Aeromonades

J Microbiol Methods, 2002 May, 49(3), 335 - 8
Immuno-capture PCR for detection of Aeromonas hydrophila; Peng X et al.; In this report, we describe the use of universal primer PCR (UPPCR) for the detection of 16S ribosomal RNA (rRNA) genes from Aeromonas hydrophila captured by anti-A . hydrophila antibody coupled to a microplate . The approach combining immuno-capture with UPPCR provides a quick, sensitive, and reproducible way for the detection of bacterial cells.

Fish Shellfish Immunol, 2002 Jan, 12(1), 35 - 48
Temperature dependent activation of leucocyte populations of rainbow trout, Oncorhynchus mykiss, after intraperitoneal immunisation with Aeromonas salmonicida; Kollner B et al.; The temperature dependence of in vivo activation of rainbow trout Oncorhynchus mykiss, leucocyte populations after intraperitoneal injection (i.p.) of fish with a T-cell independent antigen Aeromonas salmonicida (strain MT423) was investigated using a proliferation assay and flow cytometric analysis with mab specific for trout leucocyte surface markers . In trout kept at 15-17 degrees C a prominent activation of blood and spleen leucocytes was found . Also, drastic changes of the percentage of the leucocyte populations in blood and spleen occurred: the amount of monocytes in the blood increased between day 2 and day 7 post injection (p.i.), whereas in spleen the amount of monocytes stayed at a high level (approximately 35%) after a depression between day 4 and day 7 p.i . The percentage of B-lymphocytes was increased first in spleen and then in blood . The percentage of granulocytes in blood was elevated during the whole experiment compared to control fish . In trout kept at 10-12 degrees C only blood leucocytes showed a weak activation after i.p . injection of A . salmonicida, whereas spleen leucocytes showed nearly no reaction . Only the percentage of granulocytes in the blood (day 2-14 p.i.) and of monocytes in the spleen (day 2 and day 8 p.i.) was changed compared to phosphate buffered saline (PBS)-injected fish . However, the development of A . salmonicida specific antibodies was contrary to the cellular reaction . Whereas antibodies could first be detected after 16-18 days p.i . in both groups the amount of antibodies was significantly higher in sera of trout kept at 10-12 degrees C at day 22 and day 28 p.i . than in sera of trout kept at 15-17 degrees C . These results indicate stronger A . salmonicida induced activation of monocytes, granulocytes and B-lymphocytes at higher temperature . However, the development of a specific antibody response against A . salmonicida seemed to be more effective at lower temperatures.

J Biol Inorg Chem, 2002 Jan, 7(1-2), 129 - 35 Epub 2001 Aug 11.
The aminopeptidase from Aeromonas proteolytica can function as an esterase; Bienvenue DL et al.; The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM . The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1 . The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations . Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation . The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively . The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1 . The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA . For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively . Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis . The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.

J Appl Microbiol, 2002, 92(2), 289 - 96
The cloning and characterization of a second alpha-amylase of A . hydrophila JMP636; Kidd SP et al.; AIMS: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA.METHODS AND RESULTS: The amylase activity of A . hydrophila JMP636 is encoded by multiple genes . A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli . AmyB is a large alpha-amylase of 668 amino acids . Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A . hydrophila JMP636 AmyA and AmyB . Significant (80%) similarity exists between amyB and an alpha-amylase of A . hydrophila strain MCC-1 . Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability) . However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present . Both enzymes were confirmed to be alpha-type amylases.CONCLUSIONS: AmyB has been isolated, characterized and then compared to AmyA.SIGNIFICANCE AND IMPACT OF THE STUDY: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A . hydrophila.

Appl Environ Microbiol, 2002 Feb, 68(2), 650 - 5
Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh; Rahman M et al.; Sparse information is available on the virulence factors of Aeromonas strains isolated from diseased fish, from the environment, and from humans . In the present study, 52 Aeromonas isolates obtained from epizootic ulcerative syndrome (EUS) lesions in fish, from the aquatic environment, and from children with diarrhea in Bangladesh were identified by biochemical phenotyping (i.e., PhenePlate {PhP} typing) and DNA fingerprinting and then characterized with respect to certain putative virulence factors . The isolates from the fish exhibiting EUS symptoms were identified to be Aeromonas veronii biovar sobria by fatty acid methyl ester analysis and amplified fragment length polymorphism fingerprinting . Biochemical phenotyping revealed that all EUS-associated isolates belonged to a unique phenotype which was not identified among more than 1,600 environmental and diarrheal isolates in a previously collected database of PhP types of Bangladeshi Aeromonas isolates . The 52 Aeromonas isolates were investigated for the production of hemolysin and cytotoxin; for hemagglutination with erythrocytes from fish, human, and rabbit sources; for the presence of a cytolytic enterotoxin gene; and for adhesion to and invasion into fish cell lines . All of the EUS isolates produced all of the virulence factors investigated, as did also some of the environmental isolates, but the isolates from EUS were unique in their ability to agglutinate fish erythrocytes . Our results suggest that a clonal group of A . veronii biovar sobria is associated with, and may be a causative agent of, EUS in fish in Bangladesh.

Epidemiol Infect, 2001 Dec, 127(3), 561 - 6
Detection of Aeromonas caviae in the common housefly Musca domestica by culture and polymerase chain reaction; Nayduch D et al.; Aeromonas caviae has been implicated in diarrhoeal disease of livestock and humans . The potential role of houseflies in the epidemiology of this pathogen was investigated by examining the prevalence of A . caviae in houseflies collected from two South Carolina farms and one restaurant . Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific PCR using novel primers (APW-PCR) . All isolates cultured from houseflies were identified as A . caviae by biochemical characteristics and direct sequencing approximately 800 bp of the 16S rRNA gene . Aeromonas caviae was detected in 78% (272/349) dairy farm flies, 55% (54/99) pig farm flies and 39% (77/200) restaurant flies . Faeces from cows and pigs at the farms also were positive for A . caviae (58% and 100%, respectively) . The APW PCR method provided a rapid, convenient way to identify A . caviae from faeces and houseflies that contained hundreds of bacterial species.

Appl Environ Microbiol, 1997 Jul, 63(7), 2708 - 15
Diversity, persistence, and virulence of Aeromonas strains isolated from drinking water distribution systems in Sweden; Kuhn I et al.; The Aeromonas populations in 13 Swedish drinking water distribution systems, representing different treatments, were investigated . From each system, water samples were collected four times during the period from May to September 1994 from raw water and water after treatment and at two to five sites within the distribution system . In total, 220 water samples were collected . From samples containing presumptive Aeromonas, up to 32 colonies were analyzed by the PhenePlate Aeromonas (PhP-AE) system, which is a highly discriminating biochemical fingerprinting method . Selected isolates from different phenotypes (PhP types) were further identified by the API 20 NE system and by gas-liquid chromatography analysis of fatty acid methyl esters (FAMEs) . Selected isolates were also assayed for their potential to produce hemolysin and cytotoxin and for their ability to adhere to human intestinal cells . In total, 117 water samples (53%) contained presumptive Aeromonas which numbered up to 10(6) CFU/100 ml in raw water and up to 750 CFU/100 ml in tap water . Among the 2,117 isolates that were subjected to typing by the PhP-AE system, more than 300 distinct PhP types were found, of which the majority occurred only sporadically . Raw (surface) water samples usually contained many different PhP types, showing high diversity indices (Di) (median Di = 0.95) . The Aeromonas populations in samples collected from within the distribution systems were less diverse (median Di = 0.58) and were often dominated by one major PhP type that was found on several sampling occasions . Seventeen such major PhP types could be found and were represented in 1,037 isolates (49%) . Identification by API 20 NE and FAME analysis revealed that most of the major PhP types were Aeromonas hydrophila or belonged to unidentified Aeromonas species . Hemolysin and cytotoxin production was observed in most major PhP types (representing 87 and 54% of the assayed isolates, respectively), and adherence was found in 89% of the isolates that produced cytotoxin . Thus, the data presented here show that although raw water may contain very diverse Aeromonas populations, the populations seemed to be remarkably stable within the studied water distribution systems, and that some potentially pathogenic Aeromonas strains could persist for several months in drinking water.

Dtsch Tierarztl Wochenschr, 2001 Nov, 108(11), 465 - 7
Isolation and identification of motile Aeromonas species from chicken; Sarimehmetoglu B et al.; In this study a total of 140 broiler carcasses and carcass parts purchased at different supermarkets in Ankara including 50 whole carcass, 30 wing, 30 leg and 30 breast samples were analysed for the presence of motile Aeromonas species . According to analysis findings, motile Aeromonas spp . were isolated from 116 (82.9%) of total 140 samples . The distribution of the isolates were 94%, 86.6%, 80%, 63.3% in broiler carcass, wing, leg and breast samples, respectively . Aeromonas hydrophila was isolated the most prevalent species with 56% the range followed by Aeromonas sobria with 29.3% and Aeromonas caviae with 14.7% from all of the carcass and carcass part samples, respectively . Consequently, it was supposed that, examined broiler carcass and carcass parts have been contaminated to important level with motile Aeromonas species and it has been risk for public health.

Microb Drug Resist, 2001 Fall, 7(3), 263 - 72
Class 1 integrons mediate antibiotic resistance in the fish pathogen Aeromonas salmonicida worldwide; L'Abee-Lund TM et al.; The presence of class 1 integrons was investigated in 38 sulfonamide-resistant strains of Aeromonas salmonicida subsp . salmonicida, atypical A . salmonicida and Escherichia coli conjugants with R plasmids originating from A . salmonicida . The strains originated from Finland, France, Japan, Norway, Scotland, Switzerland, and the United States . Additional resistance determinants in strains with class 1 integrons were also determined . Of 21 strains containing a class 1 integron, 19 had a single gene cassette, 1 strain had two cassettes, and 1 strain was found to lack an integrated gene cassette . In the integrons with single cassettes, aadA2 was present in eight strains, dfr16 in five strains, and aadA1 and dfrIIc in three strains each . In the integron with two cassettes, qacG and orfD were present . Tetracycline resistance was observed in 20 of the integron-positive strains, caused by the determinants Tet A and Tet E, in which Tet A frequently was associated with Tn1721 . Class 1 integrons seem to be important in mediating antibiotic resistance also in the marine environment . The gene cassettes reported in this study are all described in bacteria associated with humans, and this demonstrates once more how the common gene pool is shared between organisms belonging to different environments.

Exp Hematol, 2001 Dec, 29(12), 1403 - 9
Circulating PIG-A mutant T lymphocytes in healthy adults and patients with bone marrow failure syndromes; Ware RE et al.; OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematological disorder with acquired PIG-A gene mutations and absent surface expression of proteins utilizing glycosylphosphatidylinositol (GPI) anchors . PNH often follows aplastic anemia, suggesting PIG-A mutant cells have relative dominance over normal hematopoietic cells . Somatic PIG-A mutations could arise after aplasia, or healthy persons could have rare PIG-A mutant cells that expand under selection pressure . METHODS: We developed an in vitro negative selection method to isolate GPI-deficient T lymphocytes using aerolysin, an Aeromonas toxin that binds GPI anchors and induces cell lysis . Peripheral blood mononuclear cells (PBMC) from normal adults and patients with PNH or other bone marrow failure syndromes were analyzed . RESULTS: From healthy adults, 166 T lymphocyte clones with deficient GPI-linked surface protein expression (CD55, CD59) were isolated . The mean mutant frequency (M(f)) of aerolysin-resistant clones was 17.8 +/- 13.8 per 10(6) PBMC, range 5.0-59.6 per 10(6) cells . Clones had a Class A complementation defect and distinct PIG-A mutations . Patients with PNH had elevated aerolysin-resistant M(f) values averaging 19 x 10(-2), a 10,000-fold difference . Two patients with Fanconi anemia and two others with mild aplastic anemia had M(f) values less than 15 x 10(-6), but two with recovering aplastic anemia had M(f) values of 20 x 10(-4), representing an intermediate value between normal persons and PNH patients . CONCLUSION: Identification of PIG-A mutant T lymphocytes in healthy adults suggests PNH could develop following intense negative selection of hematopoiesis, with clonal outgrowth of naturally occurring PIG-A mutant stem cells.

