Dis Aquat Organ, 2004 Nov 23, 62(1-2), 57 - 63 A deterministic model for the dynamics of furunculosis in chinook salmon Oncorhynchus tshawytscha; Ogut H et al.; Studies were undertaken to determine the parameters of transmission of Aeromonas salmonicida in chinook salmon Oncorhynchus tshawytscha, and to develop a deterministic model of the dynamics of experimental furunculosis . For determination of disease transmission coefficient (beta), disease-related mortality rate (alpha) and natural mortality rate (gamma), fish in 70 tanks (approximately 42 fish tank(-1)) were each exposed to a single infectious donor fish, 7 tanks were randomly selected daily and all individuals were examined for the presence of A . salmonicida in the kidney . The proportion of susceptible (S), infected (I) and removed (R, dead) individuals were determined daily . The parameters beta, alpha, gamma, reproductive ratio (R0) and threshold density were estimated to be 0.0214 infected ind . d(-1), 0.29 infected ind . d(-1), 0.00015 ind . d(-1), 3.23 and 13.56 ind., respectively . Using these parameters, a deterministic disease model of A . salmonicida infection as a cause of furunculosis was constructed . The net rate at which new individuals became infected (the incidence rate) per unit time was proportional to S x I x beta . The model-produced data for S were significantly associated with experimental data (r2 = 0.92) . In brief, a simple SIR (susceptible-infected-removed) model was successfully utilized to simulate observed data FEMS Microbiol Lett, 2005 Jan 15, 242(2), 195 - 201 A pore-forming toxin produced by Aeromonas sobria activates cAMP-dependent Cl(-) secretory pathways to cause diarrhea; Tanoue N et al.; Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A . sobria, a human pathogen that causes diarrhea . We investigated the effects of ASH on Cl(-) transport in human colonic epithelial cells . ASH increased short-circuit currents (Isc) and (125)I efflux from Caco-2 cells, indicating ASH activate Cl(-) secretion . Additions of inhibitors of cyclic AMP dependent Cl(-) channels, glybenclamide and NPPB suppressed the Isc and (125)I efflux increases induced by ASH . And ASH increased the intracellular cyclic AMP concentration . Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation . Thus, cAMP-dependent Cl(-) secretory pathway could be related with diarrhea induced by A . sobria. FEMS Microbiol Lett, 2005 Jan 1, 242(1), 59 - 63 Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida; Casas C et al.; Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America . These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb . The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried {tet(A) or tet(B)}, incompatibility of the plasmid {IncU or other}, whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C . Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E . coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient . The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate . The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E . coli recipient . Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria. Dis Aquat Organ, 2004 Nov 4, 61(3), 257 - 62 Genetic diversity among A-proteins of atypical strains of Aeromonas salmonicida; Lund V et al.; The virulence array protein gene A (vapA) encoding the A-protein subunit of the surface layer of 23 typical and atypical strains of Aeromonas salmonicida from salmonids and marine fish species were sequenced, and the deduced A-protein sequences compared . The A-proteins of the typical A . salmonicida ssp . salmonicida strains were shown to be identical, while amino acid variability was revealed among A-proteins of atypical strains . The highest amino acid variability appears to be in a predicted surface exposed region and is believed to result in antigenic differences among the atypical strains of A . salmonicida. J Mol Biol, 2005 Jan 28, 345(4), 785 - 95 A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem; Garau G et al.; One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases . CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available . The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis . The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site . These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam . This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. Aquat Toxicol, 2004 Dec 20, 70(4), 345 - 55 Effects of treated sewage effluent on immune function in rainbow trout (Oncorhynchus mykiss); Hoeger B et al.; In this study, the immune reactions of rainbow trout (Oncorhynchus mykiss) were examined, after exposure to 10, 30 and 70% of tertiary-treated municipal sewage effluent for 27 days . Exposures were conducted concurrently with and without an immune challenge using intraperitoneal injections of inactivated Aeromonas salmonicida salmonicida . Due to the time required to prepare and analyse samples, fish sampling was conducted over two consecutive days . There was no trout mortality for any of the experimental treatments . The exposure to effluent increased in vitro lymphocyte proliferation, decreased circulating lymphocytes and increased degrading erythrocytes in peripheral blood samples . Circulating lymphocytes were only decreased in the sham-injected, but not in the A . salmonicida-injected group . In addition to effluent effects, circulating lymphocytes and lymphocyte proliferation were decreased on day 2 of sampling as compared to day 1 . Concentration-dependent degradation of erythrocytes was only observed on day 2 of sampling . Capture and removal of trout on day 1 of sampling presumably caused low-level stress that affected some results on day 2 . Oxidative burst, phagocytosis, lysozyme, leucocyte populations other than lymphocytes and A . salmonicida-specific IgM production were not affected by exposure to effluent, and of these parameters, only oxidative burst and total leucocytes showed sampling day effects . From these results it can be observed, that with the exception of oxidative burst, those variables affected by effluent exposure were also significantly changed by the low-level sampling stress imposed by staggered sampling . Elevated liver mixed-function oxygenase activity as measured by 7-ethoxyresorufin-O-deethylase activity, and increased bile polycyclic aromatic hydrocarbon (PAH) metabolites were observed in response to sewage effluent exposure . As both PAHs and stress are known immune suppressors, it is difficult to conclude whether or not changes in immune parameters due to effluent exposure were caused by the direct action of chemicals, or were due to a general stress response. Res Microbiol, 2004 Dec, 155(10), 827 - 9 An unusually high level of quinolone resistance associated with type II topoisomerase mutations in quinolone resistance-determining regions of Aeromonas caviae isolated from diarrhoeal patients; Sinha S et al.; We examined 158 strains belonging to different Aeromonas species isolated from hospitalized acute diarrhoea cases for susceptibility to quinolones . Compared to other species, a high percentage of the A . caviae strains were resistant to ciprofloxacin and norfloxacin . Based on MIC values, 6 A . caviae strains were selected and the nucleotide sequences for the quinolone-resistant-determining regions (QRDRs) of gyrA, gyrB and parC genes were analysed . In resistant strains, double mutations (Ser(83)-->Ile and Asp(87)-->Asn) and a single mutation (Ser(80)-->Ile) were detected in the QRDR of gyrA and parC, respectively. Eur J Biochem, 2004 Nov, 271(22), 4507 - 16 Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions; Wang Z et al.; Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon . In the course of this study, it was found that when grown in vitro on tryptic soy agar, A . salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide . A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides . Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-{(N-acetyl-L-alanyl)amido}-3,6-dideoxy-D-glucose{3-{(N-acetyl-L-alanyl)amido}-3-deoxy-D-quinovose, Qui3NAlaNAc} and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: {-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-}n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group . CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form . Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2073 - 8 Aeromonas molluscorum sp . nov., isolated from bivalve molluscs; Minana-Galbis D et al.; Five Aeromonas strains (848T(T), 93M, 431E, 849T and 869N), which were isolated from bivalve molluscs and were recognized previously by numerical taxonomy as members of an unknown Aeromonas taxon, were subjected to a polyphasic taxonomic study . DNA-DNA hybridization experiments showed that DNA of strain 848T(T) was <70 % similar (27-45 %) to that of the type/reference strains of the current Aeromonas hybridization groups (HGs), but 93 % similar to that of strain 93M . The DNA G+C content of the five strains ranged from 59.0 to 59.4 mol% . 16S rRNA gene sequence analysis confirmed that the strains belonged to the genus Aeromonas and showed high similarity to Aeromonas encheleia . Amplified fragment length polymorphism fingerprinting clustered the novel strains in a homogeneous group with low genotypic relatedness to other Aeromonas species . Useful phenotypic features for differentiating the five isolates from other Aeromonas species include their negative reactions in tests for indole production, lysine decarboxylase, gas from glucose and starch hydrolysis . From the results of this study, the name Aeromonas molluscorum sp . nov . is proposed for these strains, with the type strain 848T(T) (=CECT 5864(T)=LMG 22214(T)). Eur J Mass Spectrom (Chichester, Eng), 2004, 10(5), 715 - 30 Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry; Banoub J et al.; The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry . The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: {structure: see text} After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility . The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis . CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined . Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids. Vaccine, 2004 Dec 9, 23(4), 450 - 9 Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads; Maurice S et al.; This study was focused on the utilization of a recombinant expression system to produce a unique modified subunit vaccine possessing a self-contained delivery system which could potentially improve the uptake and delivery of vaccine products as well their immunogenic potential . For this purpose the A-layer protein (At-R) associated with the fish pathogen atypical Aeromonas salmonicida was cloned and modified by the genetic fusion of the protein transduction domain (MTS) derived from Kaposi fibroblast growth factor (At-MTS) . The potential for these proteins to be employed as antigens for oral immunization of goldfish was examined by encapsulation of At-R, At-MTS and the control, BSA, into biodegradable alginate gel macrospheres which were fed to goldfish in place of standard pellet fish feed . The bead physical properties were modified only in the presence of At-R and the temporal release of proteins was significantly less when At-MTS was employed . Western blot analysis of serum samples collected from fish following intubation with the recombinant proteins determined that the rate of protein uptake from the digestive tract into the blood system improved considerably when MTS was fused to At-R . Experimental fish were fed one of three protein-alginate formulae on a schedule of 3 days/week or 5 days/month for a period of 2 months . After 1 month, animals fed on the 5-day protocol demonstrated increased serum antibody titers while following an additional month of feeding this level decreased and titers were found to be higher in fish maintained on the 3-day regime . Fish fed At-MTS maintained the highest titer at the end of 2-month period . To determine whether the diminished antibody titers were a result of oral tolerance fish were injected intraperitoneally with the At-R antigen . Only experimental groups which had been fed At-R or At-MTS demonstrated increased antibody titers which paralleled a typical secondary humoral response . In spite of the presence of an increased titer to A-protein, vaccinated fish did not demonstrate resistance to infection with atypical A . salmonicida. Dis Aquat Organ, 2004 Sep 8, 60(3), 247 - 52 Important disease conditions of newly cultured species in intensive freshwater farms in Greece: first incidence of nodavirus infection in Acipenser sp; Athanassopoulou F et al.; We describe here the main pathological conditions of freshwater fish recently introduced for intensive rearing (open ponds and recirculating freshwater systems) in Greece . Sturgeon were susceptible to skeletal abnormalities of the spine (scoliosis and lordosis) of unknown aetiology . Horizontal transmission of nodavirus from infected sea bass to sturgeon was detected for the first time . This caused serious pathology and clinical signs, such as lethargy and imbalance, leading to secondary infections with Aeromonas hydrophila and Trichodina sp . and chronic, but steady, mortality . Sea bass were very susceptible to nodavirus infection, monogenean infections and gas bubble disease . Mullet reared under recirculated and open-flow conditions were very sensitive to Chilodonella sp . infection, whereas catfish were susceptible to infection with Ichthyophthirius sp . leading to secondary infections with A . hydrophila, Saprolegnia sp . and Myxobacteria spp . Tilapia were very susceptible to gas bubble disease and A . hydrophila . This bacterium was associated with management manipulations for all species and fully responsive to corrective hygiene methods. Fish Shellfish Immunol, 2005 Mar, 18(3), 235 - 42 Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes; Irie T et al.; Liposome-entrapped atypical Aeromonas salmonicida antigen was prepared to investigate the potential protective efficacy for A . salmonicida infection . Carp (Cyprinus carpio) were immunised orally with liposome-entrapped A . salmonicida antigen . After immunisation, significantly higher antigen-specific antibodies were detected in serum, intestinal mucus and bile than non-immunised control group . Furthermore, immunised carp were challenged by immersion with 1 x 10(6) cfu ml(-1) of A . salmonicida for 60 min . Of the eight non-immunised carp, three carp died (62.5% survival), whereas five out of six (83.5%) immunised survived . Furthermore, the development of skin ulcers was significantly inhibited in carp immunised with liposomes containing A . salmonicida antigen . These results suggest that liposomes containing A . salmonicida antigen have the potential for the induction of a protective immune response against atypical A . salmonicida infection and also suggest the possibility of developing a vaccine that may ultimately be used for the prevention of fish diseases. Fish Shellfish Immunol, 2005 Mar, 18(3), 223 - 33 Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption; Vivas J et al.; The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss . In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared . Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB) . Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility . O . mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria . Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A . hydrophila aroA vaccine. J Fish Dis, 2004 Nov, 27(11), 645 - 55 Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects; Bjornsdottir B et al.; Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic . The bacterium has also been isolated from various marine fish species . Bacterial diseases have been a limiting factor in farming of turbot, but M . viscosa has not so far been isolated . In this study, turbot was shown to be sensitive to M . viscosa infection in experimental challenges . Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon . A multivalent commercial salmon vaccine, containing M . viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination . Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination . Vaccination did not induce a specific anti-M . viscosa response, while elevated anti-M . viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge . The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component . Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant . The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups. Cell Tissue Res, 2004 Nov, 318(2), 305 - 11 Epub 2004 Aug 03. Damaging effect of the fish pathogen Aeromonas salmonicida ssp . salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.); Ringo E et al.; In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract . Bacteria can cross the gastrointestinal lining in three different ways . In undamaged tissue, bacteria can translocate by transcellular or paracellular routes . Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering . Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp . salmonicida, the causative agent of furunculosis . The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen . In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions . Based on these findings, we suggest that A . salmonicida may obtain entry to the fish by seriously damaging the intestinal lining . Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A . salmonicida infections in Atlantic salmon. Int Microbiol, 2004 Sep, 7(3), 207 - 11 Resistance to beta-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss); Saavedra MJ et al.; Bacterial infections caused by members of the genus Aeromonas, with a relatively high antibiotic resistance, are among the most common and troublesome diseases of fish raised in ponds with recirculation systems . In this study, carried out at an experimental aquaculture station in northern Portugal, 51 strains identified as belonging to the genus Aeromonas were isolated from 20 rainbow trout (Oncorhynchus mykiss) skin and kidney samples, as well as from raceway water samples . Macro- and microscopic examination of the fish tissues revealed lesions or cellular alterations in skin and kidney that seemed to correlate with the presence of those isolates . The sensitivity of all isolated strains to different groups of beta-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) was evaluated using the disc diffusion method . The highest rates of resistance were to amoxicillin, carbenicillin and ticarcillin . Unexpected resistance to imipenem, an antibiotic of clinical usage, was also detected, which suggests that resistance may have been transferred to the Aeromonas population from the environment. Syst Appl Microbiol, 2004 Sep, 27(5), 549 - 57 Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp; laganowska M et al.; We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species . A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study . ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI . The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species . The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA) . The isolates were allocated into 15 groups, three strains were unclustered . The strains belonging to the following genomic species: A . hydrophila, A . bestiarum, A . salmonicida, A . caviae, A . media, A . schubertii, A . allosaccharophila, A . popoffii, and A . culicicola formed distinct clusters . Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group . Similarly, the strains of A . jandaei (HG 9) and the type strain of A . trota were allocated into one cluster . Two isolates of HG 14 formed distinct cluster . We noticed a genetic diversity among A . veronii isolates, the strains were clustered in two groups . Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas. Immunogenetics, 2004 Nov, 56(8), 572 - 84 Epub 2004 Nov. Sequence and expression of C-type lectin receptors in Atlantic salmon (Salmo salar); Soanes KH et al.; The diverse receptors of the C-type lectin superfamily play key roles in innate immunity . In mammals, cell surface receptors with C-type lectin domains are involved in pathogen recognition and in immune response, and in some cases are exploited by pathogens to gain entry into cells . This study reports on sequence and expression analysis of three paralogous group II C-type lectins from the teleost fish Atlantic salmon (Salmo salar) . Each of the receptors showed similarity to immune-relevant mammalian receptors in terms of amino acid sequence and overall organization within the C-type lectin-like domain (CTLD) . Two of the three have cytoplasmic motifs consistent with the immunoreceptor tyrosine-based activation motifs (ITAM), which are known to modulate downstream functions in leukocytes . All three C-type lectin receptors were expressed in multiple tissues of healthy fish, including peripheral blood leukocytes and salmon head kidney cells (SHK-1) . Each receptor was up-regulated in salmon liver in response to infection by Aeromonas salmonicida and one receptor was substantially up-regulated in cultured SHK-1 cells in response to lipopolysaccharide (LPS) . Putative binding sites for the CAAT-enhancer-binding protein (C/EBP) family of transcription factors in the regulatory regions of these C-type lectin genes may mediate their response to bacteria and LPS in salmon leukocytes . The identification of these types of receptors in distinct populations of cells within the immune system will provide important markers for identifying and categorizing the state of differentiation or activation of these cells and lead to further understanding of the interaction between the salmon host and multiple pathogens. J Appl Microbiol, 2004, 97(5), 1077 - 86 Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification; Sen K et al.; AIMS: To examine whether Aeromonas bacteria isolated from municipally treated water had virulence factor genes . METHODS AND RESULTS: A polymerase chain reaction-based genetic characterization determined the presence of six virulence factors genes, elastase (ahyB), lipase (pla/lip/lipH3/alp-1) flagella A and B (flaA and flaB), the enterotoxins, act, alt and ast, in these isolates . New primer sets were designed for all the target genes, except for act . The genes were present in 88% (ahyB), 88% (lip), 59% (fla), 43% (alt), 70% (act) and 30% (ast) of the strains, respectively . Of the 205 isolates tested only one isolate had all the virulence genes . There was a variety of combinations of virulence factors within different strains of the same species . However, a dominant strain having the same set of virulence factors, was usually isolated from any given tap in different rounds of sampling from a single tap . CONCLUSIONS: These results show that Aeromonas bacteria found in drinking water possess a wide variety of virulence-related genes and suggest the importance of examining as many isolates as possible in order to better understand the health risk these bacteria may present . SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a rapid method for characterizing the virulence factors of Aeromonas bacteria and suggests that municipally treated drinking water is a source of potentially pathogenic Aeromonas bacteria. Macromol Biosci, 2004 Mar 15, 4(3), 255 - 61 Metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydrophila; Qiu YZ et al.; Aeromonas hydrophila 4AK4 was able to synthesize copolyesters consisting of 3-hydroxybutyrate (3HB) and about 15 mol-% 3-hydroxyhexanoate (3HHx) (PHBHHx) when grown in long chain fatty acids such as dodecanoate regardless of growth conditions . To regulate the unit fraction in PHBHHx, phbA and phbB genes encoding beta-ketothiolase and acetoacetyl-CoA reductase in Ralstonia eutropha, were introduced into A . hydrophila 4AK4 . When gluconate was used as cosubstrate of dodecanoate, the recombinant produced PHBHHx containing 3-12 mol-% 3HHx, depending on the gluconate concentration in media . Vitreoscilla hemoglobin gene, vgb, was also introduced into the above recombinant, resulting in improved PHBHHx content from 38 to 48 wt.-% in shake flask study . Fermentor studies also showed that increased gluconate concentration in medium containing dodecanoate promoted the recombinant strain harboring phbA and phbB genes to incorporate more 3HB unit into PHBHHx, resulting in reduced 3HHx fraction . Recombinant A . hydrophila harboring phbA, phbB and vgb genes demonstrated better PHBHHx productivity and higher conversion efficiency from dodecanoate to PHBHHx than those of the recombinant without vgb in fermentation study . Combined with the robust growth property and simple growth requirement, A . hydrophila 4AK4 appeared to be a useful organism for metabolic engineering. Macromol Biosci, 2004 Mar 15, 4(3), 238 - 42 Biosynthesis and compositional regulation of poly{(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)} in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes; Tsuge T et al.; A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB(-)4 harboring mutated PHA synthase genes (phaC(Ac)) from Aeromona caviae was investigated . The strain harboring wild-type phaC(Ac) gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate {P(3HB-co-3HHx)} with 3.5 mol-% of 3HHx fraction from soybean oil . When the mutants of phaC(Ac) gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0-5.1 mol-% . Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes. Appl Environ Microbiol, 2004 Oct, 70(10), 5898 - 904 Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer; Esteve C et al.; Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme . They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10(4.61) to 10(7.11)), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy . A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A . hydrophila TF7 (O:11 reference strain) . Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein . The S-layer proteins of the O:14 and O:81 A . hydrophila strains seemed to be primarily different from those previously purified from strains A . hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA . This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1332 - 6 Molecular cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli; Lu XY et al.; The polyhydroxyalkanoate synthesis operon was cloned from Aeromonas hydrophila CGMCC 0911 . Heterogeneous expression of the cloned polyhydroxyalkanoate synthesis operon in Escherichia coli resulted in accumulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) consisting of 13.9 mol % 3-hydroxyhexanoate up to 29.2 wt % of cell dry weight when grown in lauric acid . The cell dry weight of recombinant E . coli harboring the polyhydroxyalkanoate synthesis operon was improved to 1.7 g L (-1), which was much higher than that of 0.3 g L (-1) of the wild type E . coli . Coexpression of acyl-CoA dehydrogenase gene (yafH) from E . coli and Vitreoscilla hemoglobin gene (vgb) from Vitreoscilla together with the whole A . hydrophila CGMCC 0911 polyhydroxyalkanoate synthesis operon facilitated cell growth and polyhydroxyalkanoate accumulation in E . coli . When yafH was coexpressed together with the polyhydroxyalkanoate synthesis operon, the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) content was increased from 29.2 to 52.1 wt %, and the cell dry weight was also increased slightly from 1.70 to 1.86 g L (-1) . Coexpression of vgb gene could further enhance the cell dry weight to 2.0 g L(-1) and the polyhydroxyalkanoate content to 60.7 wt %. FEMS Microbiol Lett, 2004 Oct 1, 239(1), 195 - 201 Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition; Han J et al.; Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate . To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A . hydrophila 4AK4 . The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect . Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol% . When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol% . Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture . The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content . Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much . In addition, inhibition of beta-oxidation by acrylate in A . hydrophila 4AK4 enhanced PHBHHx content . However, no monomers longer than 3HHx were detected . The results show that genetic modification of A . hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1511 - 9 Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes; Soler L et al.; The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the sigma70 factor . This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus . Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (< 2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB) . Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date . However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa . At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp . HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A . salmonicida from A . bestiarum . The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species. J Water Health, 2004 Jun, 2(2), 115 - 22 Phenotypic and genotypic identification of Aeromonas spp . isolated from a chlorinated intermittent water distribution system in Lebanon; Tokajian S et al.; Aeromonas spp . were detected in samples collected from both untreated groundwater and treated drinking water in Lebanon . Aeromonas spp . levels ranged between 2 and 1,100 colonies per 100 ml in the intake underground well and between 3 and 43 colonies per 100 ml in samples from the distribution system . Samples positive for Aeromonas spp . from the network had a free chlorine level ranging between 0 and 0.4 mg l(-1) . Multiple antibiotic-resistance was common among the isolated aeromonads; all were resistant to amoxycillin while 92% showed resistance to cephalexin . Haemolysis on blood agar was detected in 52% of the isolates recovered from the distribution network and 81% of isolates from the untreated underground source . The Biolog microbial identification system assigned identities to all of the isolated presumptive aeromonads (at least at the genus level), which was not the case with the API 20NE strips . Differences at the species level were observed when results from the Biolog system were compared with identification based on the MicroSeq 500 16S rDNA sequence analysis . The presence of Aeromonas spp . in drinking water can be an important threat to public health, thus greater awareness of Aeromonas strains as potential enteropathogens is warranted. Proteomics, 2004 Oct, 4(10), 3203 - 13 Rapid screening of highly efficient vaccine candidates by immunoproteomics; Chen Z et al.; Diseases caused by microorganisms can be controlled by vaccines, which require neutralizing antigens . Therefore, it is very important to identify highly efficient immunogens for immune prevention . By combining immunoproteomics and bacterial challenge after immunization, we developed a rapid method for screening protected antigens of pathogenic bacteria in aquaculture . Our approach may be divided into three consecutive steps . First, dominant immunogens of outer membrane proteins are screened by immunoproteomics . Second, proteins with the ability to induce production of neutralizing antibodies are identified from the immunogens by virulent bacterium challenge following vaccination . Third, vaccine candidates are determined by evaluation of neutralizing abilities . Information on the candidates has been obtained for further gene cloning by mass spectrometry . Our results indicate that highly efficient protected antigens were identified from the outer membrane proteome of Aeromonas hydrophila, in which an immunogen showed 71.4% protective ability with multivalent functions to A . hydrophila and Aeromonas sobria . In summary, we have developed a high-throughout, accurate, rapid and highly efficient method which will play an active role in immune prevention for microbiological diseases. J Biol Chem, 2004 Dec 3, 279(49), 51275 - 81 Epub 2004 Dec 3. Crystal structure of a dodecameric tetrahedral-shaped aminopeptidase; Russo S et al.; Protein turnover is an essential process in living cells . The degradation of cytosolic polypeptides is mainly carried out by the proteasome, resulting in 7-9-amino acid long peptides . Further degradation is usually carried out by energy-independent proteases like the tricorn protease from Thermoplasma acidophilum . Recently, a novel tetrahedral-shaped dodecameric 480-kDa aminopeptidase complex (TET) has been described in Haloarcula marismortui that differs from the known ring- or barrel-shaped self-compartmentalizing proteases . This complex is capable of degrading most peptides down to amino acids . We present here the crystal structure of the tetrahedral aminopeptidase homolog FrvX from Pyrococcus horikoshii . The monomer has a typical clan MH fold, as found for example in Aeromonas proteolytica aminopeptidase, containing a dinuclear zinc active center . The quaternary structure is built by dimers with a length of 100 A that form the edges of the tetrahedron . All 12 active sites are located on the inside of the tetrahedron . Substrate