonas hydrophila (vAh) in channel catfish, Ictalurus punctatus Adipose fin clipped catfish were challenged with vAh using a waterborne challenge method, and the distribution of vAh over a time course was detected and quantified using real-time polymerase chain reaction. The results showed that 77.8% of fish died within 48 h post challenge with mean day to death of 1.5 days. At 2 h post challenge, vAh (inferred from genomic DNA copies or genome equivalents) was detected in all external and internal tissues sampled. Gill had the highest vAh cells at 1 h post challenge. Spleen harbored the most vAh cells among internal organs at 4 h post challenge. The tissues/organs with most vAh cells detected at 8 h post challenge were adipose fin, blood, intestine, kidney and skin, while liver showed the highest vAh cells at 24 h post challenge. These results suggest that vAh was able to rapidly proliferate and spread, following wound infection, through the fish blood circulation system and cause mortality within 8-24 h., (Published by Oxford University Press on behalf of FEMS 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The mechanisms of resistance to levofloxacin for Aeromonas hydrophila isolated from diseased fish and selected in vitro were examined in this study. Levofloxacin-resistant mutants were obtained by selection of A. hydrophila in vitro. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were sequenced in Lev(R) strains and reverse mutation strains. All Lev(R) strains carried a point mutation at codon 83 (Ser → Ile), and one strain (25 %) harbored a mutation at position 92 (Leu → Met) in the GyrA-QRDR. After being transferred in a levofloxacin-free medium, one strain of the mutants was successfully reversed and the reversion was related with mutations of GyrA-QRDR at positions 81 (Gly → Asp) and 83 (Ile → Ser). No amino acid alteration was found in the ParC-QRDR. In addition, the minimum inhibitory concentration (MIC) of levofloxacin for the mutants was lower in the presence of reserpine, and all mutants were also resistant to some of the other quinolones. It was found that the mechanism of levofloxacin resistance of A. hydrophila selected in vitro was related to gyrA of type II topoisomerase, and an efflux mechanism was involved in the resistance as well.

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Aeromonas are autochthonous inhabitants of aquatic environments, including chlorinated and polluted waters, although they can also be isolated from a wide variety of environmental and clinical sources. They cause infections in vertebrates and invertebrates and are considered to be an emerging pathogen in humans, producing intestinal and extra-intestinal diseases. Most of the clinical isolates correspond to A. hydrophila, A. caviae, and A. veronii bv. Sobria, which are described as the causative agents of wound infections, septicaemia, and meningitis in immunocompromised people, and diarrhoea and dysenteric infections in the elderly and children. The pathogenic factors associated with Aeromonas are multifactorial and involve structural components, siderophores, quorum-sensing mechanisms, secretion systems, extracellular enzymes, and exotoxins. In this study, we analysed a representative number of clinical and environmental strains belonging to the A. hydrophila species complex to evaluate their potential pathogenicity. We thereby detected their enzymatic activities and antibiotic susceptibility pattern and the presence of virulence genes (aer, alt, ast, and ascV). The notably high prevalence of these virulence factors, even in environmental strains, indicated a potential pathogenic capacity. Additionally, we determined the adhesion capacity and cytopathic effects of this group of strains in Caco-2 cells. Most of the strains exhibited adherence and caused complete lysis.