Biomacromolecules, 2001 Spring, 2(1), 248 - 54
Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains; Park SJ et al.; Recombinant Escherichia coli strains harboring a plasmid containing a novel artificial polyhydroxyalkanoate (PHA) operon consisting of the Aeromonas PHA biosynthesis related genes and Ralstonia eutropha reductase gene were developed for the production of poly(3-hydroxybutyrate-co-hydroxyhexanoate) {P(3HB-co-3HHx)} from dodecanoic acid . By applying stepwise reduction of dissolved oxygen concentration (DOC) during the fermentation, the final dry cell weight, PHA concentration, and PHA content of 79 g/L, 21.5 g/L, and 27.2 wt %, respectively, were obtained in 40.8 h, which resulted in the PHA productivity of 0.53 (g/L)/h . The 3HHx fraction slowly increased during the fed-batch culture to reach a final value of 10.8 mol % . The 3HHx fraction in the copolymer could be increased by 3-fold when the Aeromonas hydrophila orf1 gene was coexpressed with the PHA biosynthesis genes.

Biomacromolecules, 2001 Spring, 2(1), 148 - 53
Characterization of 13 kDa granule-associated protein in Aeromonas caviae and biosynthesis of polyhydroxyalkanoates with altered molar composition by recombinant bacteria; Fukui T et al.; Analysis of native poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) {P(3HB-co-3HHx)} inclusions from Aeromonas caviae FA440 revealed that ORF1 (a 348-bp gene located immediately upstream of phaC(Ac)) encodes a 13-kDa granule-associated protein, which was referred to as phaP(Ac) . Several recombinant strains of A . caviae were constructed and conducted to analyze their PHA-producing abilities . A transconjugant of FA440 harboring additional copies of phaPCJ(Ac) genes accumulated P(3HB-co-3HHx) copolyesters with much higher 3HHx composition (46-63 mol %) than wild-type strain from alkanoates or olive oil . Deletion analysis revealed that overexpression of phaJ(Ac) encoding monomer-supplying (R)-hydratase was not a reason for the compositional change in the recombinant strains . PHA synthase activity in PHA inclusion fraction from the transconjugant composed of 60 mol % of 3HHx was 10-fold higher than that from the strain FA440 with 13 mol % of 3HHx, suggesting an importance of the level of PHA synthase activity for controlling the PHA composition in vivo.

J Bacteriol, 2002 Jan, 184(1), 142 - 51
Dissimilatory Fe(III) and Mn(IV) reduction by Shewanella putrefaciens requires ferE, a homolog of the pulE (gspE) type II protein secretion gene; DiChristina TJ et al.; Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron {Fe(III)} and manganese oxide {Mn(IV)} . Previous studies demonstrated that a 23.3-kb S . putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T . DiChristina and E . DeLong, J . Bacteriol . 176:1468-1474, 1994) . In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems . Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors . Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems . A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S . putrefaciens) MR-1 . A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates . Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction . These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S . putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).

J Food Prot, 2001 Nov, 64(11), 1836 - 40
Behavior of aeromonas hydrophila in bottled mineral waters; Croci L et al.; The growth and survival of Aeromonas hydrophila in three types of natural mineral waters were investigated . Mineral waters with different levels of mineral content (low, medium, and high) were experimentally contaminated with A . hydrophila, stored at different temperatures (10 degrees C and 20 degrees C), and analyzed at intervals over a 60-day period . Water samples that were not experimentally contaminated were investigated for indigenous A . hydrophila . The results confirmed that A . hydrophila may occur naturally in mineral waters and showed that the level of mineral content, temperature, length of storage, and, in some cases, the type of container used may favor the growth of A . hydrophila . The greatest proliferation was observed in water with a low mineral content stored in PET bottles at 10 degrees C, in which A . hydrophila peaked at day 28 (4.47 +/- 0.01 log CFU/100 ml) . At 20 degrees C, the same load was observed at day 60 . The presence of high densities of A . hydrophila in bottled mineral water can constitute a risk for some groups of consumers, such as elderly and immunocompromised persons.

Appl Environ Microbiol, 2001 Dec, 67(12), 5675 - 82
Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment; Schmidt AS et al.; A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A . S . Schmidt, M . S . Bruun, I . Dalsgaard, K . Pedersen, and J . L . Larsen, Appl . Environ . Microbiol . 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance . Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains) . Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates) . In addition, 23 isolates had "empty" integrons without inserted gene cassettes . As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE) . Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron . Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings . Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes . While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTC-resistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids . These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 50 - 5
Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); Chen GQ et al.; Large scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) {P(3HB-co-3HHx)} by Aeromonas hydrophila 4AK4 was examined in a 20,000 l fermentor . Cells were first grown using glucose as a carbon source, and polyhydroxyalkanoate (PHA) biosynthesis was triggered by the addition of lauric acid under conditions of limited nitrogen or phosphorus . When cells first grown in a medium containing 50 g glucose l(-1) were further cultivated after the addition of 50 g lauric acid l(-1) under phosphorus limitation, a final cell concentration, PHA concentration and PHA content of 50 g l(-1), 25 g l(-1), and 50 wt%, respectively, were obtained in 46 h, equivalent to PHA productivity of 0.54 g l(-1)t h(-1) . The copolymer produced was found to be a random copolymer, and the 3HHx fraction was 11 mol%.

Dev Comp Immunol, 2002 Jan, 26(1), 63 - 72
Antimicrobial peptide defenses against pathogens associated with global amphibian declines; Rollins-Smith LA et al.; Global declines of amphibian populations are a source of great concern . Several pathogens that can infect the skin have been implicated in the declines . The pathogen most frequently associated with recent die-offs is a chytrid fungus, Batrachochytrium dendrobatidis . A second fungus, Basidiobolus ranarum, was isolated from declining populations of Wyoming toads . A third pathogen, Aeromonas hydrophila, is an opportunistic bacterium found in healthy frogs, but capable of inducing disease . Among the immune defense mechanisms used by amphibians is the production of antimicrobial peptides in granular glands in the skin . These packets of natural antibiotics can be emptied onto the skin when the amphibian is injured . To determine whether antimicrobial skin peptides defend against these amphibian pathogens, six peptides (magainin I, magainin II, PGLa, CPF, ranalexin, and dermaseptin), from three species, and representing three structurally different families of peptides, were tested in growth inhibition assays . We show here that the peptides can kill or inhibit growth of both fungi but not Aeromonas . Although each peptide varied in its effectiveness, at least one from each species was effective against both fungi at a concentration of about 10-20 microM . This is the first direct evidence that antimicrobial peptides in the skin can operate as a first line of defense against the organisms associated with global amphibian declines . It suggests that this innate defense mechanism may play a role in preventing or limiting infection by these organisms.

Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2001, 12(1), 15 - 21
{Aeromonas species infection with severe clinical manifestation in okinawa, Japan -association with gas gangrene-}; Nakasone I et al.; We experienced two patients having Aeromonas species infection with severe clinical manifestations . The one patient was a 15-year-old high school girl student, who had been healthy in her school life, was admitted to the hospital with a sudden onset of left thigh muscle pain and swelling . She subsequently went into septic shock and died one day after admission . Pathological examination on autopsy revealed massive gas formation, skin bullas and ulcers, and extensive severe soft tissue damage throughout the body . Also, all the specimens, including brain, liver, spleen, thigh muscle, and blood in cardiac cavity, were positive for A . veronii biovar sobria . The other patient was 35-year-old man, who suffered from multiple bone fractures during the work in the harbor . One day after admission, he became febrile and went into septic shock . With the presumptive diagnosis of sepsis and gas gangrene, amputation of left thigh was performed . The exudate and aspirate of the amputated portion were repeatedly positive for A . hydrophila . Through the surveillance in Okinawa, Kagoshima, Miyazaki, and Kumamoto Prefectures, a total of 426 isolates from blood cultures were collected in the period from August, 1999 to February, 2000 . Of these, 14 isolates (3.3%) were the species of Aeromonas . Of 14 isolates of Aeromonas, 13 were reported from Okinawa and the remaining one was from Kumamoto . Most patients had underlying diseases, particularly liver diseases including liver cirrhosis . The mortality rate was extremely high at 62.5%, and the patients died in short terms after blood culture became positive . With these, Aeromonas species infection is unique to Okinawa, and positive blood culture for Aeromonas species potentially indicates a high-risk, particularly among the patients with underlying diseases.

Inorg Chem, 1998 Sep 7, 37(18), 4578 - 4583
Coordination Chemistry of the Amonabactins, Bis(catecholate) Siderophores from Aeromonas hydrophila(1); Telford JR et al.; The amonabactins are a series of four bis(catecholate) siderophores isolated from the pathogenic organism, Aeromonas hydrophila . As tetradentate ligands, they cannot singly satisfy the octahedral coordination sphere of iron . The solution coordination chemistry of the amonabactins has been elucidated using potentiometric and spectrophotometric titrations, circular dichroism, and mass spectroscopy . They form 2:3 metal:ligand complexes at high pH and excess ligand . Their complexation behavior is essentially identical to one another, with log beta(230) = 86.3 . At lower pH, they preferentially form a 1:1 bis(catecholato)bis(aqua) iron(III) species, with log beta(110) = 34.3 . The 2:3 complexes show a very slight Delta preference in chirality at the metal center, while the 1:1 complexes are achiral . The biological implications of these properties are discussed.

Scand J Infect Dis, 2001, 33(9), 718 - 9
Three cases of bacteraemia caused by Aeromonas veronii biovar sobria; Thomsen RN et al.; Septicaemia caused by Aeromonas species is a life-threatening condition, arising primarily in immunocompromised patients, which has rarely been reported in Scandinavia . Herein we describe 3 cases of Aeromonas sobria bacteraemia from Denmark . All the patients were male and all 3 cases occurred during the summer . Two patients had acute leukaemia and HIV infection, respectively, while the third patient had colorectal cancer diagnosed several years later . The clinical presentation in all patients was chest and/or abdominal pain with fever developing into sepsis without any known infectious focus . All patients responded well to antibiotic therapy.

Med Secoli, 1995, 7(3), 485 - 503
{Drinking water: use, consume and biological hazard}; Boccia A et al.; The Authors drew the attention to the rising danger of water crisis also in countries usually immune from this problem . The most important causes of these crisis are presented along with some of the most important laws concerning civil use of water . The Authors underline the biological risk induced by new aetiologycal agents (i.e . aeromonas hydrophyla, Criptococcus, Escherichia coli (O:127 H7) found in drinking water too . Some of the health problems correlated to the civil use of water are also presented: water supply of hospitals, schools and hotels . The Authors conclude underlining that the answer to this problem must be found in a good management of water sources which requires efficient monitoring systems.