access is granted by pores with a maximal diameter of 10 A, allowing only small peptides and unfolded proteins access to the active site. Ann Ig, 2004 Jul-Aug, 16(4), 523 - 30 Effect of growth conditions on extracellular products (ECPs) of Aeromonas hydrophila; Di Pietro A et al.; Owing to the significant role in gastrointestinal illness of A . hydrophila, frequently detected in various raw and ready-to eat foods, its pathogenetic mechanisms are particularly studied . In this paper we report the results obtained studying in vitro the effect of O2 tension and inoculum age on the extra cellular products (ECPs) of seven strains food-borne isolated and cultured at 37 degrees . The considered factors influenced markedly bacterial growth as well as ECPs production and the more notable differences were detected among 15 hours old strains let grown slowly shaking (15SH), that showed the highest bacterial yield, and 24 h old strains cultured statically (24ST), whose haemolysin and cytotoxin production was favoured . Wilcoxon test shows as, in these latter conditions, the strains needed short time to adapt the haemolysin and cytotoxin production . The oxygen tension reduction, extending the replication time, induces the bacterial metabolism toward secondary products, as verified by Spearman test applied to ECPs indexes . The increased production per cell of virulence-associated factors could be responsible of gastrointestinal disorders caused by food-borne A . hydrophila strains, even without a massive gut colonization, especially when immunocompromised individuals ingest contaminated foods. Appl Environ Microbiol, 2004 Sep, 70(9), 5685 - 7 Increasing the carbon flux toward synthesis of short-chain-length--medium-chain-length polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae through modification of the beta-oxidation cycle; De Oliveira VC et al.; Short-chain-length-medium-chain-length polyhydroxyalkanoates were synthesized in Saccharomyces cerevisiae from intermediates of the beta-oxidation cycle by expressing the polyhydroxyalkanoate synthases from Aeromonas caviae and Ralstonia eutropha in the peroxisomes . The quantity of polymer produced was increased by using a mutant of the beta-oxidation-associated multifunctional enzyme with low dehydrogenase activity toward R-3-hydroxybutyryl coenzyme A. J Vet Med B Infect Dis Vet Public Health, 2004 Jun, 51(5), 222 - 4 Role of Ichthyophthirius multifiliis in the Infection of Aeromonas hydrophila; Liu YJ et al.; Aeromonas hydrophila and Ichthyophthirius multifiliis are important fish pathogens . To clarify their relationship, two host models, goldfish with trauma and those infected with I . multifiliis, were used to study infection kinetics of A . hydrophila 4332 tagged with plasmid pGFPuv (Ah4332(GFP)) . The results showed that in fish suffering from ichthyophthiriosis, the bacterial infection process was accelerated and more persistent than in the other groups . It is suggested that I . multifiliis plays an important role in the infection of A . hydrophila . Mechanical trauma caused by I . multifiliis might act as a portal of entry for A . hydrophila present in the water, thereby facilitating the invasion of bacteria . The ectoparasite might also serve as a vector and reservoir of pathogenic bacteria. BMC Infect Dis . 2004 Aug 25;4(1):28. External decontamination of wild leeches with hypochloric acid; Aydin A et al.; BACKGROUND: Medicinal leech, Hirudo medicinalis, has been used in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps . In many countries, wild leeches are still provided from local markets and utilised with antibiotic prophylaxies . In this research, results of identification of bacteria in the transport fluid is reported, oral and intestinal floras and the antibiograms of the identified microorganisms are investigated . Also, to avoid possible infections, the ability of hypochloric acid, a disinfectant, to suppress the relevant microorganisms without changing the life style and behavior of leeches in terms of sucking function, is investigated . METHODS: Bacterial identifications and antibiograms of oral and intestinal flora and transport medium were performed for 10 leeches . The optimum concentration of hypochloric acid which eliminated microorganisms without affecting the viability and sucking function of the leeches were determined by dilution of hypochloric acid to 100, 50, 25, 12.5, 6.25 ppm concentrations in different groups of 25 leeches . Finally, 20 leeches were applied atraumatically to the bleeding areas of rats, the duration of suction was determined and compared statistically between the leeches treated and not treated with hypochloric acid solution . RESULTS: Aeromonas hydrophilia was the most commonly identified microorganism and found to be resistant to first generation cephalosporins, frequently used in prophylaxis at surgical wards . In the next stages of the study, the leeches were subjected to a series of diluted hypochloric acid solutions . Although disinfection of the transport material and suppression of the oral flora of hirudo medicinalis were successful in 100, 50, 25, 12.5, 6.25 ppm concentrations; 12.5 ppm solution was the greatest concentration in which hirudo medicinalis could survive and sucking function was not affected significantly . CONCLUSIONS: External decontamination of wild leeches with 12.5 ppm hypochloric acid enables bacterial suppression without causing negative effects on leech sucking function and life. Infect Immun, 2004 Sep, 72(9), 5439 - 45 Microarray analysis of Aeromonas hydrophila cytotoxic enterotoxin-treated murine primary macrophages; Galindo CL et al.; We performed microarray analyses of murine peritoneal macrophages to examine cellular transcriptional responses to a cytotoxic enterotoxin of Aeromonas hydrophila . While 66% of altered genes were common to both primary macrophages and the murine macrophage cell line RAW 264.7, Act caused expression changes of 28 genes specifically in murine peritoneal macrophages. J Med Microbiol, 2004 Sep, 53(Pt 9), 895 - 901 Mechanisms of quinolone resistance and clonal relationship among Aeromonas salmonicida strains isolated from reared fish with furunculosis; Giraud E et al.; The mechanisms of resistance to quinolone and epidemiological relationships among A . salmonicida strains isolated from diseased fish in French marine farms from 1998 to 2000 were investigated . The quinolone resistance-determining regions of the gyrA and parC genes of 12 clinical A . salmonicida isolates with different levels of quinolone susceptibility were sequenced . MICs were determined in the presence of the efflux pump inhibitor (EPI) Phe-Arg beta-naphthylamide and E(max) values (MIC without EPI/MIC in the presence of EPI) were calculated . Isolates fell into two classes: (i) those that had a wild-type gyrA gene with oxolinic acid MIC </= 0.5, flumequine MIC </= 1 and ciprofloxacin MIC </= 0.25 micro g ml(-1); and (ii) those that had a single mutation in gyrA encoding Asp-87 --> Asn with oxolinic acid MIC >/= 2, flumequine MIC >/= 4 and ciprofloxacin MIC >/= 0.125 micro g ml(-1) . No mutations were found in parC . High E(max) values obtained for flumequine and oxolinic acid (up to 16 and 8, respectively, for the most resistant isolates of the two classes) indicated an important contribution of efflux to the resistance phenotype . Flumequine accumulation experiments confirmed that high E(max) values were associated with a much lower level of accumulation . PCR/RFLP assays conducted on 34 additional isolates showed the presence of a mutation at codon 87 of gyrA in nearly all the quinolone-resistant isolates . This finding, together with PFGE typing results, strongly suggests a common clonal origin of these quinolone-resistant isolates. Epidemiol Infect, 2004 Aug, 132(4), 627 - 36 Biological characterization of Aeromonas spp . isolated from the environment; Rahim Z et al.; Cytotoxic enterotoxin (Act) is a key virulence factor in the pathogenesis of infections caused by Aeromonas spp . The cytotoxic enterotoxin gene (act) was detected in 32 out of 69 environmental isolates of Aeromonas spp . by hybridization with the act gene probe . To evaluate the pathogenic potential of the act gene probe-positive isolates, 32 act gene probe-positive and 31 randomly selected act gene probe-negative isolates were tested for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a Vero cell line . The act gene probe-positive isolates significantly differed from the toxin gene probe-negative ones with respect to enterotoxicity in the SMA (P=0.009) and haemolytic activity (P=0.005) . The CAMP-haemolysin phenotype was significantly associated with the rabbit ileal loop assay (P= 0.08), Vero cell assay (P= 0.064), and haemolysin production under the microaerophilic conditions (P= 0.056) of the act gene probe-positive isolates of Aeromonas spp . These data indicated the role of Act in the pathogenesis of Aeromonas infections and that the enterotoxic potential of Aeromonas spp . could be assessed by simply performing a CAMP-haemolysin assay. Eur J Mass Spectrom (Chichester, Eng), 2004, 10(4), 541 - 54 Structural reinvestigation of the core oligosaccharide of a mutant form of Aeromonas salmonicida lipopolysaccharide containing an O-4 phosphorylated and O-5 substituted Kdo reducing end group using electrospray QqTOF-MS/MS; Banoub J et al.; The molecular structure of the mutant form of the lipopolysaccharide of Aeromonas salmonicida was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and is proposed as the following: {molecular structure: see text} It was established that during the cleavage of this LPS with 1% acetic acid, to release the core oligosaccharide from the Lipid A portion, we obtained a degraded core oligosaccharide which eliminated its phosphate group with extreme facility . The precise molecular structure of this dephosphorylated core was deduced by electrospray mass spectrometry and is proposed as the following:{molecular structure: see text} Low energy collision ESI-QqTOF-MS/MS analysis of the dephosphorylated core oligosaccharide confirmed the presence of the O-5 glycosylated 4,8- and 4,7-anhydro derivatives of the enolizable alpha-keto-acids . The CID tandem mass spectrometric analysis of the heterogeneous mixture of the permethylated core oligosaccharide established the unreported methylation reaction on the diastereomeric 4,8- and 4,7-anhydro alpha-keto-acids and the complete permethylation and addition reaction of the O-5 glycosylated open chain reducing end terminal D-arabino-3-en-2-ulonic acid . The stereo-specific fragmentation routes obtained during the tandem mass spectrometric analysis permitted the precise sequencing of this dephosphorylated rough core oligosaccharide of the mutant LPS of A . salmonicida. Curr Microbiol, 2004 Aug, 49(2), 79 - 83 Effect of different temperature downshifts on protein synthesis by Aeromonas hydrophila; Imbert M et al.; The psychrotrophic bacterium Aeromonas hydrophila 7966 was subjected to cold shocks from 30 degrees C to 20 degrees C, 15 degrees C, 10 degrees C, or 5 degrees C, or were incubated at low temperature to determine its adaptative response . The cell protein patterns analyzed by two-dimensional electrophoresis revealed that only a few proteins were underexpressed, whereas numerous new proteins appeared with the decrease of temperature, and some others were overexpressed . Among them, a few constituted cold shock proteins because they were transiently induced, whereas others belong to the acclimatation family proteins . Two cold shock proteins of 11 kDa were synthesized at low level because they were visualized only after radiolabeling or silver staining . Moreover, under our experimental conditions, no major cold shock protein of a molecular mass similar to that of E . coli (7.4 kDa) could be identified. Cell Tissue Res . 