Listeria monocytogenes exhibits symbiotic codependence with the dominant commensal bacteria, which may help it avoid being removed or inactivated by disinfectants in local environments. In this study, we investigated L. monocytogenes-positive biofilms at food production facilities, and the dominant bacterial species of the biofilms were identified to determine the properties of the microbiological background. For this purpose, the ISO 11290 method was used for the detection and isolation of L. monocytogenes, and the species were further identified based on 16S rRNA and hly genes. 16S rRNA gene-based cloning, terminal restriction fragment length polymorphism, and denaturing gradient gel electrophoresis were combined to evaluate the dominant bacteria of the drain biofilms. Out of 100 drain samples, 8 were naturally contaminated with L. monocytogenes. Three molecular methods consistently showed that Pseudomonas psychrophila, Pseudomonas sp., and Klebsiella oxytoca were dominant species in 3Q, 5Q, and 6Q samples; Aeromonas hydrophila and Klebsiella sp. were significantly dominant in 1-2, 1-3, and 3-2 samples; A. hydrophila and K. oxytoca were dominant in the 2-3 sample; and A. hydrophila and Pseudomonas sp. were prominent in the 3-3 sample. Different biofilms from the same plant shared common bands, suggesting that similar bacteria can be found and can be dominant in different biofilms. This study provides a better understanding of the dominant compositions in these bacterial communities. Further studies to determine the mechanism of co-culture with L. monocytogenes will be of critical importance in predicting effective disinfection strategies.

Objective: To investigate the infection status and virulent genes of Aeromonas in patients with acute diarrhea in Pudong New Area, Shanghai., Methods: In 2012, stool samples were collected from diarrhea patients in 12 sentinel hospitals in Pudong for the detections of 13 pathogens causing diarrhea, and the detections of 5 diarrhea related virulent genes were conducted for Aeromonas isolates., Results: A total of 101 patients were infected with Aeromonas in 2533 patients (4.0%). A total of 101 Aeromonas strains were isolated, including 17 Aeromonas hydrophila strains (18.8%), 44 Aeromonas veronii biovar sobria strains (52.5%) and 12 Aeromonas caviae strains (29.7%). And 44 coinfections with other pathogens were detected. Aeromonas infection mainly occurred in summer and in people aged ≥20 years. Among the patients infected with Aeromonas, 71 (70.3%) had watery diarrhea, 20 (19.8%) had vomiting and 11 (10.9%) had fever. Virulent genes detection showed that 95.0% of the Aeromonas. strains carried virulent genes, and the detection rates of hlyA, aerA, act, alt, and ast genes were 5.9%, 6.9%, 67.3%, 42.6% and 13.9%, respectively., Conclusions: High incidence of Aeromonas infection was found in the patients with acute diarrhea in Pudong, and a high proportion of coinfections with other pathogens was detected too. Most Aeromonas strains carried virulent genes, and the distribution varied.

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Two gram-negative, facultatively anaerobic, motile and rod-shaped bacteria, strains P2G1(T) and P1A11, were isolated from the Ter River in Ripoll, Spain. Phylogenetic analysis based on 16S rRNA gene sequences showed that both strains are closely related to each other and that their closest relatives were Aeromonas media ATCC 33907(T) (99.4%) and Aeromonas hydrophila ATCC 7966(T) (99.3%). Multilocus sequence analysis (MLSA) based on the partial sequences of gyrA, gyrB, rpoD, recA, and dnaJ genes suggested that these two strains represent a novel species that clustered with A. media ATCC 33907(T). This was further supported by DNA-DNA hybridization analysis between P2G1(T) and A. media LMG 9073(T). Phenotypic features also allowed their differentiation from closely related species. These two strains should, therefore, be considered to represent a novel species within the genus Aeromonas, for which the name Aeromonas rivipollensis sp. nov. is proposed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: The nucleotide sequence analysis of the gyrB gene indicated that the fish Aeromonas spp. isolates could be identified as Aeromonas hydrophila and Aeromonas veronii biovar sobria, whereas chicken Aeromonas spp. isolates identified as Aeromonas caviae. PCR data revealed the presence of Lip, Ser, Aer, ACT and CAI genes in fish Aer. hydrophila isolates, ACT, CAI and Aer genes in fish Aer. veronii bv sobria isolates and Ser and CAI genes in chicken Aer. caviae isolates. All chicken isolates showed variable resistance against all 12 tested antibiotic discs except for cefotaxime, nitrofurantoin, chloramphenicol and ciprofloxacin, only one isolate showed resistance to chloramphenicol and ciprofloxacin. Fish Aeromonads were sensitive to all tested antibiotic discs except amoxicillin, ampicillin-sulbactam and streptomycin., Significance and Impact of the Study: Many integrated fish farms depend on the application of poultry droppings/litter which served as a direct feed for the fish and also acted as pond fertilizers. The application of untreated poultry manure exerts an additional pressure on the microbial world of the fish's environment. Aeromonas species are one of the common bacteria that infect both fish and chicken. The aim of this study was to compare the phenotypic traits and genetic relatedness of aeromonads isolated from two diverse hosts (terrestrial and aquatic), and to investigate if untreated manure possibly enhances Aeromonas dissemination among cohabitant fish with special reference to virulence genes and antibiotic resistant traits., (© 2015 The Society for Applied Microbiology.)