Int J Food Microbiol, 2001 Sep 28, 69(3), 191 - 8
Growth and survival of clinical vs . environmental species of Aeromonas in tap water; Mary P et al.; The ability of four species of Aeromonas (two of clinical and two of environmental origin) to survive and/or grow in tap water microcosms supplemented with sodium thiosulphate was tested . After bottling, the autochthonous microflora reached 6 x 10(5) cfu ml(-1) after a 5-day incubation period in tap water unfiltered and which was non-autoclaved . In filtered tap water, "ultramicrocells" were detected and final populations of ca . 10(6) cfu ml(-1) after 7 days were obtained . Aeromonas was inoculated at an initial cell concentration of ca . 10(4) cfu ml(-1) . All strains were able to grow in tap water samples, which were filtered and autoclaved, and a final concentration of 10(5)-10(6) cfu ml(-1) was observed . Any inherent capability of Aeromonas to grow in tap water was eliminated by the presence of autochthonous microflora and "ultramicrocells" bacteria . Survival rates were strain- and microcosm-dependent . In unfiltered-non-autoclaved water, viable counts declined to below the detection limit (i.e . 1 log cfu ml(-1)) in 1.5 to 20 days . The declines in viable counts were even more pronounced in the filtered microcosm . Although inoculation ratios (100/1 in unfiltered-non-autoclaved and 1,000/1 in filtered microcosms) were favourable for aeromonads, at least for I to 3 days, the organisms disappeared in these microcosms . Thus, competition for nutrients was an unlikely cause of the limitation of aeromonads . The bacteriolytic effect of enzymes released by membrane vesicles from the autochthonous microflora and of "tail phage-like particles" bacteriocins were suggested as an in situ control of aeromonad populations . The present study showed that environmental strains of Aeromonas had no ecological advantage over clinical isolates . Thus, waterborne infections and contaminations of foods by pathogenic Aeromonas species could not be discounted.

Toxicon, 2001 Nov, 39(11), 1637 - 45
Not as simple as just punching a hole; Fivaz M et al.; Like a variety of other pathogenic bacteria, Aeromonas hydrophila secretes a pore-forming toxin that contribute to its virulence . The last decade has not only increased our knowledge about the structure of this toxin, called aerolysin, but has also shed light on how it interacts with its target cell and how the cell reacts to this stress . Whereas pore-forming toxins are generally thought to lead to brutal death by osmotic lysis of the cell, based on what is observed for erythrocytes, recent studies have started to reveal far more complicated pathways leading to death of nucleated mammalian cells.

Dis Aquat Organ, 2001 Aug 22, 46(1), 23 - 9
Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China?
Nielsen ME, Hoi L, Schmidt AS, Qian D, Shimada T, Shen JY, Larsen JL.
The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated . Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs . PCR and traditional biochemical methods were used for identification of A . hydrophila . Out of 69 motile aeromonads, 35 isolates were identified as A . hydrophila by biochemical tests . However, 6 of those were not identified as A . hydrophila by a species specific PCR method . Serotyping revealed 2 dominant serotypes (O9 and O97) among A . hydrophila isolates . The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A . hydrophila . It is noteworthy that A . hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia . Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp . were also found . The PCR assay was useful in preventing misidentification of A . hydrophila, which may occur when only phenotypic tests are employed.

J Bacteriol, 2001 Oct, 183(20), 5956 - 63
Type II secretion by Aeromonas salmonicida: evidence for two periplasmic pools of proaerolysin; Burr SE et al.; Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway . Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated . All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock . Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin . Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking . Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded . Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction . We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein . Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A . salmonicida.

Vet Immunol Immunopathol, 2001 Sep 28, 82(1-2), 121 - 35
Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA; Wagner U et al.; An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida . This new assay permits a specific determination of anti-protease-antibodies, without antigen purification . Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies . Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753) . Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease . A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective . Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity . The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.

East Mediterr Health J, 2000 Mar-May, 6(2-3), 497 - 9
An outbreak of acute gastroenteritis due to Aeromonas sobria in Benghazi, Libyan Arab Jamahiriya; Taher AA et al.; We report an outbreak of acute diarrhoea due to Aeromonas sobria in Benghazi which occurred during a 1-month period in 1997 . Of 69 patients admitted with acute gastroenteritis, 28 were positive for A . sobria based on the production of gas from glucose, the production of acetoin, hydrogen sulfide and lysine decarboxylase and on aesculin hydrolysis and fermentation of arabinose and salicin . The strains were sensitive to chloramphenicol, co-trimoxazole, tetracycline and gentamicin but resistant to ampicillin and carbenicillin . We were unable to trace the source of the infection.

Biochem Cell Biol, 2001, 79(4), 525 - 31
Induction of apoptosis in HT29 human intestinal epithelial cells by the cytotoxic enterotoxin of Aeromonas hydrophila; Falcon RM et al.; The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen . In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA . Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate . Cytoplasmic blebbing and nuclear condensation also occurred . DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis . These results show that the cytotoxic enterotoxin of A . hydrophila can induce apoptosis in human intestinal cells in culture.

Indian J Med Res, 2001 Mar, 113, 85 - 97
Typing of Aeromonas isolates from children with diarrhoea & water samples by randomly amplified polymorphic DNA polymerase chain reaction & whole cell protein fingerprinting; Alavandi SV et al.; BACKGROUND & OBJECTIVES: Aeromonas spp . are water-borne organisms, often associated with childhood diarrhoea . The present study was conducted to examine the epidemiological relationship among the Aeromonas spp . isolated from water and children with acute diarrhoea in Chennai . METHODS: Thirty six Aeromonas isolates inclusive of 16 from children with diarrhoea, 15 from domestic water samples and 5 reference strains were studied by randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) . Twenty eight Aeromonas isolates, 15 from children with diarrhoea, 10 from domestic water samples and three reference strains were analysed by SDS-PAGE for their whole cell protein profiles . RESULTS: The 36 Aeromonas isolates examined by RAPD-PCR generated RAPD fingerprints with majority of the bands ranging from about 250 to 2800 bp . The RAPD fingerprints did not correspond with the phenospecies and varied greatly among the strains within the phenospecies . Cluster analysis revealed two major groups at 75 per cent hierarchical level, comprising 18 Aeromonas isolates, mainly recovered from domestic water samples, while the clinical isolates were scattered in different hierarchical levels in the dendrogram . The whole cell protein fingerprints examined by SDS-PAGE did not correspond with the phenospecies . Only four isolates of A . caviae were found to produce similar protein fingerprints allowing them to form a cluster at about 90 per cent hierarchical level, while the rest of the isolates were scattered at various hierarchical levels in the dendrogram . INTERPRETATION & CONCLUSIONS: In the present study, RAPD fingerprinting was found to be useful in distinguishing Aeromonas isolates recovered from clinical and domestic water supplies . However, RAPD-PCR could not distinguish the phenospecies of the genus Aeromonas . Whole cell protein fingerprinting and cluster analysis could neither differentiate isolates from clinical and domestic water sources nor the phenospecies of the genus Aeromonas.

Syst Appl Microbiol, 2001 Jul, 24(2), 177 - 82
New DNA-DNA hybridization and phenotypic data on the species Aeromonas ichthiosmia and Aeromonas allosaccharophila: A . ichthiosmia Schubert et al . 1990 is a later synonym of A . veronii Hickman-Brenner et al . 1987; Huys G et al.; Previously, a DNA fingerprinting study based on Amplified Fragment Length Polymorphism (AFLP) analysis has revealed a possible genotypic resemblance of the species Aeromonas ichthiosmia and Aeromonas allosaccharophila to Aeromonas veronii (Huys et al., Int . J . Syst . Bacteriol . 46, 572-580 {19961) . Currently, two genotypically indistinguishable biovars are known to exist in the latter species, namely A . veronii biovar sobria and A . veronii biovar veronii . In the current study, new DNA-DNA hybridization experiments showed that the type strain of A . ichthiosmia, LMG 12645T (= DSM 6393T), and that of A . allosaccharophila, LMG 14059T (= CECT 4199T), were 84-96% and 78-82% related to A . veronii strain LMG 9075T (= ATCC 35624T), respectively . Based upon phenotypic characterization including a total of 151 tests, the type strain of A . ichthiosmia could be clearly allocated to A . veronii biovar sobria . On the other hand, the three strains constituting the species A . allosaccharophila were found to be phenotypically heterogeneous . None of these strains clearly fitted the biochemical description of either of the two A . veronii biovars or tightly clustered with any of the A . veronii reference strains . On the basis of published taxonomic evidence (including AFLP and phylogenetic data) and the newly reported results, there is compiling evidence to conclude that A . ichthiosmia Schubert et al . 1990 is a later synonym of A . veronii Hickman-Brenner et al . 1987 . However, due to the lack of agreement encountered between the new DNA reassociation results and previously reported DNA homology and phylogenetic data, a conclusive proposal on the genotypic position of A . allosaccharophila should await further studies.

Diagn Microbiol Infect Dis, 2001 Jul, 40(3), 91 - 4
Rapid iodometric detection of Aeromonas amylase and its diagnostic significance; Emele FE; Twenty-five Aeromonas hydrophila isolates from different sources (Food, 13; Clinical, 6 and Environmental, 6) were studied for the mode of production of Amylase and rapid iodometric detection of the enzyme in vitro . All twenty-five of the isolates produced the enzyme constitutively at 37 degrees C . Amylase producing ability was not dependent on the source of isolation of Aeromonas (F = 0.1069; p > 0.05) . Using iodometric technique, in a microtitration tray, the enzyme was fully demonstrated in 10(40%) of the isolates within 30 min, in 22(88%) within 60 min and in all (25 or 100%) within 90 min . The rapid detection of Aeromonas amylase will, no doubt, be of great value in routine diagnostic microbiology.

Vet Immunol Immunopathol, 2001 Aug 30, 81(1-2), 71 - 83
Induction of inflammatory cytokines by extracellular products and LPS of the fish pathogen Aeromonas salmonicida ssp . achromogenes in mice and mouse cell cultures; Gudmundsdottir S et al.; Balb/c mice, injected i.p . with extracellular products (ECP) of Aeromonas salmonicida subsp . achromogenes (Asa), displayed symptoms similar to toxic shock syndrome . The LD(50) observed was between 1.5 and 2.0 microg g(-1) and the mice died within 19 h . Four inflammatory cytokines were measured in mice receiving sublethal ECP doses . TNF-alpha and IL-6 showed a sharp peak in the serum while IL-1 beta and IL-2 were not detected . When peritoneal macrophages were cultivated in the presence of ECP, AsaP1 (a toxic caseinolytic metallo-protease purified from ECP) or LPS, all cultures produced TNF-alpha, IL-6 and IL-1 beta . The same antigens were mitogenic in spleen cell cultures . Furthermore, IL-2 production, which is a normal T-cell response to ConA stimulation, was downregulated in spleen cell cultures from mice injected with ECP.

Yakugaku Zasshi, 2001 Jul, 121(7), 481 - 5
{Maturation pathway of hemolysin of Aeromonas sobria and the mechanism of action of the hemolysin}; Nomura T; Aeromonas sobria has been recognized as pathogens associated with acute gastroenteritis in both adults and children . The major virulence factor has been proposed to be hemolysin which possesses both hemolytic and enterotoxic activities . Mature (bio-active) hemolysin secreted out of cells binds to the target cells of the host and injure the cells . However, hemolysin remained in bacteria can not express such toxicity . It means that the maturation and secretion pathway of hemolysin is closely related to the pathogenicity of bacteria . Therefore, I examined the pathway and clarified the following events . Hemolysin synthesized in cytoplasm translocates across the inner membrane and appears in a periplasmic space . Hemolysins appeared in the space associates to form dimer in the space . The C-terminal region of hemolysin functions as a trigger in the association . Dimerized hemolysin crosses the outer membrane and emerges in milieu, but monomer can not cross it . Therefore, the C-terminal region of hemolysin attributes not only to the formation of the dimer but also to its secretion into milieu . Hemolysin emerged in milieu is inactive . Inactive hemolysin is converted to bio-Active hemolysin by deleting its carboxyl-terminal 42-amino-acid peptide . Active hemolysin generated binds to the receptor of the target cell and stimulates the production of cyclic AMP by the cell . I assume that this stimulation closely relates to the induction of diarrhea by hemolysin.