2004 Aug 3; {Epub ahead of print} Damaging effect of the fish pathogen Aeromonas salmonicida ssp . salmonicida on intestinal enterocytes of Atlantic salmon ( Salmo salar L.); Ringo E et al.; In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract . Bacteria can cross the gastrointestinal lining in three different ways . In undamaged tissue, bacteria can translocate by transcellular or paracellular routes . Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering . Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon ( Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp . salmonicida, the causative agent of furunculosis . The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen . In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions . Based on these findings, we suggest that A . salmonicida may obtain entry to the fish by seriously damaging the intestinal lining . Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A . salmonicida infections in Atlantic salmon. Can J Microbiol, 2004 Jun, 50(6), 397 - 404 Population patterns and antimicrobial resistance of Aeromonas in urban playa lakes; Warren WJ et al.; Bacteria belonging to the genus Aeromonas are indigenous to aquatic environments . Once regarded as unimportant human pathogens, reports of opportunistic infections caused by these organisms have appeared increasingly in the medical literature . To estimate the potential for human infection by Aeromonas where limited water resources are being used intensively, we studied the spatial and temporal variation and incidence of antimicrobial resistance among environmental isolates of Aeromonas from two urban playa lakes in Lubbock, Texas . Aeromonas population densities varied seasonally, with the highest densities occurring from mid-April to late October . The greatest range of densities was 100-fold, from 2.50 to 255.17 colony-forming units per 0.1 mL of water sample . Densities also varied with water depth, although the variation did not display a consistent pattern . One hundred fifty-one Aeromonas isolates were divided into 10 species or subspecies groups by using the BIOLOG identification system . Nine isolates displayed resistance to co-trimoxazole, tetracycline, and cefuroxime, and none was resistant to more than one of these antimicrobial agents . In summary, the results of this study showed that the densities of Aeromonas peak in the late spring and again in late summer, times when human activity around the playa lakes is also high . Thus, we infer that human exposure to these potential pathogens varies seasonally . Compared to other published studies, the incidence of antimicrobial-resistant Aeromonas is relatively low in urban playa lakes in Lubbock, Texas . Nevertheless, resistant organisms were detected. J Appl Microbiol, 2004, 97(3), 557 - 65 Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature; Maalej S et al.; AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state . METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells . Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test . However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer . hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained . During the temperature-induced resuscitation, constant total cell counts were observed . Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase . We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells . CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer . hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated . SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer . hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn . Infect Immun, 2004 Aug, 72(8), 4848 - 58 Aeromonas hydrophila beta-hemolysin induces active chloride secretion in colon epithelial cells (HT-29/B6); Epple HJ et al.; The diarrheal mechanisms in Aeromonas enteritis are not completely understood . In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6) . Ion secretion was determined as a short-circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers . Transepithelial resistance (R(t)) served as a measure of permeability . A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I(SC) (39 +/- 3 microA/cm(2)) and decreased the R(t) to approximately 10% of the initial value . A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I(SC) in totally stripped human colon . Tracer flux and ion replacement studies revealed the I(SC) to be mainly accounted for by electrogenic Cl(-) secretion . Supernatant applied serosally completely abolished basal I(SC) . The supernatant-induced I(SC) was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca(2+) chelator (BAPTA-AM) had no effect . Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin . Accordingly, identical I(SC) and R(t) responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin . Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp . strain Sb . In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl(-) secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C. FEMS Microbiol Lett, 2004 Aug 1, 237(1), 127 - 32 Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae; Nakasone N et al.; A novel protease produced by Aeromonas caviae was purified and characterized . The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis . The protease activity was not inhibited completely by heating at 100 degrees C for 15 min . The proteolytic activities were inhibited by metalloprotease inhibitor . The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN . AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test . A high concentration of the protease showed cytotoxic activity against Vero cells. Protein Expr Purif, 2004 Aug, 36(2), 272 - 9 Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv . sobria; Crawford PA et al.; The gene from Aeromonas veronii bv . sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography . This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions . The biochemical properties of recombinant ImiS were compared with those of native ImiS . Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures . Gel filtration chromatography revealed that both enzymes exist as monomers in solution . MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca . 0.7 mol of Zn(II) . Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc . Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme . This over-expression protocol now allows for detailed spectroscopic and mechanistic studies on ImiS as well as site-directed mutants of ImiS to be prepared for future structure/function studies. J Environ Biol, 2003 Oct, 24(4), 373 - 9 Influence of pH, salt concentration and temperature on the growth of Aeromonas hydrophila; Vivekanandhan G et al.; The influence of environmental factors on the growth of Aeromonas hydrophila was investigated Four isolates (AH 37, AH 79, AH 86 and AH 100) were exposed to various environmental factors such as pH, salt concentration and temperature in laboratory condition . All the four isolates showed more or less similar growth at pH 7.0, 8.0 and 9.0 at 30 degrees C and 5 degrees C . At pH 5.0, 6.0 and 10.0, the log number of cells was found to be lesser than that of pH 7.0, 8.0 and 9.0 at both 30 degrees C and 5 degrees C . The results of the influence of salt concentration on the growth of Aeromonas hydrophila revealed that NaCl concentration of 0.5%, 1.0% and 2.0% favored the growth of this organism at both 30 degrees C and 5 degrees C . Increase in the salt concentration resulted in the growth of the decrease of this organism . Three percentages and 4% salt concentration moderately supported the growth of the organisms in the medium whereas at 5.0% NaCl concentration, there was no growth . Moderate growth of A . hydrophila at 5 degrees C is an interesting observation . The ability to grow at salt concentration between 0.5%, 4.0% under acidic and alkaline conditions pose a problem in the preservation of seafoods . These criteria may account for modified preservation techniques. J Appl Microbiol, 1998 Jan, 84(1), 37 - 46 The development of random DNA probes specific for Aeromonas salmonicida; Oakey HJ et al.; RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida . DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous . However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer . salmonicida . Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences . All five fragments were evaluated as DNA probes for the specific detection of Aer . salmonicida DNA: two hybridized specifically to DNA of all Aer . salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida, achromogenes and masoucida, but not subspecies smithia; one hybridized to subspecies salmonicida and achromogenes, but not subspecies masoucida or smithia; and one hybridized to subspecies salmonicida, achromogenes and smithia, but not subspecies masoucida . It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens. Appl Environ Microbiol, 2004 Jul, 70(7), 3925 - 32 Analysis and validation of a predictive model for growth and death of Aeromonas hydrophila under modified atmospheres at refrigeration temperatures; Pin C et al.; Specific growth and death rates of Aeromonas hydrophila were measured in laboratory media under various combinations of temperature, pH, and percent CO(2) and O(2) in the atmosphere . Predictive models were developed from the data and validated by means of observations obtained from (i) seafood experiments set up for this purpose and (ii) the ComBase database .Two main reasons were identified for the differences between the predicted and observed growth in food: they were the variability of the growth rates in food and the bias of the model predictions when applied to food environments . A statistical method is presented to quantitatively analyze these differences . The method was also used to extend the interpolation region of the model . In this extension, the concept of generalized Z values (C . Pin, G . Garcia de Fernando, J . A . Ordonez, and J . Baranyi, Food Microbiol . 