The multifunctional-autoprocessing repeats-in-toxin (MARTX(Vv)) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the 'Makes caterpillars floppy-like' effector domain (MCF(Vv)) is previously unstudied. Using transient expression and protein delivery, MCF(Vv) and MCF(Ah) from the Aeromonas hydrophila MARTX(Ah)) toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCF(Vv) identified an Arg-Cys-Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF(Vv). The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF(Vv), indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCF(Vv) within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins., (© 2015 John Wiley & Sons Ltd.)

Motile aeromonad septicemia (MAS) caused by Aeromonas hydrophila is one of the common bacterial causes of fish mortalities. Prophylactic vaccination against this and other diseases is essential for continued growth of aquaculture. The type VI secretion system (T6SS) plays a crucial role in the virulence of A. hydrophila. The hemolysin co-regulated protein (Hcp) is an integral component of the T6SS apparatus and is considered a hallmark of T6SS function. Here, the T6SS effector Hcp was expressed and characterized, and its immunogenicity and protective efficacy were evaluated in common carp (Cyprinus carpio). Hcp secretion was found to be strongly induced by low temperature in A. hydrophila. Immunoblot analysis demonstrated that Hcp is conserved among A. hydrophila strains of different origins. The vaccination with recombinant Hcp resulted in an increased survival (46.67%) in common carp during a 10-day challenge time compared to non-vaccinated fish (7.14%). The vaccinated fish also showed the significantly increased levels of IgM antibody in serum and cytokines such as inerleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in kidney, spleen and gills. The recombinant Hcp shows promise as a vaccine candidate against A. hydrophila., (Copyright © 2015. Published by Elsevier Ltd.)

The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by Aeromonas hydrophila on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30 °C for 24 h and 96 h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but Aeromonas-selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain Chromobacterium violaceum CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with Vibrio harveyi BB170. The biofilm structure at various salinities (0-3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of A. hydrophila grown at 0-0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1-3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0-0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1-3%) induced stress in A. hydrophila. The present study provides insight into the ecology of A. hydrophila growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Unlabelled: Aeromonas hydrophila has been increasingly implicated as the aetiologic agent of various human diseases. Therefore, reliable laboratory detection and identification of this bacterium has become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method for rapid detection of Aer. hydrophila using a cross-priming amplification (CPA) assay with the desA gene as the target. The desA gene is crucial for the survival and growth of Aer. hydrophila under iron starvation. The results can be visualized as colour changes without opening the reaction tubes. No false-positive results were observed for the 33 non-Aer. hydrophila strains tested to evaluate assay specificity. The limit of detection for Aer. hydrophila was approximately 200 copies of desA per reaction (on reference plasmids) and 5 × 10(3)  CFU g(-1) Aer. hydrophila in simulated human stool, which is the same sensitivity as a qPCR assay. The performance of the CPA assay was also evaluated with 100 stool specimens from diarrhoea patients and 40 environmental water samples. In conclusion, the simplicity, cost-effectiveness and nearly instrument-free platform of the CPA assay make it practical for use in primary care facilities and smaller clinical laboratories., Significance and Impact of the Study: Aeromonas hydrophila is a human pathogen that infects via exposed wounds or ingestion of contaminated water and food. In this study, a CPA-based PCR method was developed for specific, rapid, cost-effective detection of Aer. hydrophila, and the test results could be visualized without opening the reaction tubes. This is the first report on the application of the CPA method for the detection of Aer. hydrophila. This novel method could be practical for use in primary care facilities and smaller clinical laboratories., (© 2015 The Society for Applied Microbiology.)