Microbios, 2001, 106(413), 69 - 73
Enzymatic activity of micro-organisms isolated from cork wine stoppers; Centeno S et al.; The production of enzymes by micro-organisms which are found on vegetal substrates is important due to their ability to decompose cellulose, lignin and other components, which guarantee the integrity of the vegetal cell . The objective of this study was to determine the enzymatic activity of filamentous fungi, yeasts and bacteria, isolated from natural cork stoppers for bottles of still and sparkling wines . Suspensions of fungal conidia, yeasts and bacterial cells of micro-organisms were established in concentrations of 10(6) CFU/ml . The enzymatic activity of these micro-organisms was evaluated by means of the API ZYM system, with which it was possible to determine and semi-quantify nineteen enzymatic activities simultaneously . The enzymes produced by all of the species were esterase (C1), esterase lipase and naphthol-AS-BI-phosphohydrolase . The micro-organisms with the greatest enzymatic activity were Monilia sitophila, Alternaria alternata, Aspergillus niger and Aeromonas sp.

Proteins, 2001 Sep 1, 44(4), 490 - 504
Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism; Gilboa R et al.; Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues . It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions . The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products . Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies . These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate . The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution) . These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions . A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction . These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme . Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex . Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon . The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs . These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP .

J Chromatogr A, 2001 Jul 6, 921(2), 197 - 205
Functional polymer affinity matrix for purifying hexahistidine-tagged recombinant protein; Zeng Q et al.; A functional polyacrylic acid (PAA) adsorbent has been prepared for metal chelate affinity chromatography . It has been found to chelate nickel ion Ni2+ strongly, and was evaluated for the ability to bind proteins containing neighbouring histidine residues . The principle of the technique was illustrated with Aeromonas hydrophila outer membrane protein OmpTS . DNA elements coding for adjacent histidines were fused to the Aeromonas hydrophila ompTS gene . Subsequent expression in E . coli resulted in the production of hybrid protein His6-OmpTS that could be purified by Ni2+-PAA affinity chromatography . The remarkable specificity found makes it an attractive addition to the range of adsorbents for metal chelate affinity chromatography.

Crit Care Med, 2001 Jul, 29(7), 1445 - 51
Prostacyclin is neither sufficient alone nor necessary to cause pulmonary dysfunction: results from infusions of prostacyclin and antiprostacyclin antibody in porcine septic shock; Tran HS et al.; OBJECTIVE: This study evaluated whether prostacyclin is a necessary mediator of inflammation in graded bacteremia or is sufficient alone in pathophysiologic concentrations to cause the pulmonary derangement of bacteremic shock . DESIGN: Experimental . SETTING: Laboratory . SUBJECTS: Twenty-three anesthetized adult swine . INTERVENSIONS: Swine were studied in four groups for 4 hrs: a) an anesthesia control group (n = 6); b) a septic control group (n = 6), in which 1010/mL Aeromonas hydrophila was infused intravenously at 0.2 mL.kg-1.hr-1 and increased to 4.0 mL.kg-1.hr-1 over 3 hrs; c) a prostacyclin infusion group (n = 6), which received prostacyclin infusion to match septic control plasma concentrationsclm without bacteremia; and d) an antiprostacyclin antibody group (n = 5), which received continuous Aeromonas hydrophila infusion plus antiprostacyclin antibody infusion . MEASUREMENTS AND MAIN RESULTS: Pulmonary hemodynamics, arterial blood gases, and plasma concentrations of arachidonate metabolites were measured hourly over a 4-hr period . In the septic control group and antiprostacyclin antibody group, elevated pulmonary vascular resistance index and pulmonary artery pressure with decreased Pao2, as well as lower pH, were documented after 1 and 3 hrs of graded bacteremia compared with the anesthesia control group and prostacyclin infusion group (p <.05) . Thromboxane B2 concentration increased significantly in all groups during septic shock . In the antiprostacyclin antibody group, leukotriene B4 increased immediately after starting antiprostacyclin antibody infusion and reached significance at 3 hrs compared with the septic control group (p <.05) . The prostacyclin infusion group had consistently lower concentrations of leukotrienes C4, D4, and E4 than all other groups . CONCLUSIONS: Prostacyclin does not mediate blood gas changes, alterations of pulmonary hemodynamics, or platelet abnormalities in porcine septic shock, because antiprostacyclin antibody infusion did not change the pulmonary hypertension and hypoxemia, and infusion of prostacyclin to pathophysiologic blood concentrations did not reproduce such changes . Antiprostacyclin blockade during bacteremia significantly increased concentrations of leukotrienes C4, D4, and E4 and leukotriene B4, whereas prostacyclin infusion suppressed concentrations of leukotrienes C4, D4, and E4, suggesting that endogenous prostacyclin may blunt leukotriene release.

Am J Trop Med Hyg, 2001 Mar-Apr, 64(3-4), 159 - 61
Case report: recovery of Calliphora vicina first-instar larvae from a human traumatic wound associated with a progressive necrotizing bacterial infection; Delhaes L et al.; Human myiasis caused by Calliphora vicina is rare in Europe . Here we report a case of C . vicina infection occurring in the traumatic leg wound of a healthy 21-year-old man . Firstly, a progressive necrotizing infection developed in the wound despite administration of antibiotics . Aeromonas hydrophila was isolated from the wound samples . Secondly, during debridement, C . vicina first-instar larvae were isolated from the wound . To our knowledge, this is the first European case of C . vicina wound myiasis associated with severe A . hydrophila infection.

Eur J Biochem, 2001 Jul, 268(13), 3840 - 50
Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam; Zervosen A et al.; Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products . As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively . The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity . The mechanism of inactivation by moxalactam was studied in detail . Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site . Interestingly, this reaction is catalyzed by cacodylate.

FEMS Microbiol Lett, 2001 May 1, 198(2), 183 - 8
The cell division genes (ftsE and X) of Aeromonas hydrophila and their relationship with opsonophagocytosis; Merino S et al.; A transposon mutant from Aeromonas hydrophila AH-3 was obtained which was highly resistant to opsonophagocytosis . The mutation was identified in the ftsE gene and we characterised the operon ftsY, E and X from this bacterium . These genes, as in enteric bacteria, are neighbours to rpoH . The A . hydrophilia ftsE and X genes were fully able to complement Escherichia coli ftsE mutants, and also complement the opsonophagocytosis-resistant phenotype of the A . hydrophila mutant strain . This phenotype seems to be related to the filamentous phenotype at 37 degrees C exhibited by the A . hydrophila ftsE mutant.

Fish Shellfish Immunol, 2001 May, 11(4), 347 - 55
Effect of ascorbic acid on the immune response of the catfish, Mystus gulio (Hamilton), to different bacterins of Aeromonas hydrophila; Anbarasu K et al.; Ascorbic acid is one of the essential vitamins for normal physiological activities of any organism . The present study demonstrates an immunostimulatory effect of vitamin C on the humoral and cell mediated immunity of the bagrid catfish, Mystus gulio, determined using different bacterins of Aeromonas hydrophila . Humoral as well as cell mediated immune responses were elucidated in the vitamin supplemented, vaccinated and unvaccinated groups . The vitamin supplemented lipopolysaccharide (LPS) vaccinated group exhibited greater immune (both humoral and cell mediated) responses than its formalin killed (FK) and heat killed (HK) bacterin vaccinated counterparts . Nevertheless, in the challenge study, the relative percent survival (RPS) was found to be the same for both FK and LPS immunised vitamin treated groups while lower for the HK immunised vitamin treated group.

Fish Shellfish Immunol, 2001 May, 11(4), 281 - 91
Immunological parameters of Javanese carp Puntius gonionotus (Bleeker) exposed to copper and challenged with Aeromonas hydrophila; Shariff M et al.; This study was to determine the median lethal concentration (LC50) of copper to Javenese carp, Puntius gonionotus (Bleeker), and the immune response after the fish were exposed to sublethal levels of copper and challenged with formalin killed Aeromonas hydrophila . The LC50 of copper on P . gonionotus at 24, 48, 72, 96 and 120 h were estimated as 2.17, 0.91, 0.57, 0.53 and 0.42 mg l(-1), respectively . To determine the effect of copper on the immune system, fish were exposed for 66 days to 0.05, 0.10 and 0.15 mg Cu l(-1) . After 56 days of initial exposure to copper, fish were challenged with 0.1 ml of 4.5 x 10(5) cfu ml(-1) formalin killed A . hydrophila and maintained in the same concentration of copper . After the challenge, the immune response was monitored for 2 weeks using haematological and serological assays . During the initial phase of exposure to copper, significant changes were noted in the white blood cell, lysozyme, potential killing activity, total plasma protein, total immunoglobulin and haematocrit levels between the control and treated fish . One week after challenge with A . hydrophila, there was a significant increase in the values of white blood cells, total protein and total immunoglobulin compared to the values before the challenge . However, these values were not significantly different (P>0.05) between the control and the treated fish . In contrast, NBT and lysozyme assays exhibited a significant difference (P<0.05) in fish exposed to 0.10 mg Cu l(-1) (0.525 +/- 0.17; 24.42 +/- 3.35 x 10(2) micromg ml(-1)) and 0.15 mg Cu 1(-1) (0.536 +/- 0.19; 21.78 +/- 1.29 x 10(2) micromg ml(-1)) compared to the control (0.746 +/- 0.31; 30.73 +/- 5.42 x 10(2) micromg ml(-1)) after the bacterial challenge (day 61) . There was however no significant difference (P>0.05) in NBT and lysozyme levels in fish exposed to lower level of copper (0.05 mg Cu l(-1)), suggesting the absence of immunosuppressive effects at lower level of exposure.

Rev Argent Microbiol, 2001 Jan-Mar, 33(1), 47 - 51
{Beta-lactam antibiotic sensitivity in Aeromonas spp . of clinical, animal, and environmental origin}; Benassi FO et al.; Susceptibility to beta-lactam antibiotics was investigated in Aeromonas spp . Microorganisms were isolated from both, clinical and water creek samples, as well as from processed raw chicken carcasses . Aeromonas like colonies were identified by means of Aerokey II and API 20 E System (Bio-Merieux) . A . hydrophila prevailed both of human origin (44%) and water creek samples (41%), while A . caviae ranked first among raw chicken samples (65%) . Dilution testing by Agar Method was performed to determine minimum inhibitory concentration (MIC), following NCCLS standards . All tested microorganisms were susceptible to third generation cephalosporin, cefepime, imipenem, aztreonam, and resistant to ampicillin . Only with cefepime and aztreonam exceptions, strains of human origin showed higher values of MIC90 than environmental ones . These results suggest that antibiotic resistance is mainly due to a steady environmental pressure, on account of the widely used above mentioned compounds.

J Commun Dis, 2000 Sep, 32(3), 169 - 74
Occurrence of enterotoxigenic Aeromonas species in foods; Kumar A et al.; Out of a total of 246 food samples of animal origin screened for isolation of Aeromonas spp., 33 (13.41%) were positive for these organisms . Maximum positivity was shown by the samples from fish (28.57%), followed by poultry meat (16.67%), poultry eggs (12.50%), goat meat (12%), buffalo meat (7.69%) and cow milk (5.56%) . A . hydrophila was the predominant species (51.52%) followed by A . sobria (39.39%) and A . caviae . Of these, 70.59% A . hydrophila, 69.23% A . sobria and 33.33% A . caviae showed enterotoxigenic reaction in mouse paw oedema test.