18:539-545, 2001) played an important role . The extension depended partly on the density of the model-generating observations and partly on the accuracy of extrapolated predictions close to the boundary of the interpolation region . The boundary of the growth region of the organism was also estimated by means of experimental results for growth and death rates. J Environ Qual, 2004 May-Jun, 33(3), 1041 - 7 Bacterial removal and protozoan grazing in biological sand filters; Bomo AM et al.; The objective of the study was to investigate the importance of protozoan predation as a biological removal mechanism in sand filters used for purification of bacteria from wastewater . Eleven sand filter columns were seeded with a high dose of wastewater (70 mm d(-1)) and a high concentration (10(8) colony forming units {CFU} mL(-1)) of Aeromonas hydrophila (American Type Culture Collection {ATCC} 14715) for a period of 30 d . Water samples from three filter outlets were analyzed for the concentration of A . hydrophila . In addition, one filter column was sacrificed each sampling day for the quantification of A . hydrophila, culturable bacteria (heterotrophic plate counts, HPC), total bacterial counts, and protozoa in the sand . The mean removal efficiency of A . hydrophila in the sand filter columns was 4.7 log units . The concentration of A . hydrophila in the sand filter effluent, however, had a clearly time-dependent pattern from high (log 6) and unstable concentrations to low and more stable levels (log 2) . The removal efficiency of A . hydrophila correlated significantly (P = 0.0005, r2 = 0.6) with numbers of protozoa in the sand filters . Significantly higher (P < 0.05) concentrations of A . hydrophila were observed in sand filter effluents from columns treated with the protozoan inhibitor cycloheximide, compared with nontreated columns . Results from the present study show that protozoan grazing plays an important role as a bacterial removal mechanism in sand infiltration systems. Vet Microbiol, 2004 Jul 14, 101(3), 167 - 76 Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine; Vivas J et al.; Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence . We have previously described the characterisation and use of an auxotrophic aroA mutant of the A . hydrophila AG2 strain as a live attenuated vaccine against A . hydrophila infections in rainbow trout (Oncorhynchus mykiss) . In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula . The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline . Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines . On the other hand, the production of serine- and metalloprotease activities in A . hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing . A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula . The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production. Indian J Med Res, 2004 May, 119(5), 186 - 9 Cytotoxin testing of environmental Aeromonas spp . in Vero cell culture; Balaji V et al.; BACKGROUND & OBJECTIVES: Aeromonas spp . are known to cause a variety of infections in humans and this organism has been isolated from a variety of sources including environmental sources . The pathogenicity of the environmental isolates in and around Vellore has not been studied . This study was conducted to determine the cytotoxicity of the Aeromonas spp . isolated from water bodies, soil sediments, plankton and sewers in and around Vellore . METHODS: Aeromonas spp . isolated from environmental sources were identified by standard procedures . Representative isolates of Aeromonas spp . were tested for cell free cytotoxic factor in tissue culture system . Undiluted and diluted cell free filtrates of isolates and known toxigenic and non-toxigenic bacteria were added to Vero cell monolayer in microtitre plates . After appropriate incubation in 5 per cent CO2 atmosphere, the microtitre plate was examined for cytopathic effect . Cell detachment and shrinkage of Vero cells were recorded as toxic changes . RESULTS: All 36 environmental isolates demonstrated cytopathic effect of which 41.7, 50 and 8.3 per cent belonged to A . hydrophila, A . veronii biotype sobria and A . caviae respectively . INTERPRETATION & CONCLUSION: The results demonstrated the presence of potentially pathogenic environmental aeromonads in and around Vellore and they produced cytotoxin. J Biol Chem, 2004 Sep 3, 279(36), 37597 - 612 Epub 2004 Jun 23. Aeromonas hydrophila cytotoxic enterotoxin activates mitogen-activated protein kinases and induces apoptosis in murine macrophages and human intestinal epithelial cells; Galindo CL et al.; A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages . In this study, we utilized five target cell types (a murine macrophage cell line (RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase (MAPK) pathways and mechanisms leading to apoptosis . As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells . Act also induced phosphorylation of upstream MAPK factors (MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun) . Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells . In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased . Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor . However, mitochondrial membrane depolarization was not detected in response to Act . Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells . Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells. Syst Appl Microbiol, 2004 May, 27(3), 343 - 9 Polyphasic taxonomic study of "Aeromonas eucrenophila-like" isolates from clinical and environmental sources; Demarta A et al.; In the current study, a group of 17 "Aeromonas eucrenophila-like" isolates from clinical and environmental origins was subjected to a polyphasic taxonomic characterization including ribotyping, 16S rDNA sequencing, FAFLP fingerprinting, and biochemical testing . Genotypic, phylogenetic, and phenotypic results were consistent with the conclusion that this group of 17 isolates may represent a currently undescribed Aeromonas taxon most closely related to A . eucrenophila HG6 . In addition, comparison of newly obtained 16S rDNA sequencing data with published sequences demonstrated a high phylogenetic heterogeneity among isolates currently classified in the species A . encheleia. Int J Food Microbiol, 2004 Jul 15, 94(2), 113 - 21 The usefulness of molecular techniques to assess the presence of Aeromonas spp . harboring virulence markers in foods; Bin Kingombe CI et al.; A total of 78 raw and 123 processed and ready-to-eat retail food samples were used to assess the presence of motile Aeromonas spp . harboring virulence genes (cytotoxic enterotoxin and hemolysin genes) using a recently described PCR method in comparison with the conventional cultivation method based on the use of Ampicillin-Dextrin Agar (ADA) medium . With the ADA-based method, 65/201 (32.3%) samples showed presumptive Aeromonas spp . colonies whereas the PCR method revealed the presence of Aeromonas spp . harboring the targeted virulence genes in 51/201 (25.4%) of the tested samples . The rate of contaminated samples and the presence of pathogenic Aeromonas were significantly lower with both methods for processed than in case of raw samples . A polyphasic identification approach including biochemical and molecular techniques was applied to a selection of 34 PCR-positive presumptive Aeromonas isolates . Following fatty acid methyl ester (FAME) analysis and amplified fragment length polymorphism (AFLP) fingerprinting, a total of 33 isolates (97%) could be identified to the DNA hybridization group (HG) level . The majority of these isolates belonged to the species Aeromonas hydrophila HG3 (50%) and Aeromonas veronii biovar sobria (HG8/10) (38%) . Molecular characterization of PCR amplicons obtained from these strains by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) fingerprinting and PCR-Amplicon Sequence Analysis (PCR-ASA) allowed classification of all strains in a known PCR-RFLP and PCR-ASA type . In conclusion, the current findings demonstrate that the combined use of PCR-based virulence marker detection, PCR-RFLP and PCR-ASA offers a rapid, sensitive, and specific system to assess the presence and prevalence of Aeromonas spp . harboring virulence markers in food samples. Carbohydr Res, 2004 Jun 22, 339(9), 1631 - 6 Chemical structure of aeromonas gum--extracellular polysaccharide from Aeromonas nichidenii 5797; Xu X et al.; Aeromonas (A) gum, an extracellular heteropolysaccharide produced by the bacterium Aeromonas nichidenii strain 5797, was studied by 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H, 13C HMQC, HMBC and ROESY experiments after O-deacetylation and Smith degradation . These investigations revealed the presence of an O-acetylated pentasaccharide repeating unit composed of mannose, glucose, xylose and glucuronic acid, and it has the following structure: {Image: see text} Biosci Biotechnol Biochem, 2004 May, 68(5), 1082 - 90 Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9; Lan X et al.; A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9 . The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source . Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc) . Most of the activity, however, was not detected in culture fluid but was associated with cells . A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication . The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product . The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide . A gene coding for the purified beta-N-acetylglucosaminidase was isolated . The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues . The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases. J Med Microbiol, 2004 Jun, 53(Pt 6), 527 - 34 Prevalence, serotype distribution, antibiotic susceptibility and genetic profiles of mesophilic Aeromonas species isolated from hospitalized diarrhoeal cases in Kolkata, India; Sinha S et al.; A comprehensive study was performed to examine incidence, species distribution, drugs sensitivity, virulence genes and molecular fingerprints of Aeromonas species isolated from patients with acute diarrhoea over a period of 2 years in Kolkata, India . Following the Aerokey II scheme, more than 95 % of strains were identified to species level . Seven different species were encountered in this study, with Aeromonas caviae being dominant, followed by Aeromonas hydrophila and Aeromonas veronii biovar sobria . Thirty different serotypes were encountered, with O16, O83 and O85 being dominant, but no serotype was associated specifically with a single species . The majority of Aeromonas strains exhibited multidrug resistance . The alt and act genes, which encode heat-labile cytotonic and cytotoxic enterotoxins, were respectively found in 71.9 and 20.1 % of strains examined . Only 2.4 % of strains carried the heat-stable cytotonic enterotoxin (ast) gene . The hlyA gene was found in 28 % of Aeromonas strains . With few exceptions, genomic diversity of Aeromonas strains belonging to the same serotype was observed by random amplification of polymorphic DNA PCR and ribotyping . Different species of Aeromonas and different clones of Aeromonas species seem to be associated with hospitalized cases of diarrhoea in Kolkata, India. J Med Microbiol, 2004 Jun, 53(Pt 6), 477 - 82 Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria; Song T et al.; Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia . An A . veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized . Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors . Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases . An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation . Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain . The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type . AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins . Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity. J Biol Chem, 2004 Jul 23, 279(30), 31018 - 25 Epub 2004 May 11. The catalytic role of glutamate 151 in the leucine aminopeptidase from Aeromonas proteolytica; Bzymek KP et al.; Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP) . However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP . In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate . The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%) . Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP . The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change . Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively . Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base. Mar Biotechnol (NY) . 2004 Apr 15; {Epub ahead of print} Identification of Immune-Relevant Genes from Atlantic Salmon Using Suppression Subtractive Hybridization; Tsoi SC et al.; In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon ( Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis . Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process . Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059) . Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries . These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others . A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection. Appl Environ Microbiol, 2004 May, 70(5), 2702 - 8 Behavior of an Aeromonas hydrophila aroA live vaccine in water microcosms; Vivas J et al.; Genetically modified auxotrophic mutants of different fish pathogens have been used as live vaccines in laboratory experiments, but the behavior of the strains after release into aquatic ecosystems has not been characterized . We previously constructed and characterized an aroA mutant of Aeromonas hydrophila and studied the protection afforded by this mutant as a live vaccine in rainbow trout . In this work, we describe the survival of this strain in aquatic microcosms prepared from fish water tanks . The aroA mutant disappeared rapidly in nonfiltered, nonautoclaved fish tank water, declining below detection levels after 15 days, suggesting an inhibitory effect of the autochthonous microflora of the water . When the aroA strain was used to inoculate sterilized water, its culturability was lower than that of wild-type strain A . hydrophila AG2; after long periods of incubation, aroA cells were able to enter a viable but nonculturable state . Entry into this nonculturable state was accompanied by changes in the cell morphology from rods to spheres, but the cells appeared to remain potentially viable, as assessed by the preservation of cell membrane integrity . Supplementation of the culture medium with sodium pyruvate favored the culturability and resuscitation of the two A . hydrophila strains at low temperatures (6 and 16 degrees C) . These results contribute to a better understanding of the behavior of the aroA strain in natural environments and suggest that the inactivation of the aroA gene may be beneficial for the safety of this live vaccine for aquacultures. Fish Shellfish Immunol, 2004 Feb, 16(2), 227 - 39 Isolation and characterization of complement component C3 from Atlantic cod (Gadus morhua L.) and Atlantic halibut (Hippoglossus hippoglossus L.); Lange S et al.; Complement component C3 was isolated from the plasma of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.) . Fast protein liquid chromatography (FPLC) techniques, involving ion exchange and gel filtration columns, were used . The purified proteins were analysed by SDS-PAGE which showed a two-chain structure, alpha- and beta-chains, as seen in higher vertebrates . Both proteins had intra-chain thioesters located within their alpha-chains and N-terminal amino acid sequencing confirmed their identity with reference to known C3 amino acid sequences from other species . Specific antibodies were prepared against cod and halibut C3 and tested in Western blotting on sera and purified C3 . The proteolytic fragmentation of C3 was tested with trypsin, pepsin, papain and the extracellular product (ECP) from the bacterium Aeromonas salmonicida ssp . achromogenes (Asa) . Both trypsin and papain were successful in cleaving C3 whereas pepsin and ECP had no effect . Carbohydrate moieties were detected in the alpha- and beta-chains of cod and halibut C3 and N-linked oligosaccharides were removed from the C3 with PNGase treatment, revealing a difference in C3 glycosylation between the two species. Fish Shellfish Immunol, 2004 Feb, 16(2), 193 - 206 The auxotrophic aroA mutant of Aeromonas hydrophila as a live attenuated vaccine against A . salmonicida infections in rainbow trout (Oncorhynchus mykiss); Vivas J et al.; An auxotrophic aroA mutant of the Aeromonas hydrophila AG2 strain is a live attenuated vaccine against A . hydrophila infection in rainbow trout (Oncorhynchus mykiss) . The protection conferred by the live attenuated vaccine against A . salmonicida strains is reported here, and several parameters of the specific and non-specific immune response in vaccinated trout were characterised . Vaccination with a dose of 10(7)cells/fish of the aroA mutant elicited significant protection against the Hooke and DK30 strains of A . salmonicida (relative percent survival RPS >60%) . This cross-protection correlated moderately with the activation of the humoral and cellular specific immune responses, which show cross-reactivity against antigens shared by the two bacterial species, and a moderate increase in the lysozyme and antiprotease activities in the serum of vaccinated trout. Fish Shellfish Immunol, 2004 Mar, 16(3), 437 - 45 The effect of various Aeromonas bestiarum vaccines on non-specific immune parameters and protection of carp (Cyprinus carpio L.); Kozinska A et al.; Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish . Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland . Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination . Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days . The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group . Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI) . The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control . Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish. Fish Shellfish Immunol, 2004 Mar, 16(3), 271 - 85 Vaccination and immune responses against atypical Aeromonas salmonicida in spotted wolffish (Anarhichas minor Olafsen) juveniles; Grontvedt RN et al.; To study the effect of early vaccination, wolffish juveniles of size 50 and 90 mm, respectively, were vaccinated with an oil-adjuvanted atypical A . salmonicida bacterin . Vaccination resulted in significant protection after challenge with the homologous bacterial strain and specific antibody responses were demonstrated against whole bacteria as well as purified A-layer protein and LPS by ELISA and Western blotting but individual variation in immune responses was apparent . The A-protein was the most immunogenic bacterial component . In addition, higher numbers of immunoglobulin producing cells were detected by in situ hybridisation in kidney and spleen of vaccinated fish compared to non-vaccinated fish . Plasma cells were also present in gut and gills in equal numbers irrespective of treatment . No plasma cells were found in the skin . Finally, the frequencies of expressed V(H)families and C(L)isotypes of wolffish immunoglobulins were shown by PCR . The relative expression of the three variable regions of the Ig heavy chain and the three isotypes of the Ig light chain in the spotted wolffish spleen seemed to be unaffected by immunisation with a complex antigen like the A . salmonicida bacterin. Biochemistry, 2004 May 11, 43(18), 5414 - 27 Aeromonas proteolytica aminopeptidase: an investigation of the mode of action using a quantum mechanical/molecular mechanical approach; Schurer G et al.; The aminopeptidase of Aeromonas proteolytica (AAP) belongs to the group of metallo-hydrolases that require two divalent cations for full activity . Such binuclear metal centers are found in several aminopeptidases, raising the question whether a common mechanism, at least partly, is likely . We have used a quantum mechanical/molecular mechanical (QM/MM) approach to investigate the reaction mechanism of AAP . Among several possibilities, one reaction path was found to be clearly the most favorable . Beside the chemical transformation steps, effects of the enzyme environment and the influence of the solvent on the catalytic reaction were included in the study . The results are in good agreement with experimental studies and correspond to a high degree to our previous QM/MM calculations on the reaction mechanism of the related binuclear bovine lens leucine aminopeptidase (blLAP), which, although related to the AAP, has different Zn(2+)-coordination spheres and a different catalytic residue . The mechanisms of the two enzymes as suggested in the literature differ on the mode of coordination of the nucleophile and the identity of the general base . However, the results of this and our previous work on blLAP allow us to identify a common mechanism for the two enzymes . This mechanism is probably quite general for binuclear zinc enzymes. Fish Shellfish Immunol, 2004 May, 16(5), 645 - 58 Cloning, characterisation and expression of Aeromonas hydrophila major adhesin; Fang HM et al.; Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms . The adhesion is a prerequisite for successful invasion . In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed . Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da) . The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A . hydrophila . Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli . The purified recombinant adhesin could competitively inhibit A . hydrophila from invading fish epithelial cells in vitro . Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas . When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A . hydrophila challenge. Fish Shellfish Immunol, 2004 May, 16(5), 633 - 44 Sequential study of antigen persistence and concomitant inflammatory reactions relative to side-effects and growth of Atlantic salmon (Salmo salar L.) following intraperitoneal injection with oil-adjuvanted vaccines; Mutoloki S et al.; The persistence of antigens at the injection site (area around pyloric caeca and spleen), concomitant inflammatory reaction and granuloma development were monitored at 3, 6 and 12 months following intraperitoneal injection with multivalent, oil-adjuvanted vaccines in Atlantic salmon . Parallel assessment of side-effect profiles and growth rate were also performed . Antigen persistence was examined by use of a monoclonal antibody that recognises Aeromonas salmonicida lipopolysaccharide in an immunohistochemical method for in situ identification of bacteria or bacterial fragments . The inflammatory reaction was monitored using standard histological techniques . The amount of persistent antigens and size of inflammation/adhesions were estimated semi-quantitatively . A steady decrease in the quantity of antigens at the injection site was observed from 3 to 12 months . Antigens were consistently found in inflamed tissues located in the pancreatic region . The size of inflammation increased during the first 6 months but declined thereafter . These findings suggest that persistent antigens at the injection site may act as inflammatory stimulants that induce and perpetuate the inflammatory reaction, eventually leading to adverse side-effects. Acta Microbiol Pol, 2003, 52(4), 343 - 54 16S to 23S rDNA spacer fragment length polymorphism of Aeromonas sp; Laganowska M et al.; We analyzed polymorphism of the PCR-amplified 16S-23S rDNA spacer of Aeromonas species . A total of 69 isolates representing 18 DNA hybridization groups were used in this study . The analysis of PCR products of 16S-23S rDNA spacers revealed patterns consisting of two to eight DNA fragments . The fragment sizes ranged from 730 to 1050 bp . DNA patterns revealed a considerable genetic diversity between species and within a species . When a procedure to eliminate heteroduplex formation was performed, the number of bands was reduced to 2-5 . Nevertheless the homoduplex ISR (intergenic spacer region) patterns obtained were not useful for species distinguishing. Clin Infect Dis, 2004 Apr 15, 38(8), 1084 - 9 Epub 2004 Mar 29. Outbreak of Aeromonas hydrophila wound infections associated with mud football; Vally H et al.; On 16 February 2002, a total of 26 people presented to the emergency department of the local hospital in the rural town of Collie in southwest Western Australia with many infected scratches and pustules distributed over their bodies . All of the patients had participated in a "mud football" competition the previous day, in which there had been ~100 participants . One patient required removal of an infected thumbnail, and another required surgical debridement of an infected toe . Aeromonas hydrophila was isolated from all 3 patients from whom swab specimens were obtained . To prepare the mud football fields, a paddock was irrigated with water that was pumped from an adjacent river during the 1-month period before the competition . A . hydrophila was subsequently isolated from a water sample obtained from the river . This is the first published report of an outbreak of A . hydrophila wound infections associated with exposure to mud. J Appl Microbiol, 2004, 96(5), 994 - 1001 Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase); Esteve C et al.; AIMS: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity . METHODS AND RESULTS: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used . In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin . In contrast, proteases were mainly released during the stationary phase . Serine protease activity in EO63 culture supernatants was four times greater than that caused by metalloproteases . Two main proteases were partially purified from EO63 culture supernatants by isoelectrophoresis: a serine protease (68 kDa) active against casein; a mixture of different protein bands (60, 44 and 31 kDa) representing a thermostable metalloprotease active against elastin and casein . This metallo-elastase was also inhibited by dithiothreitol and showed a pH optimum of 8.0 . Both exoenzymes were toxic for eels at LD50 doses of 1.1 and 3.5 microg (g fish)(-1), respectively . CONCLUSIONS: A serine caseinase and a metallo-elastase may play a role in the pathogenicity of EO63 for eels . These toxins are excreted in vitro by EO63 in the ratio of 4:1 during the stationary phase of growth . Strain EO63 also produced beta-haemolysins in vitro which could correspond to aerolysin . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the purification of a metallo-elastase excreted by a wild-type A . hydrophila strain. J Chin Med Assoc, 2004 Jan, 67(1), 51 - 3 Aeromonas hydrophila orbital cellulitis in a patient with myelodysplastic syndrome; Chou SY et al.; Orbital cellulitis caused by Aeromonas hydrophila developed in a 73-year-old male with a history of myelodysplastic syndrome . He was admitted because of fever, general malaise, pain as well as periorbital swelling in the right eye . Four days later, a yellowish pustule with purulent material was noted over right lower eyelid . Aeromonas hydrophila was isolated from the discharge . After administering intravenous cefuroxime 1,500 mg every 8 hours and topical ofloxacin eye oint, his symptoms subsided gradually . We present the first known case of orbital cellulitis from Aeromonas hydrophila in a patient with myelodysplastic syndrome . In patients with myelodysplastic syndrome, Aeromonas hydrophila should be listed as an important pathogen in any soft tissue infection including eyelid infection . Culture and adequate antimicrobial therapy are recommended, because rapid worsening may result in orbital cellulitis or even septicemia in patients with suppressed immune system. Proteomics, 2004 Apr, 4(4), 1074 - 85 Differential proteomic analysis of Aeromonas salmonicida outer membrane proteins in response to low iron and in vivo growth conditions; Ebanks RO et al.; Aeromonas salmonicida subsp . salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids . Bacterial phenotypes are known to change in vivo compared to the in vitro state . Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass . Using an in vivo growth chamber model, the pathogenic fish bacterium A . salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis . Growth of A . salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions . Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor . When cultured in vivo, A . salmonicida up-regulated the identical 73, 76 and 85 kDa proteins . The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A . salmonicida within the peritoneal cavity of salmon. J Formos Med Assoc, 2004 Jan, 103(1), 53 - 7 Nosocomial infection of Aeromonas hydrophila presenting as necrotizing fasciitis; Cheng NC et al.; Aeromonas hydrophila is infrequently reported as a causative organism of necrotizing fasciitis . We report a case of necrotizing fasciitis due to A . hydrophila in a 44-year-old man with Marfan syndrome who underwent valve replacement surgery twice . He was admitted due to a 2-day history of fever . The fever was attributed to hepatitis, and ingestion of herbal medication was suspected to be the cause . The fever relapsed on the 28th day of hospitalization with rapidly progressive erythematous patches on the bilateral lower extremities . Septic shock developed within a few hours, and 2 small diagnostic incisions on the skin lesions suggested necrotizing fasciitis . Surgical exploration further revealed extensive necrosis of the subcutaneous tissue and fascia, but the muscle was spared . Blood cultures and cultures of the debrided tissue all yielded A . hydrophila . Pathological examination showed necrosis and degeneration of the soft tissue . Although appropriately managed with broad-spectrum antibiotics, fasciotomies and debridement, the patient's condition deteriorated rapidly and resulted in death 11 hours after the surgery . This case indicates that A . hydrophila can be a causative organism of nosocomial necrotizing fasciitis. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 481 - 5 Aeromonas simiae sp . nov., isolated from monkey faeces; Harf-Monteil C et al.; Two Aeromonas strains, IBS S6874(T) and IBS S6652, were isolated from the faeces of two healthy monkeys (Macaca fascicularis) from Mauritius that were kept in quarantine in the Centre for Primatology, Strasbourg, France . Phylogenetic analysis based on 16S rRNA gene sequences showed that the two isolates formed an unknown genetic lineage within the genus Aeromonas . The two isolates had nearly identical sequences (0.1 % nucleotide substitution) that were related closely to those of recognized Aeromonas species (1.7-3.5 % nucleotide substitution) . DNA-DNA hybridization showed that strains IBS S6874(T) and IBS S6652 had high DNA-DNA similarity (89 %) to each other and a low level of DNA-DNA similarity to closely related taxa (18 % relatedness to Aeromonas trota and 16 % relatedness to Aeromonas schubertii) . Phenotypically, the two monkey isolates differed from most previously described mesophilic Aeromonas species by their lack of haemolysis on sheep-blood agar and inability to produce indole, gas from glucose or acid from mannitol . They differed from the most closely related species, A . schubertii, by their ability to produce acid from D-cellobiose and D-sucrose and by their pyrazinamidase activity . The name Aeromonas simiae sp . nov . is proposed for these isolates; strain IBS S6874(T) (=CIP 107798(T)=CCUG 47378(T)) is the type strain. FEMS Microbiol Lett, 2004 Mar 12, 232(1), 61 - 6 Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101; Chang MC et al.; Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence . In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII) . Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan . In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII) . In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis . The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding. Ophthalmology, 2004 Feb, 111(2), 348 - 51 Aeromonas caviae keratitis associated with contact lens wear; Pinna A et al.; OBJECTIVE: We report the first case of bilateral contact lens-related Aeromonas caviae keratitis associated with A . caviae contamination of the contact lens case . The presence of virulence factors produced by Aeromonas species was also investigated . DESIGN: Case report . INTERVENTION AND TESTING: Conjunctival swabs and corneal scrapings from both eyes were inoculated for culture . The contact lens case was also cultured . The isolate was analyzed for the presence of virulence properties, such as gelatinase and protease production . The presence of virulence genes, such as the cytolytic enterotoxin (AHCYTOEN) and type IV Aeromonas pilus (tap) genes, was investigated using polymerase chain reaction . The susceptibility of A . caviae to 6 commercial contact lens disinfecting solutions was tested . MAIN OUTCOME MEASURES: Culture results, protease activity, and gelatinase production were analyzed . Polymerase chain reaction amplification products were visualized in ethidium bromide-stained agarose gel . Bacterial growth after exposure to contact lens disinfecting solutions was assessed . RESULTS: Aeromonas caviae was grown bilaterally from the conjunctiva, cornea, and contact lens case . The organism showed protease and gelatinase production . Polymerase chain reaction amplification revealed that the A . caviae strain contained the AHCYTOEN and tap virul