Two new natural CphA metallo-β-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

For a better understanding of the genitic regulation of the adhesion of Aeromonas hydrophila, a mini-Tn10 transposon mutagenesis system was introduced to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOF/Km) and the recipient A. hydrophila strain W. Out of 332 individual colonies, 7 mutants with significantly attenuated adhesion ability were selected. The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant strain M45 was amplified by TAIL-PCR. The results showed that an ORF of approximately 870 bp (GenBank accession number KP271930) of the mutant strain M45 was inserted by mini-Tn10. This ORF putatively encodes a deduced 289 amino acid protein that displays the highest identity (100%) with the CobQ/CobB/MinD/ParA family protein of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type W, the mutant strain M45 and the complemented strain C45 were investigated. The results indicated that mutation of MinD gene in A. hydrophila could lead to abnormal cell division and reduce the ability of adhesion, biofilm formation and bacterial motility., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Adhesion to the host mucus is a crucial step in the early infection stage of pathogenic bacteria. To investigate the mechanisms of the adhesion of Aeromonas hydrophila to its host mucus, a mutant library was constructed using the mini-Tn10 transposon mutagenesis system. Of 276 individual colonies, the mutant strain with the most attenuated adhesion ability in this study was screened out and designated A77. Molecular analysis showed that a 414-bp sequence flanking mini-Tn10 in A77 had the highest identity (97%) with the bacterial flagellar protein gene flgN. A complemented strain flgN+ was constructed and the biological characteristics of the wild-type, mutant A77, and complemented flgN+ strains were investigated. The results showed that the decreased abilities of motility, adhesion to mucus, and biofilm formation in the mutant strain were partially recovered in the complemented flgN+ strain, which suggested that flgN plays an important role in the adhesion of A. hydrophila to its host.

Outbreaks in fish of motile Aeromonad septicemia (MAS) caused by Aeromonas hydrophila have caused a great concern worldwide. Here, for the first time, we provide two complete genomes of epidemic A. hydrophila strains isolated in China. To gain an insight into the pathogenicity of epidemic A. hydrophila, we performed comparative genomic analyses of five epidemic strains belonging to sequence type (ST) 251, together with the environmental strain ATCC 7966(T). We found that the known virulence factors, including a type III secretion system, a type VI secretion system and lateral flagella, are not required for the high virulence of the ST251 clonal group. Additionally, our work identifies three utilization pathways for myo-inositol, sialic acid and L-fucose providing clues regarding the factors that underlie the epidemic and virulent nature of ST251 A. hydrophila. Based on the geographical distribution and biological resources of the ST251 clonal group, we conclude that ST251 is a high-risk clonal group of A. hydrophila which may be responsible for the MAS outbreaks in China and the southeastern United States.

To explore the antibacterial activity and mechanism of total alkaloids and berberine from Coptidis Rhizoma on Aeromonas hydrophila, and determine the effect of total alkaloids and berberine from Coptidis Rhizoma on minimum inhibitory concentrations, permeability and fluidity of cell membrane, conformation of membrane proteins and virulence factors of A. hydrophila. The results showed that both total alkaloids and berberine from Coptidis Rhizoma had antibacterial activities on A. hydrophila, with minimum inhibitory concentrations of 62.5 and 125 mg · L(-1), respectively. Total alkaloids and berberine from Coptidis Rhizoma could increase the fluidity of membrane, change the conformation of membrane porteins and increase the permeability of bacteria membrane by 24.52% and 19.66%, respectively. Besides, total alkaloids and berberine from Coptidis Rhizoma significantly decreased the hemolysis of exotoxin and the mRNA expressions of aerA and hlyA (P < 0.05, P < 0.01), the secretion of endotoxin and the mRNA expression of LpxC (P < 0.05, P < 0.01). The results suggested that the antibacterial activity of total alkaloids and berberine from Coptidis Rhizoma on A. hydrophila may be related to the bacteria membrane injury. They inhibited the bacterial growth by increasing membrane lipid fluidity and changing conformation of membrane proteins, and reduced the secretion of virulence factors of A. hydrophila to weaken the pathogenicity.