Infect Immun, 2001 Jul, 69(7), 4257 - 67
Motility and the polar flagellum are required for Aeromonas caviae adherence to HEp-2 cells; Rabaan AA et al.; Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among the young . The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood . Initial investigations demonstrated that adherence of A . caviae to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella . To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the A . caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH, and flaJ . Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells . N-terminal amino acid sequencing, mutant analysis, and Western blotting demonstrated that A . caviae has a complex flagellum filament composed of two flagellin subunits encoded by flaA and flaB . The predicted molecular mass of both flagellins was approximately 31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 35,500 Da . This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays . Single mutations in either flaA or flaB did not result in loss of flagella but did result in decreased motility and adherence by approximately 50% . Mutation of flaH, flaJ, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence . However, mutation of flaG did not affect motility but did significantly reduce the level of adherence . Centrifugation of the flagellate mutants (flaA, flaB, and flaG) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (flaH, flaJ, and flaA flaB) increased adherence slightly . We conclude that maximum adherence of A . caviae to human epithelial cells in vitro requires motility and optimal flagellar function.

Biochemistry, 2001 Jun 19, 40(24), 7035 - 46
Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid . Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis; Stamper C et al.; The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated . LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM . Electronic absorption spectra, recorded at pH 7.5 of {CoCo(AAP)}, {CoZn(AAP)}, and {ZnCo(AAP)} upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site . EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both {CoZn(AAP)} and {ZnCo(AAP)} become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions . The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution . The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent . Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine . A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases . On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.

Indian J Exp Biol, 2000 Nov, 38(11), 1143 - 6
L-asparaginase activity in Aeromonas sp . isolated from freshwater mussel; Pattnaik S et al.; Aeromonas sp . from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C . The optimum enzyme activity was at pH 9 when temperature was 45 degrees C . Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively . Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively . The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1 . Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations . Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.

J Antimicrob Chemother, 2001 Jun, 47(6), 735 - 43
Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas; Schmidt AS et al.; Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America . Three different dhfr genes, conferring trimethoprim resistance, and one ant(3")1a aminoglycoside resistance gene were identified as gene inserts . The gene cassettes tended to be conserved among isolates from a particular geographical area . Nineteen isolates transferred R-plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred . Transferable sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons . It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids.

Singapore Med J, 2001 Feb, 42(2), 057 - 60
Retrospective study of Aeromonas infection in a Malaysian urban area: a 10-year experience; Lee WS et al.; AIMS: To describe the patterns of isolation of Aeromonas spp . and the resulting spectrum of infection, intestinal and extra-intestinal,from infants and children in an urban area in a hot and humid country from SoutheastAsia . MATERIALS AND METHODS: Retrospective review of all bacterial culture records from children below 16 years of age, from the Department of Medical Microbiology, University of Malaya Medical Centre, Kuala Lumpur, from January 1988 to December 1997 . Review of all stool samples and rectal swabs obtained from children during the same period were carried out to ascertain the isolation rate of Aeromonas sp . from stools and rectal swabs . The case records of those with a positive Aeromonas culture were retrieved and reviewed . RESULTS: During the study period, 84 culture samples were positive of Aeromonas spp . (stools 48, rectal swabs 36) . During the same period, 1,352 stool samples were positive for at least one enteropathogen . Aeromonas spp . constituted 0.62% of all stool samples . Of the 61 cases reviewed,four patterns of colonization were observed: (a) 17 cases of mostly asymptomatic nursery newborns with a positive rectal swab; (b) 9 children with no diarrhoea; (c) 23 cases, of who seven were immunocompromised, had acute, brief watery diarrhoea without severe dehydration or disturbances of serum electrolytes . No chronic diarrhoea or bacteraemia was noted . (d) 12 cases had a mixed infection with a second enteropathogen isolated from stool samples . Three had chronic diarrhoea No extra-intestinal infection attributed to Aeromonas spp . was identified in this study . CONCLUSION: Aeromonas was a rare cause of gastroenteritis in urban Malaysian children . It was isolated almost exclusively from gastro-intestinal tract, caused mostly by mild gastroenteritis with no serious complications . Asymptomatic stool carriage among newborns admitted to special care nursery and older children with no diarrhoea were observed.

Arch Microbiol, 2001 Mar, 175(3), 220 - 5
G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues; Lin FP et al.; A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin . The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A . caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system . The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE . The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C) . Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity . However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose {4-MU-(GlcNAc)3} as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates . Results obtained from this study suggest that the active region of A . caviae ChiA is located in the region before G561 of the protein molecule.

Antimicrob Agents Chemother, 2001 Jun, 45(6), 1868 - 71
CENTA as a chromogenic substrate for studying beta-lactamases; Bebrone C et al.; CENTA, a chromogenic cephalosporin, is readily hydrolyzed by beta-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme . Although it cannot practically be used for the detection of beta-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Biotechnol Bioeng, 2001 Jul 5, 74(1), 81 - 6
Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains; Park SJ et al.; The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) {P(3HB-co-3HV-co-3HHx)} from dodecanoic acid plus odd carbon number fatty acid . In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer .

J Appl Microbiol, 2001 May, 90(5), 797 - 802
The occurrence of aerolysin-positive Aeromonas spp . and their cytotoxicity in Norwegian water sources; Ormen O et al.; AIMS: The aim of the study was to investigate the occurrence of Aeromonas spp . and their aerolysin status in Norwegian natural water sources . METHODS AND RESULTS: Seventy-one samples from 33 Norwegian water sources were examined for the presence of Aeromonas spp . From most of the sample sites, the strains were isolated on blood-ampicillin-agar and Difco Aeromonas agar simultaneously . The majority of the samples (73/77) contained Aeromonas spp., with an average of 35-100 cfu 100 ml(-1) . The highest counts were found in faecally-contaminated water . Using PCR, 445 isolates were screened for the presence of aerolysin, and 79% of them were found to be carriers of the aerolysin gene . A selection of the isolated strains was tested on Vero cell cultures and 83% of them showed cytotoxicity . CONCLUSION: There is widespread occurrence of aerolysin-positive cytotoxic Aeromonas spp . in many different Norwegian natural waters, including drinking water sources . SIGNIFICANCE AND IMPACT OF THE STUDY: The widespread occurrence of potentially-pathogenic Aeromonas spp . in the environment demands that these bacteria should not be ignored in drinking water supplies and in the food industry.

J Food Prot, 2001 May, 64(5), 687 - 91
Mesophilic aeromonads in wild and aquacultured freshwater fish; Gonzalez CJ et al.; Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish specimens . Although no significant differences were observed in water samples, initial levels for skin, gill, and intestines were significantly lower in farmed rainbow trout (Oncorhynchus mykiss) than in wild brown trout (Salmo trutta) and pike (Esox lucius) . During storage of wild specimens, naturally occurring aeromonads grew fairly well on the surfaces of skin and body cavity . Of 171 strains assigned to the genus Aeromonas, 88% were identified to phenospecies and putative genospecies level by using comprehensive biochemical schemes . The isolates were allocated to putative hybridization groups (HGs) 1 and 3 Aeromonas hydrophila (29%); putative HG 8 Aeromonas veronii biovar sobria (19%); putative HG 2 Aeromonas bestiarum (18%); putative HG 9 Aeromonas jandaei (16%); putative HGs 4 and 5a Aeromonas caviae (2%); putative HG 12 Aeromonas schubertii (2%); and putative HG 11 (unnamed, 0.6%) . The remaining 20 isolates (12%) resembled A . schubertii but could not be allocated to currently recognized phenospecies or to putative HGs . Although cultured rainbow trout yielded strains of putative HGs 1, 4, and 8, which appear to be of major clinical importance, most isolates assigned to putative HGs 1 and 8 were recovered from pike . Differences among HGs found in wild animals could be related to their origin (unpolluted rivers for brown trout and urban rivers for pike) . The recovery of these aeromonads species was not related to sampling site . The initial levels of motile aeromonads, their behavior during storage, and the strong potential spoilage activity of most isolates confirm that these bacteria can contribute to deterioration of iced wild freshwater fish . Although adequate cooking would inactivate motile aeromonads, the high incidence of isolates belonging to gastroenteritis-associated HGs should be regarded as a potential health concern, particularly for susceptible populations when there is a possibility of cross-contamination.

Enferm Infecc Microbiol Clin, 2001 Apr, 19(4), 161 - 4
{Aeromonas spp bacteremia: study of 12 cases and review of the literature}; Campo C et al.; Twelve cases of Aeromonas spp . bacteremia are here reviewed in adult patients occurred at our institution during a 6-year period . Three cases corresponded to patients with hematological disease and four had a solid neoplasm . The source of infection in seven patients was extra-nosocomial; infections in the five remaining patients were considered to be acquired in the hospital . In seven patients, potential portals of entry were found . The usual clinical presentation was febrile syndrome in all cases and only in two patients did the clinical picture evolve to fulminant septic shock . Speciation of microorganisms was determined in only four cases: 2 A . hydrophilia, 1 A . caviae, and 1 A . veronii . Most isolates were susceptible to aminoglycosides, cotrimozazol, phosphomycin, and quinolonos, and resistant to ampicillin . Three patients (25%) died as a result of the infection . Aeromonas spp . bacteremia represented 0.12% of blood cultures in our hospital and occurs in immunosuppressed patients although it may be reported in previously healthy individuals.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 487 - 94
Cloning, expression, and characterization of a family 52 beta-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1; Suzuki T et al.; A lambda phage genomic library of Aeromonas caviae ME-1, a multiple-xylanase-producing bacterium, was screened for xylan degradation activities . We isolated one clone, B65, which had weak xylanase activity, by the DNS method, but gave no visible bands on zymogram assay using SDS-xylan-PAGE . Based on TLC analyses of enzymatic products and some glycosidase assays using p-nitrophenyl substrates, we established that pB65 encodes a beta-xylosidase gene . In the nucleotide sequence analysis, we found a 2190-bp open reading frame (ORF) named xysB . XysB protein is similar to some beta-xylosidases, which are categorized in the glycosyl hydrolase family 52 . Another ORF (xyg), that showed similarity to the family 67 alpha-glucuronidase, was also found downstream of the xysB gene . The xysB ORF and its promoter region were cloned into the pT7-Blue vector and the transformant cells had beta-xylosidase activity . The relative molecular mass were estimated to be 75 kDa by SDS-PAGE and 159 kDa by gel filtration . These data showed that XysB has a dimeric structure of 80,697 Da subunits . This enzyme showed optimal activity at 50 degrees C and pH 6.0 . It was stable below 40 degrees C and pH 5-8 . The Km and Vmax were calculated to be 0.34 mM and 33 nmol x min(-1) x microg(-1), respectively . This enzyme also showed transglycosylation activity against X3 and produced X4 and X5.

Dis Aquat Organ, 2001 Mar 9, 44(2), 109 - 20
Histopathology of viremia-associated ana-aki-byo in combination with Aeromonas hydrophila in color carp Cyprinus carpio in Japan; Miyazaki T et al.; A disease in which 'viremia-associated ana-aki-byo' is combined with an Aeromonas hydrophila infection currently occurs and is highly transmissible in color carp Cyprinus carpio in Japan . In the present study, to determine the interrelation between a corona-like virus and A . hydrophila, we conducted transmission trials by cohabiting naturally diseased carp with healthy carp with skin that had been slightly damaged artificially . Experimentally exposed fish successfully replicated the combination of a corona-like viral viremia and A . hydrophila infection . Diseased carp displayed scale-sac edema, ascites and exophthalmus adding to the formation of skin ulcers . In addition to pathological changes due to the corona-like virus infection, various changes due to the A . hydrophila infection occurred . Anasarcous skin lesions exhibited a separated epidermis, expanded scale-sacs, and an edematous dermis accompanied by hemorrhage and necrosis . The underlying lateral musculature was edematous and possessed markedly atrophied muscle fibers . Hepatocytes were either atrophied or swollen and had a granular appearance . Renal tubular cells showed vacuolar degeneration, cloudy swelling, necrosis and destruction . Hemosiderin deposition occurred within macrophages in the spleen and hematopoietic tissue, and within hepatocytes . Cardiac muscle fibers exhibited degeneration and necrosis accompanied by hemorrhage in the myocardium of heart . These changes appeared to be induced by bacterial toxins because bacterial cells did not directly invade these affected tissues.

FEMS Microbiol Ecol, 2001 May, 35(3), 295 - 304
Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot); Edwards ML et al.; The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m) . Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis . The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February . The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index . All three indices detected a number of significant differences with depth demonstrating stratification . The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity . Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure . Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera . Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates . Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Fish Shellfish Immunol, 2001 Feb, 11(2), 127 - 42
A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes; Kollner B et al.; The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described . The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6) . No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected . In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained . The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma . In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26 . However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A . salmonicida particles . Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A . salmonicida . The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.

Fish Shellfish Immunol, 2001 Feb, 11(2), 115 - 26
Mucosal immune response of spotted sand bass Paralabrax maculatofasciatus (Steindachner, 1868) orally immunised with an extracellular lectin of Aeromonas veronii; Merino-Contreras ML et al.; To assess the immunogenic and immunoprotective role of the extracellular lectin from Aeromonas veronii (MCBP), which has affinity for mucosal constituents such as mucin, lactoferrin, immunoglobulins and collagen, spotted sand bass (Paralabrax maculatofasciatus) were orally immunised either with soluble MCBP, adjuvant-conjugated MCBP or immobilised MCBP on latex microspheres . The results suggest that the MCBP is capable of eliciting protective immunity against A . veronii infections when administered orally . The highest mucosal immune response was elicited in fish immunised with MCBP covalently linked to cholera toxin B subunit (CTB) or to Escherichia coli heat-labile toxin (hLT) . MCBP-CTB was found to elicit immunoprotection against a challenge with live Aeromonas cells with a relative percent survival of almost 70% and without the expression of the severe histopathological alterations induced by A . veronii.

Fish Shellfish Immunol, 2001 Feb, 11(2), 101 - 13
Roles of an endogenous serum lectin in the immune protection of blue gourami, Trichogaster trichopterus (Pallus) against Aeromonas hydrophila; Fock WL et al.; The serum of blue gourami, Trichogaster trichopterus (Pallus), contains a calcium-dependent, N-acetyl-galactosamine-binding lectin (BGL) which efficiently activates and enhances the non-specific immune response of fish towards a virulent strain of Aeromonas hydrophila . In the in vitro studies, a lectin concentration range of 0.05-1.0 ng ml(-1) was found to significantly promote phagocytic uptake of the bacteria by macrophages . This effect was further augmented when purified lectin was combined with laminarin (beta-1,3-D-glucan) . Supernatants obtained from these lectin-stimulated macrophage cultures also exhibited significant bacteria-killing activities . In addition, complement from naive fish serum, in the presence of purified BGL, was able to kill A . hydrophila . Finally, challenge experiments demonstrated that BGL could confer effective immune protection to naive blue gourami against an Aeromonas infection.

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 420 - 3
Characterization of the pro-aminopeptidase from Aeromonas caviae T-64; Zhang ZZ et al.; The pro-aminopeptidase from Aeromonas caviae T-64 (pro-apAC) had maximal activity at 60 degrees C and was more stable than mature apAC at temperature up to 65 degrees C for 1 hour . The pH stability of pro-apAC ranged from 4.0 to 8.0, which is broader than the range for the mature apAC . The kcat/Km of pro-apAC was 1.4% to 24% of that of mature apAC.

Intern Med, 2001 Feb, 40(2), 118 - 23
Fulminant pneumonia due to Aeromonas hydrophila in a man with chronic renal failure and liver cirrhosis; Murata H et al.; A 40-year-old man on hemodialysis was admitted due to dyspnea and chest pain and was diagnosed with pneumonia and pericarditis . Ampicillin was administered, but thereafter severe septic shock developed . The fulminant type of pneumonia progressed rapidly, and he died only 48 hours after the onset of symptoms . The autopsy and sputa culture revealed pneumonia due to Aeromonas hydrophila . The source of this infection remained unkown . Interestingly, there were two types of A . hydrophila found during such a short period . The physician should suspect this disease by questioning the patient's history . Early treatment with adequate antibiotics is the only means of saving such a patient's life.

Proc R Soc Lond B Biol Sci, 2001 Mar 7, 268(1466), 479 - 85
Association between major histocompatibility complex class IIB alleles and resistance to Aeromonas salmonicida in Atlantic salmon; Langefors A et al.; We have tested the importance of genetic variation in the major histocompatibility complex (MHC) class IIB in Atlantic salmon (Salmo salar) for survival after challenge with a highly virulent bacterial pathogen . Forty juvenile full siblings from each of 120 families were infected with the bacterium Aeromonas salmonicida, which causes high mortality in salmon due to furunculosis . Fishes from high-resistance (HR, < 35% mortality) and low-resistance (L,R, > 80% mortality) families were screened for their MHC class IIB genotypes using the denaturing gradient gel electrophoresis (DGGE) technique . The exon 2 sequences, encoding the major part of the peptide-binding region, were established for each DGGE fragment . One allele, e, containing a missense single base substitution was significantly more prevalent in HR families than in LR families . An odds-ratio test showed that broods carrying this allele had a 12-fold higher chance of being HR than broods without the e allele . A second allele, i, showed significantly higher frequencies in uninfected and surviving individuals than in infected dead individuals . A third allele, j, tended to more prevalent both in LR families and in individuals that had died of the infection . There was no correlation between MHC heterozygosity and resistance to A . salmonicida . Our results support the hypothesis that MHC polymorphism is maintained through pathogen-driven selection acting by means of frequency-dependent selection rather than heterozygous advantage.

J Med Microbiol, 2001 Apr, 50(4), 313 - 9
Identification of a 43-kDa outer-membrane protein as an adhesin in Aeromonas caviae; Rocha-De-Souza CM et al.; Aeromonas spp . are associated with intestinal and extra-intestinal infections . However, the virulence factors of A . caviae remain, for the most part, poorly known . This study examined the interactions involved in the adherence of A . caviae isolates Ae56, Ae391 and Ae398 to HEp-2 cells . All strains expressed high levels of aggregative adherence . Maximum adhesion occurred with bacteria grown at 22 degrees C, but transmission electron microscopy did not reveal the presence of fimbrial structures on the bacterial cell surface . Outer-membrane proteins (OMPs) extracted from isolate Ae398, grown at 22 degrees C and 37 degrees C, showed similar SDS-PAGE protein profiles . Most proteins were < 60 kDa . A major 43-kDa protein was seen only in the boiled OMP extract . The biotinylated 43-kDa protein bound specifically to HEp-2 cells . Microbeads coated with the 43-kDa protein were also adherent to HEp-2 cells, and anti-43-kDa protein antibody blocked adherence of 43-kDa protein-coated latex beads . These data suggest that the 43-kDa OMP functions as an adhesin in A . caviae.

J Med Microbiol, 2001 Apr, 50(4), 303 - 12
Enhancement of the virulence of Aeromonas caviae diarrhoeal strains by serial passages in mice; Krzyminska S et al.; Thirteen clinical Aeromonas caviae isolates from the faeces of 13 children with mild to severe diarrhoea were tested for enhancement of mouse lethality, adhesion ability, siderophore and cholera toxin cross-reactive (CTC) factor production, by four consecutive passages through mice by intraperitoneal injection of A . caviae suspensions . The passaged A . caviae strains were re-isolated from monomicrobic cardiac blood samples and inocula were prepared for the next passage . All A . caviae isolates possessed the ability to adhere to the mucosal epithelial surface of the rabbit small intestine . Serial passage in mice showed that the virulence of some isolates for mice was increased in terms of percentage mortality and a lowering of the LD50 . For some of the isolates, but not all, serial passage appeared to increase siderophore production and adhesion to rabbit small intestinal cells . For the A . caviae isolates tested, increased values of the CTC factor were observed after passage . A clear correlation was observed between the lowering of LD50 and the enhancement of CTC factor production after passage in mice . These results indicate that the A . caviae isolates possessed virulence factors.

Mem Inst Oswaldo Cruz, 2001 Feb, 96(2), 169 - 73
Prevalence, species differentiation, haemolytic activity, and antibiotic susceptibility of aeromonads in untreated well water; Ghenghesh KS et al.; The use of untreated water for drinking and other activities have been associated with intestinal and extraintestinal infections in humans due to Aeromonas species . In the present study aeromonads were isolated from 48.7% of 1,000 water samples obtained from wells and other miscellaneous sources . Aeromonas species were detected in 45% of samples tested in spring, 34.5% in summer, 48% in autumn and 60% of samples tested in winter . Speciation of 382 strains resulted in 225 (59%) being A . hydrophila, 103 (27%) A . caviae, 42 (11%) A . sobria and 11 (3%) atypical aeromonads . Of 171 Aeromonas strains tested for their haemolytic activity, 53%, 49%, 40% and 37% were positive in this assay using human, horse, sheep and camel erythrocytes respectively . The results obtained indicate that potentially enteropathogenic Aeromonas species are commonly present in untreated drinking water obtained from wells in Libya (this may also apply to other neighbouring countries) which may pose a health problem to users of such water supplies . In addition, ceftriaxone and ciprofloxacin are suitable drugs that can be used in the treatment of Aeromonas-associated infections, particularly in the immunocompromised, resulting from contact with untreated sources of water.

Int J Hyg Environ Health, 2001 Mar, 203(3), 281 - 7
Improved method for the fluorimetric detection of beta-D-galactosidase in water; Hattenberger M et al.; A very convenient method to quantify coliform bacteria in water can probably be designed via the determination of the activity of the enzyme beta-D-galactosidase, whose natural occurrence is, apart from less frequently occurring aeromonads mainly restricted to this type of microorganisms . 4-methylumbelliferyl-beta-D-galactoside is used as substrate, which is hydrolyzed during the enzymatic reaction; the released 4-methylumbelliferone can be quantified fluorimetrically . In the present study the influence of various physical and chemical parameters on the determination is investigated and the experimental conditions are optimized . Most important entities are the pH value during hydrolysis, the presence of nutrients and co-factors in the sample, and the modification of the substrate . Statistical evaluation of the results obtained by changing single or multiple parameters reflects clearly their positive or negative influence on the enzyme activity . Thus, deliberate addition of surfactants, specific nutrients, salts and co-enzymes results in a significantly increased activity of beta-D-galactosidase towards the substrate, which can be advantageously exploited to increase the sensitivity of the analytical method together with a decrease of the detection limit . The influence of the parameters and the optimized conditions of the improved analytical methods are presented.

J Food Prot, 2001 Feb, 64(2), 195 - 200
Antibacterial effects of different food-related phosphates using Aeromonas hydrophila; Velazquez LD et al.; Aeromonas hydrophila is considered to be an emergent food-related bacterium . Phosphates are used as additives, mainly in meat products, to improve the quality of these foods . The antibacterial properties of phosphates are also well known . In this work, two A . hydrophila strains in early exponential phase were used: (A) A . hydrophila ATCC 7965 and (B) A . hydrophila derived from food, isolated in our laboratory . MIC and MBC studies were performed to assess the antibacterial effects of four phosphates assayed in brain heart infusion broth (BHI) and modified complete defined synthetic medium (mCDS) as compared to cooked ground meat medium (CM) . The MBC values of the phosphates in CM were significantly higher than MIC values in BHI broth and mCDS medium (P < 0.05) . In the two latter media, the growth of both A . hydrophila strains was totally inhibited by concentrations between 0.5 and 3.0% . Although all the assayed phosphates proved to have bactericidal effects on A . hydrophila, 0.5% sodium acid pyrophosphate (SAPP) exhibited greater effects in both strains and was selected for subsequent experiments . The bacteriolytic effect of SAPP was spectrophotometrically determined (260 nm of absorbance) by means of the leakage of intracellular nucleotides and microscopically confirmed by the presence of massive gelatinous aggregates . These were identified by enzymes (RNase, DNase, and proteinase) that hydrolyzed the nucleotides and proteins released during cellular lysis in the presence of SAPP . It was concluded that 0.5% SAPP can have bactericidal and bacteriolytic effects in early exponential phase A . hydrophila cells.