Aeromonas hydrophila is an aquatic bacterium responsible for several human illnesses. The aim of this work was to investigate the survival ability and virulence expression of two strains from different sources (fish, strain 87 and surface water, strain LS) maintained in a seawater microcosm. The strains were analyzed for the total and viable bacterial counts, adhesion ability to Hep-2 cells and aerA gene expression by qPCR throughout the experiment (35 days). Both strains reached a putative VBNC state and lost adhesive properties but exhibited a different behavior in the expression of aerA. This could be due to the different origin of the two strains; the former adapted to a habitat rich of nutrient and the latter already used to survive in a more hostile environment. Moreover, our results indicate that the quantitative determination of aerA mRNA can be a useful indicator of virulence expression under stress conditions.

Background: Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers., Results: In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS=67.0%) or E. tarda EIB202 (RPS=72.3%)., Conclusions: Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts.

Unlabelled: The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA&#95;0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries., Significance and Impact of the Study: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes., (© 2014 The Society for Applied Microbiology.)

Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in aquatic environments and can cause septicemia in both fish and humans. However, the underlying mechanisms leading to severe infection are not well understood. In this study, an A. hydrophila nuclease (ahn) deletion mutant was constructed to investigate its contribution to pathogenesis. This mutant did not differ from the wild-type strain in terms of its growth or hemolytic phenotype. However, the ahn-deficient mutant was more susceptible to being killed by fish macrophages and mouse blood in vitro. Furthermore, evidence obtained using both fish and murine infection models strongly indicated that the inactivation of Ahn impaired the ability of A. hydrophila to evade innate immune clearance in vivo. More importantly, the virulence of the mutant was attenuated in both fish and mice, with reductions in dissemination capacities and mortality rates. These findings implicate Ahn in A. hydrophila virulence, with important functions in evading innate immune defenses.

Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.

The development of resistance to antimicrobials used in aquatic animals is an increasing concern for aquaculture and public health. To monitor the occurrence of antimicrobial resistance and resistance genes in Aeromonas, a total of 106 isolates were collected from cultured freshwater animals in China from 1995 to 2012. Antimicrobial susceptibilities were determined by the disk diffusion method. The highest resistance percentage occurred with ampicillin, rifampin, streptomycin, and nalidixic acid. Most strains were sensitive to fluoroquinolones, doxycycline, cefotaxime, chloramphenicol, and amikacin. The isolates from turtle samples had the highest levels of resistance to 11 of the 12 tested antimicrobials when compared with those from fish or shrimp. Polymerase chain reaction and DNA sequence results showed that all trimethoprim/sulfamethoxazole-resistant strains contained sul1, and 37.0% were positive for tetA in tetracycline-resistant strains. ant(3″)-Ia was identified in 13 (24.5%) streptomycin-resistant strains. Plasmid-borne quinolone resistance genes were detected in five Aeromonas hydrophila (4.7%), two of which carried qnrS2, while the other three strains harbored aac(6')-Ib-cr. Two cefotaxime-resistant A. hydrophila were positive for bla(TEM-1) and bla(CTX-M-3). To our knowledge, this is the first report characterizing antimicrobial resistance in Aeromonas isolated from cultured freshwater animals in China, and providing resistance information of pathogen in Chinese aquaculture.