J Microbiol Immunol Infect, 2000 Dec, 33(4), 241 - 7
Outcomes of Aeromonas bacteremia in patients with different types of underlying disease; Lau SM et al.; Over a 6-year period, 42 patients with different underlying diseases developed Aeromonas bacteremia in our hospital . The male to female ratio was 2:1 . The vast majority of these patients had underlying diseases, including various types of neoplasm (n = 14), liver cirrhosis (n = 11), biliary tract disorder (n = 10) and other illnesses (n = 7) . Community-acquired bacteremia was predominant (33 cases, 79%) . Aeromonas hydrophila was the most common species isolated (88%) . Monomicrobial bacteremia was more common than polymicrobial bacteremia (64% vs 36%) . Monomicrobial bacteremia was associated with neoplasm or liver cirrhosis in 80% of patients . Polymicrobial bacteremia was more common in patients with biliary tract disorder than in patients from other groups (60% vs 40%) . Escherichia coli (60%) was the predominant concomitant organism isolated . The major clinical manifestations were fever (74%), jaundice (57%), and abdominal pain (45%) . Recognized infection sites included biliary tract, soft tissue involvement, peritoneal involvement, while 50% of patients had no recognized infection site . Eight patients (80%) received cholecystectomy due to gall stone with acute cholecystitis . However, none of the cirrhotic patients with necrotizing fasciitis received surgical treatment . The mortality attributed to Aeromonas bacteremia was 70% . Patients with liver cirrhosis or malignancy had a higher acute mortality (death within 7 days after admission) than the other patients (89% vs 11%) . We conclude that Aeromonas bacteremia can cause a rapidly fatal outcome and should be considered an important pathogen for septicemia in patients with liver cirrhosis or neoplasm.

Antimicrob Agents Chemother, 2001 Apr, 45(4), 1281 - 3
In vitro and in vivo combinations of cefotaxime and minocycline against Aeromonas hydrophila; Ko WC et al.; The activities of cefotaxime and minocycline against Aeromonas hydrophila were investigated . Cefotaxime (4 times the MIC) plus minocycline (0.75 times the MIC) elicited an inhibitory effect for 48 h in a time-kill study, and more infected mice treated with both drugs survived (91%) than survived after treatment with cefotaxime (9%) or minocycline (44%) alone, suggesting that cefotaxime and minocycline act synergistically against A . hydrophila.

Intern Med, 2000 Dec, 39(12), 1128 - 30
Rapidly progressive pneumonia due to Aeromonas hydrophila shortly after near-drowning; Miyake M et al.; An 87-year-old woman died of rapidly progressive pneumonia due to Aeromonas hydrophila shortly after a near-drowning event . Autopsy showed necrotizing pneumonia and postmortem cultures of both blood and lung revealed the organism . Fulminant pneumonia should be considered in patients of a near-drowning event.

Antimicrob Agents Chemother, 2001 Mar, 45(3), 837 - 44
Metallo-beta-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum; Rossolini GM et al.; Eleven environmental samples from different sources were screened for the presence of metallo-beta-lactamase-producing bacteria by using a selective enrichment medium containing a carbapenem antibiotic and subsequently testing each isolate for production of EDTA-inhibitable carbapenemase activity . A total of 15 metallo-beta-lactamase-producing isolates, including 10 Stenotrophomonas maltophilia isolates, 3 Chryseobacterium spp., one Aeromonas hydrophila isolate, and one Janthinobacterium lividum isolate (a species in which production of metallo-beta-lactamase activity was not previously reported), were obtained from 8 samples . In the J . lividum isolate, named JAC1, production of metallo-beta-lactamase activity was elicited upon exposure to beta-lactams . Screening of a JAC1 genomic library for clones showing a reduced imipenem susceptibility led to the isolation of a metallo-beta-lactamase determinant encoding a new member (named THIN-B) of the highly divergent subclass B3 lineage of metallo-beta-lactamases . THIN-B is most closely related (35.6% identical residues) to the L1 enzyme of S . maltophilia and more distantly related to the FEZ-1 enzyme of Legionella gormanii (27.8% identity) and to the GOB-1 enzyme of Chryseobacterium meningosepticum (24.2% identity) . Sequences related to bla(THIN-B), and inducible production of metallo-beta-lactamase activity, were also detected in the J . lividum type strain DSM1522 . Expression of the bla(THIN-B) gene in Escherichia coli resulted in decreased susceptibility to several beta-lactams, including penicillins, cephalosporins (including cephamycins and oxyimino cephalosporins), and carbapenems, revealing a broad substrate specificity of the enzyme . The results of this study indicated that metallo-beta-lactamase-producing bacteria are widespread in the environment and identified a new molecular class B enzyme in the environmental species J . lividum.

ALTEX, 1998, 15(5), 79 - 82
{Development of an ELISA-system for the assessment of furunculosis vaccines}; Wagner U et al.; As an alternative method for challenge experiments in fish a sandwich-ELISA has been established for the detection of potentially protective antibody populations . Assay specificity depends on monoclonal antibodies (mabs) directed against different antigens of Aeromonas salmonicida, the causative agent of furunculosis . Comparable levels of salmon antibodies against LPS and the A-layer protein were recorded by ELISA and antigen binding assay . These studies, together with the first analysis of trout sera refer to the specificity and suitability of the ELISA-system for monitoring of individual antibody populations . A study of different mabs against the A-layer protein points to the possibility for detecting at least partially different antibody populations against this virulence factor.

J Appl Microbiol, 2001 Feb, 90(2), 190 - 200
Miniaturized tests for computer-assisted identification of motile Aeromonas species with an improved probability matrix; Carson J et al.; AIMS: To develop miniaturized tests for the phenotypic identification of motile Aeromonas species using an improved probability matrix . METHODS AND RESULTS: Conventional tests were miniaturized for use in 96-well plates, and their performance assessed using 60 aeromonads comprising type and reference strains as well as clinical, fish and water isolates . A revised probability matrix for Aeromonas hybridization groups 1-14, including A . allosaccharophila, A . bestiarum, A . encheleia and A . popoffii, was developed . Using 26 tests, all the reference strains were correctly identified with the revised probability matrix, and 80% of the isolates were correctly identified at a Willcox probability level of 95% . CONCLUSION: The compact test format, coupled with a robust identification matrix, provides a convenient basis for identifying motile aeromonads . SIGNIFICANCE AND IMPACT OF THE STUDY: The identification system for identifying aeromonads will be of use to medical and veterinary laboratories undertaking disease diagnosis.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 143 - 7
Locomotion and feeding of Acanthamoeba at the water-air interface of ponds; Preston TM et al.; Acanthamoeba trophozoites attach to and effect amoeboid locomotion at the water-air interface of ponds . Their locomotory rate (approximately 0.8 microm s(-1)) and manner of independent movement at this interface is similar to that over solid substrata . Adhesion forces developed between amoebae and the water-air interface are greater than gravity and thus amoebae are also transported passively without detachment . Amoebae docked with the water-air interface remain and flourish here as they are shown, by using green fluorescent protein-labelled Aeromonas hydrophila, to feed on bacteria that occur at the interface, digesting them intracellularly.

Trends Microbiol, 2000 Apr, 8(4), 168 - 72
Adventures of a pore-forming toxin at the target cell surface; Abrami L et al.; The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation . This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.

Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2069 - 73
Extended method for discrimination of Aeromonas spp . by 16S rDNA RFLP analysis; Figueras MJ et al.; A previously described molecular method, based on 16S rDNA RFLP analysis, for the identification of Aeromonas spp . was unable to separate the species Aeromonas salmonicida, Aeromonas bestiarum and the recently described Aeromonas popoffii . In this study, the method has been extended with endonucleases AIwNI and PstI for the identification of these species . A molecular frame for the identification of all known Aeromonas spp . is presented.

Prev Vet Med, 2001 Jan 29, 48(2), 129 - 41
Hatchery-level predictive values for infectious pancreatic necrosis virus and Aeromonas salmonicida in Ontario, Canada; Nathalie Bruneau N et al.; The probability and uncertainty of correctly classifying the IPNV and Aeromonas salmonicida status of fish-rearing and natural sites in Ontario were estimated through Monte Carlo simulations . Propagating several uncertain inputs showed the extent to which natural variability and our present lack of knowledge affect the probability of site misclassification . For the scenarios investigated, the site-level negative predictive values (SNPVs) were high and fairly constant . The site-level positive predictive values (SPPVs) - given a test specificity ranging between 0.999 and 1.0 - were much lower, more variable, and highly affected by cut-off point and sample size . Substantial uncertainty resides in classifying the pathogen status of test-positive sites, whereas much less uncertainty resides in classifying pathogen status of test-negative sites.

Acta Crystallogr D Biol Crystallogr, 2001 Jan, 57(Pt 1), 145 - 7
Crystallization and preliminary X-ray analysis of (R)-specific enoyl-CoA hydratase from Aeromonas caviae involved in polyhydroxyalkanoate biosynthesis; Hisano T et al.; Dimeric (R)-specific enoyl-coenzyme A (CoA) hydratase from Aeromonas caviae catalyzes the hydration of trans-2-enoyl-CoAs with carbon lengths of 4-6 to yield their corresponding (R)-3-hydroxyacyl-CoAs and is essential for polyhydroxyalkanoate (PHA) biosynthesis . The enzyme has been crystallized by vapour diffusion against a reservoir solution containing 20% polyethylene glycol 4000, 5% 2-propanol and 20 mM HEPES pH 7.0 at 298 K . Crystals belong to the monoclinic space group C2, with unit-cell parameters a = 111.54 (3), b = 59.29 (1), c = 47.27 (4) A, beta = 113.04 (2) degrees and contain a dimeric molecule in the asymmetric unit . Flash-cooling of a crystal at 100 K alters its unit-cell parameters to a = 109.82 (7), b = 57.98 (6), c = 46.84 (2) A, beta = 112.71 (3) degrees . Native data to a resolution of 1.7 A have been collected with 94.5% completeness and an R(merge) of 4.0% under cryogenic (100 K) conditions using synchrotron radiation.

Dis Aquat Organ, 2000 Oct 25, 43(1), 77 - 80
Vaccination influences growth of Arctic charr; Pylkko P et al.; There is limited knowledge about the effects of oil-based vaccines on the growth of Arctic charr Salvelinus alpinus, in particular at different rearing temperatures . One-year-old Arctic charr were immunized intraperitoneally at 2.9 degrees C with a metabolizable oil-adjuvanted, bivalent vaccine containing killed typical and atypical Aeromonas salmonicida bacteria . After vaccination the non-vaccinated (controls) and vaccinated individually marked fish were held for 20 d at 10.0 degrees C and then for 7 wk at 10.3, 14.1 or 18.1 degrees C . During the first 20 d at 10.0 degrees C the growth rate (G) was higher for non-vaccinated than vaccinated fish . Thereafter vaccinated charr had higher G than control fish at 10.3 and 14.1 degrees C . In contrast, at 18.1 degrees C there was no difference in G and therefore no compensation of earlier growth suppression in vaccinated fish was observed at that temperature . The study indicates that vaccination has no ultimate negative effects on the growth of Arctic charr at temperatures ranging from 10.3 to 14.1 degrees C.