The interaction between pathogenic bacteria and the host phagocytes is complicated. It is generally believed that only obligate intracellular pathogens can invade and survive in host phagocytes. In this study, we revealed that the pathogenic Aeromonas hydrophila B11 can also invade and survive in the macrophages of its host Anguilla japonica in vitro. To further investigate the mechanisms of A. hydrophila invasion and survival in host macrophages, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient A. hydrophila B11. Out of 465 individual colonies, 13 mutants impaired in survival within macrophages were selected, and the mutant BM116 was the most seriously impaired strain. Molecular analysis showed that an ORF of approximately 1335 bp (GenBank accession numbers JQ974982) of the mutant BM116 was inserted by mini-Tn10. This ORF putatively encodes a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type B11, the mutant B116 and the complemented strain were investigated. The results reveal that the flagella of the mutant BM116 was absent and that these mutant bacteria exhibited defective motility, adhesion, and invasion and survival in host macrophages when compared with the wild type and the complemented strain. These findings indicate that flgE is required for flagellum biogenesis in A. hydrophila and that flagellar motility is required for A. hydrophila invasion and survival in the macrophages of its host. Our findings provide an important new understanding of the nonintracellular pathogenic bacteria invasion and survival in host phagocytes and the interactions between the pathogens and their host., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The type two secretion system is a large, trans-envelope apparatus that secretes toxins across the outer membrane of many Gram-negative bacteria. In Aeromonas hydrophila, ExeA interacts with peptidoglycan and forms a heteromultimeric complex with ExeB that is required for assembly of the ExeD secretin of the secretion system in the outer membrane. While the peptidoglycan-ExeAB (PG-AB) complex is required for ExeD assembly, the assembly mechanism remains unresolved. We analyzed protein-protein interactions to address the hypothesis that ExeD assembly in the outer membrane requires direct interaction with the PG-AB complex. Yeast and bacterial two hybrid analyses demonstrated an interaction between the periplasmic domains of ExeB and ExeD. Two-codon insertion mutagenesis of exeD disrupted lipase secretion, and immunoblotting of whole cells demonstrated significantly reduced secretin in mutant cells. Mapping of the two-codon insertions and deletion analysis showed that the ExeB-ExeD interaction involves the N0 and N1 subdomains of ExeD. Rotational anisotropy using the purified periplasmic domains of ExeB and ExeD determined that the apparent dissociation constant of the interaction is 1.19±0.16 µM. These results contribute important support for a putative mechanism by which the PG-AB complex facilitates assembly of ExeD through direct interaction between ExeB and ExeD. Furthermore, our results provide novel insight into the assembly function of ExeB that may contribute to elucidating the role of homologous proteins in secretion of toxins from other Gram negative pathogens.

Aeromonas hydrophila, an inhabitant of aquatic ecosystems found in most parts of the world, has considerable virulence potential. The polymerase chain reaction technique was used to assay for the presence of five virulence factor genes: haemolytic toxins aerA and ahh1, elastase ahyB, the enterotoxin act, and the polar flagella flaA/flaB in the A. hydrophila strain isolated from the River Nile. Drug screening showed high levels of resistance to β-lactam antibiotics and tetracycline. Slime production was determined by the Congo red agar plate test. The isolate produced two restriction enzymes named AehI and AehII which are isoschizomers of XhoI and StuI respectively. The complete nucleotide sequence of the cryptic plasmid pAhy2.5 (2524 bp) from this strain was determined. Sequence analysis revealed the presence of two open reading frames (ORFs) encoding putative proteins. The protein coded by ORF1 is homologous with Rep proteins of plasmids belonging to the pC194 family, which are known to replicate by the rolling-circle mechanism. The putative double-strand origin of replication and a region with palindromic sequences that could function as a single-strand origin were detected in pAhy2.5., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The genomes of 10 Aeromonas isolates identified and designated Aeromonas hydrophila WI, Riv3, and NF1 to NF4; A. dhakensis SSU; A. jandaei Riv2; and A. caviae NM22 and NM33 were sequenced and annotated. Isolates NF1 to NF4 were from a patient with necrotizing fasciitis (NF). Two environmental isolates (Riv2 and -3) were from the river water from which the NF patient acquired the infection. While isolates NF2 to NF4 were clonal, NF1 was genetically distinct. Outside the conserved core genomes of these 10 isolates, several unique genomic features were identified. The most virulent strains possessed one of the following four virulence factors or a combination of them: cytotoxic enterotoxin, exotoxin A, and type 3 and 6 secretion system effectors AexU and Hcp. In a septicemic-mouse model, SSU, NF1, and Riv2 were the most virulent, while NF2 was moderately virulent. These data correlated with high motility and biofilm formation by the former three isolates. Conversely, in a mouse model of intramuscular infection, NF2 was much more virulent than NF1. Isolates NF2, SSU, and Riv2 disseminated in high numbers from the muscular tissue to the visceral organs of mice, while NF1 reached the liver and spleen in relatively lower numbers on the basis of colony counting and tracking of bioluminescent strains in real time by in vivo imaging. Histopathologically, degeneration of myofibers with significant infiltration of polymorphonuclear cells due to the highly virulent strains was noted. Functional genomic analysis provided data that allowed us to correlate the highly infectious nature of Aeromonas pathotypes belonging to several different species with virulence signatures and their potential ability to cause NF., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide., Importance: Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens., (Copyright © 2014 Hossain et al.)

The utility of specific strains of natural algicidal bacteria isolated from shallow wetland sediments was evaluated against several strains of algae with potential immediate or future commercial value. Two strains of bacteria, Pseudomonas pseudoalcaligenes AD6 and Aeromonas hydrophila AD9, were identified and demonstrated to have algicidal activity against the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. These bacteria were further evaluated for the potential to improve lipid extraction using a mild solvent extraction approach. Aeromonas hydrophila AD9 showed a nearly 12-fold increase in lipid extraction with D. tertiolecta, while both bacteria showed a sixfold improvement in lipid extraction with N. oleoabundans., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Metagenomics is a magnificent tool to isolate genes from unknown/uncharacterized species and also from organisms that cannot be cultured. In this study, we constructed a metagenomic library from isolated DNA of soil samples collected from Palamuru University campus premises, in Mahabubnagar district of Andhra Pradesh, India. We isolated a novel lipase gene LipHim1, which has an open reading frame of 591 base pairs and encodes ∼23 kDa protein consisting of 196 amino acids. The Lipase LipHim1 showed maximum 32% homology at the protein level with the extracellular Aeromonas hydrophila lipase (Class II, GDSL family) and was significantly different from all other known lipases. The isolated lipase catalyzed the hydrolysis of fatty acid esters of polyoxyethylene sorbitan such as Tween 60. Our results indicate that the isolated lipase gene is novel.

Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen Vibrio cholerae. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by V. cholerae. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in V. cholerae membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the in vitro activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An Aeromonas hydrophila VolA homolog complements a V. cholerae VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of V. cholerae, improving the overall adaptation and survival of the organism in different environments.

Porins, the outer membrane proteins of gram negative bacteria, perform vital roles in bacterial survival and virulence, such as nutrient transportation across the membrane as well as adhesion to host cells during infection. The outer membrane proteins, OmpF and OmpC, are part of a two-component regulatory system, essential for the maintenance of solute concentrations in the cytoplasmic milieu of bacteria, and are thus considered vital for bacterial survival. Exposed on the surface of gram-negative bacteria, these channel proteins are highly immunogenic and can thus be exploited as vaccine candidates. In the present study, we have cloned, characterized, and expressed outer membrane protein OmpF of Aeromonas hydrophila, a major fish pathogen and also known to cause severe infections in humans. The cloned ompF gene of A. hydrophila consisting of an open reading frame corresponding to mature OmpF was expressed and purified from the heterologous host, E. coli. High level of expression resulted in recovery of ~120 mg/L of the purified rOmpF at shake flask level. Polyclonal antisera raised against the recombinant OmpF showed a very high endpoint titer (>1:80,000) and were able to specifically agglutinate live A. hydrophila. Further, anti-OmpF antisera cross-reacted with the cell lysates of various Aeromonas isolates, suggesting that anti-rOmpF antibodies can be used to identify different A. hydrophila isolates in infected conditions. Antibody isotyping, cytokine ELISA, and ELISPOT assay indicated predominantly Th1 type of immune response. The recombinant OmpF reported in the present study thus has the potential to be used as a vaccine candidate against A. hydrophila.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966(T) could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. β-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

The present study evaluated genetic variation in Aeromonas hydrophila strains using PCR-RAPD and their varied susceptibility to phyto-extract. Four strains of Aeromonas hydrophila isolated from skin infections of common freshwater fish, Cyprinus carpio were characterized by various biochemical methods, physiological tests and PCR- RAPD. Antimicrobial activity of the leaf extracts of three medicinal plants, Ocimum sanctum, Adathoda vasica and Calendula officinalis were tested against the four strains of A. hydrophila by disc diffusion (Kirby-Bauer) method. Antagonistic effects of leaf extracts against A. hydrophila strains were assessed by co-culture method. RAPD analysis showed that all the microbes isolated from skin infection belong to the same species but there was no 100% genetic similarity among them Dendrogram constructed by UPGMA clearly supported the PCR pattern of genetic variability among the strains. This study revealed that Aeromonas hydophila exhibits genetic variability and varied susceptibility towards phyto-extracts. Results indicated that phyto-extracts offers a promising alternative to the use of antibiotics in controlling Aeromonas hydrophila.

A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection., (Published by Elsevier B.V.)

The complete genome of Gram-negative Aeromonas hydrophila 4AK4 that has been used for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) was sequenced and annotated. Its chromosome is 4,527,993 bp in size encoding 4,272 genes, including 28 rRNA genes and 104 tRNA genes. Comparative analysis indicated that genome of A. hydrophila 4AK4 was similar to that of the A. hydrophila ATCC 7966(T), an intensively studied aeromonad for its pathogenicity related to its genomic information. Genes possibly coming from other species or even other genus were identified in A. hydrophila 4AK4. A large number of putative virulent genes were predicted. However, a cytotonic enterotoxin (Ast) is absent in A. hydrophila 4AK4, allowing the industrial strain to be different from other A. hydrophila strains, indicating possible reduced virulence of strain 4AK4, which is very important for industrial fermentation. Genes involved in polyhydroxyalkanoate (PHA) metabolism were predicted and analyzed. The resulting genomic information is useful for improved production of PHA via metabolic engineering of A. hydrophila 4AK4.

Aerolysin is one of the putative toxins in extracellular products (ECP) produced by Aeromonas hydrophila, an important pathogen of catfish. To better understand the molecular mechanism and mode of action of this toxin, proaerolysin-coding gene was cloned from the genomic DNA of an A. hydrophila strain, cultured from diseased channel catfish, and heterologously expressed in E. coli. Functional recombinant proaerolysin was obtained, revealing some unique properties. The purified recombinant proaerolysin was inactive but could be activated by treatment with furin, trypsin, and ECP although different treatments produced different cleavage profiles and resulted in differential hemolytic and cytotoxic activities. The highest activity was observed from aerolysin processed by furin while treatment of proaerolysin with trypsin and ECP resulted in reduced activities. The unprocessed proaerolysin, though not hemolytic in vitro, had the same cytopathic effect on cultured walking catfish gill cells as the furin-processed had. In in vivo assay, the recombinant proaerolysin was found to be lethal to catfish when injected via intraperitoneal (IP) route. The lethal toxicity was acute and dose-dependent, as observed in IP injection of live A. hydrophila. This is the first recombinant proaerolysin confirmed to be a virulence factor; the recombinant protein could be used to further evaluate virulence, pathogenicity and antigenicity associated with A. hydrophila infection., (Published by Elsevier B.V.)