Infect Immun, 2001 Jan, 69(1), 65 - 74
Role of flm locus in mesophilic Aeromonas species adherence; Gryllos I et al.; The adherence mechanism of Aeromonas caviae Sch3N to HEp-2 cells was initially investigated through four mini-Tn5 mutants that showed a 10-fold decrease in adherence . These mutants lost motility, flagella, and their lipopolysaccharide (LPS) O antigen (O-Ag) . Three genes, flmB-neuA-flmD, were found to be interrupted by the transposon insertions; additionally, two other genes, one lying upstream (flmA) and one downstream (neuB), were found to be clustered in the same operon . While the flmA and flmB genes were present in all mesophilic Aeromonas spp . (A . hydrophila, A . caviae, A . veronii bv . veronii, and A . veronii bv . sobria) tested, this was not the case for the neuA-flmD-neuB genes . Construction and characterization of flmB insertion mutants in five other mesophilic Aeromonas strains revealed the loss of motility, flagella, and adherence but did not alter the LPS composition of these strains . Taking the above findings into consideration, we conclude (i) that flagella and possibly the LPS O-Ag are involved in the adherence of the mesophilic Aeromonas to human epithelial cells; (ii) flmA and flmB are genes widely distributed in the mesophilic Aeromonas and are involved in flagella assembly, and thus adherence; and (iii) in A . caviae Sch3N the flmA and flmB genes are found in a putative operon together with neuA, flmD, and neuB and are involved in LPS O-Ag biosynthesis and probably have a role in flagellum assembly.

J Invertebr Pathol, 2000 Nov, 76(4), 278 - 84
Responses of giant freshwater prawn (Macrobrachium rosenbergii) to challenge by two strains of Aeromonas spp; Sung HH et al.; The virulence of two Aeromonas strains (A . veronii and A . caviae) isolated from the hepatopancreas of apparently healthy giant freshwater prawns (Macrobrachium rosenbergii) was compared using a challenge by injections . For the A . veronii strain, challenge with 3.7 x 10(5) cells/g of body weight led to 100% mortality; for the A . caviae strain, 3.8 x 10(6) cells/g produced 100% mortality . The 50% lethal doses (LD50) were 2.0 x 10(3) cells/g for A . veronii and 51.2 x 10(3) cells/g for A . caviae . Use of different culture media (trypticase soy broth vs prawn muscle extract) did not significantly affect the virulence of A . veronii . Injection of a sublethal dose (1 x 10(3) cells/g) of A . veronii led to a significant decrease in the total hemocyte count (THC) between 4 and 24 h after injection . Saline injections also caused a similar though less decrease in THC . In the first 24 h after injection of A . veronii (1 x 10(3) cells/g), the change in the percentages of granulocytes (both granular cells and semigranular cells) in the hemolymph was significantly different . After a significant initial increase, the percentage of hyaline cells fell by a factor of 4, from 9 to 2% . Phenoloxidase activity increased fourfold immediately after injection and returned to preinjection levels at 24 h.

Int J Med Microbiol, 2000 Oct, 290(4-5), 363 - 7
Surface dynamics of aerolysin on the plasma membrane of living cells; Abrami L et al.; Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes . The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active . The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore . We have examined these various steps at the surface of target cells . The toxin binds to specific receptors . Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety . The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains . We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor . The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin . The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Appl Environ Microbiol, 2000 Dec, 66(12), 5533 - 5
Functional Tn5393-like transposon in the R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subspecies salmonicida isolated in Norway; L'Abee-Lund TM et al.; Tn5393c containing strA-strB was identified as part of R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp . salmonicida . This is the first time an intact and active transposon in the Tn5393 family has been reported in an ecological niche other than an agricultural habitat.

Toxicology, 2000 Nov 16, 153(1-3), 255 - 64
Cytotoxicity assessment of chlorinated bacteria in water using the RNA synthesis inhibition method; Ferey KA et al.; Chlorination of drinking water containing organic materials is known to generate toxic by-products . We suggested that such compounds may also be produced by interactions between chlorine and bacteria present in water . To confirm this hypothesis, a method based on RNA synthesis inhibition of HeLa S3 human cells in the presence of toxic compounds was applied . This method is rapid and highly sensitive since the concentration of the samples is not required . Furthermore, it was shown to be a suitable method for measurement of the cytotoxicity of water . Aeromonas hydrophila suspensions, prepared with pyrodistilled water, devoid of any organic material, were chlorinated for a definite contact time . HeLa S3 cells were incubated (20 h, 37 degrees C) in a culture medium prepared with the chlorinated bacteria suspensions . The rate of incorporation of 3H uridine into RNA was used as a measure of RNA synthesis and was evaluated in the presence and absence of chlorinated bacteria suspension . This study showed that chlorinated bacteria suspensions are cytotoxic . We observed that 0.22 microm filters retain cytotoxic compounds but 0.45 microm filters did not . Chlorine concentration and bacteria level influence the cytotoxicity . First, the toxicity level increases with chlorine concentration, then it decreases when chlorine concentration is too high . On another hand, a dose effect relationship between bacteria concentration and cytotoxicity was established.

Lett Appl Microbiol, 2000 Nov, 31(5), 359 - 63
Incidence and identification of mesophilic Aeromonas spp . from retail foods; Neyts K et al.; Sixty-eight food samples were examined for the presence of mesophilic Aeromonas species both qualitatively and quantitatively . Aeromonads were isolated from 26% of the vegetable samples, 70% of the meat and poultry samples and 72% of the fish and shrimps . Numbers of motile aeromonads present in the food samples varied from <10(2) cfu g(-1) to >10(5) cfu g(-1) . GLC analysis of FAMEs was used to identify a selection of presumptive Aeromonas colonies to fenospecies or genomic species level . Aeromonas strains belonging to the Aer . caviae complex, which also includes the potentially pathogenic genospecies HG4, were mostly isolated from vegetables but were also found in meat, poultry and fish . In addition, three strains of the virulent taxon Aer . veronii biovar sobria HG8 were isolated from poultry and minced meat . All members of the Aer . hydrophila complex, predominant in the fish, meat and poultry samples, were classified in the non-virulent taxon HG3 . Although the significance of Aeromonas in foods remains undefined, the isolation of Aeromonas HG4 and HG8 strains from a variety of retail foods may indicate that these products can act as possible vehicles for the dissemination of food-borne Aeromonas gastroenteritis.

J Antimicrob Chemother, 2000 Nov, 46(5), 695 - 702
Aeromonas hydrophila AmpH and CepH beta-lactamases: derepressed expression in mutants of Escherichia coli lacking creB; Avison MB et al.; The class 1 cephalosporinase (CepH) and class 2d oxacillinase (AmpH) from an Aeromonas hydrophila clinical isolate, strain T429125, have been cloned and sequenced . Both enzymes are typical of their equivalents in other species of Aeromonas Both cloned beta-lactamase genes were expressed at a low level in a standard laboratory Escherichia coli strain, but when cloned into a cre deletion E . coli mutant, they were expressed at significantly higher levels . Specific disruption of the creB gene resulted in similar increased levels of beta-lactamase expression, so it was concluded that CreB represses the transcription of ampH and cepH in a cre(+) E . coli strain . The expression of cepH was four times that of ampH in the deltacre mutant because of an additional factor encoded on the cloned T429125 chromosomal fragment containing cepH . This factor was able to trans-activate expression of co-resident ampH in the deltacre mutant such that expression of the two genes was approximately equal . The entire cepH-containing fragment was sequenced, but it contained no genes that were obviously related to any known class of DNA-binding protein.

New Microbiol, 2000 Oct, 23(4), 433 - 40
Effect of sodium chloride and citric acid on growth and toxin production by A . caviae and A . sobria at moderate and low temperatures; Abu-Ghazaleh BM; The effect of sodium chloride and citric acid on hemolysin and caseinase production by Aeromonas caviae and Aeromonas sobria at 32 degrees C and 5 degrees C was investigated . At 32 degrees C, although both strains were tolerant to 3% NaCl in TSB, the production of caseinase was decreased in the presence of 1-3% NaCl, and the production of hemolysin was abolished by 2-3% NaCl . Citric acid (0.03%) was less effective than NaCl in reducing hemolysin and caseinase production by both strains at 32 degrees C . A combination of low temperature (5 degrees C) and citric acid treatment reduced hemolysin and caseinase production by both strains . A combination of low temperature (5 degrees C) and NaCl (3%) treatment was the most effective procedure in reducing growth and hemolysin and caseinase production by the tested strains.

Sheng Wu Gong Cheng Xue Bao, 2000 May, 16(3), 345 - 8
{Purification and characterization of recombinant Aeromonas punctata prolyl endopeptidase}; Li M et al.; The study of down-stream techniques of recombinant Aeromonas punctata prolyl endopeptidase (apPEP) was presented here . High cell-density fermentation of E . coli BL21/pKKH-PEP in NBS BioFlo 3000 5 L fermentor was achieved, the final cell density was 22.5 g (DCW)/L after 14 h cultivation, the yield of apPEP expressed in soluble protein was 3.0 g per litter broth . After sonication, the supernatant of free cell extract was purified by ammonium sulfate fractionation, High performance Q sepharose FF, Phenyl sepharose 6 FF, the purity of apPEP reached 96%, enzyme specific activity was 65.5 u/mg, apPEP yield reached 0.86 g/L broth . Total recovery of enzyme protein was 8.2%, actviity recovery was 24.4% . The molecular weight of apPEP was 76,464 +/- 30 Da measured by MS, N terminus amino acids sequence consistent with that deduced from DNA sequence . pI 6.0, which was similar with PEP from Aeromonas hydrophila.

J Appl Microbiol, 2000 Oct, 89(4), 607 - 16
Production and secretion of collagen-binding proteins from Aeromonas veronii; Ascencio F et al.; Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction . The results of this study show that 98% of the total CNBP produced by Aer . veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane . CNBP is specifically secreted from Aer . veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins . CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C . The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase . There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium . CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer . hydrophila . It is concluded that CNBP secretion from Aer . veronii must be achieved by a mechanism different from that reported for aerolysin secretion.

Izv Akad Nauk Ser Biol, 2000 Sep-Oct, (5), 533 - 7
{Comparative study of various biochemical parameters in pathogenic and nonpathogenic Aeromonas strains}; Bogdan VV et al.; We studied certain biochemical properties of virulent and avirulent strains of mobile aeromonads . The pathogenic strain featured a higher proportion of odd fatty acids in the lipids, increased protease activity, and a high concentration of 55 kDa protein . We propose that these compounds be used as pathogenic markers of these microorganisms.

Prikl Biokhim Mikrobiol, 2000 Sep-Oct, 36(5), 592 - 6
{Cytoplasmic protein vaccine against bacterial hemorrhagic septicemia (aeromonosis) of fish}; Smirnov LP et al.; Water-soluble proteins from Aeromonas sobria, a causative agent of bacterial hemorrhagic septicemia of fishes, were separated into six fractions by gel chromatography on Sephadex G-100 . Injections of fraction II (67 kDa) provided the highest protection of carps against the disease . Injections of proteins contained in fraction II caused stronger effects on certain biochemical parameters in the fish liver (fatty acids of phospholipids and cathepsin B and D activities) in comparison to infections of the live culture.

FEMS Immunol Med Microbiol, 2000 Oct, 29(2), 115 - 21
Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria; Francki KT et al.; TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen . Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells . Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained . Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv . tomato which confers resistance to copper and other heavy metals . The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper . Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A . veronii biovar sobria 3767 . The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P . syringae . No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned . Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence . The association between copper tolerance and adhesion in A . veronii biovar sobria requires further study.

J Appl Microbiol, 2000 Sep, 89(3), 415 - 22
The expression of proteinases and haemolysins by Aeromonas hydrophila under modified atmospheres; McMahon MA; The study estimated the proteolytic activity (against Hide Powder Azure) and haemolytic activity (against horse erythrocytes) in cell-free filtrates (CFF) from four strains of Aeromonas hydrophila growing under a range of commercially relevant modified atmospheres (2% O2, 78% N2, 20% CO2; 10% O2, 80% N2, 10% CO2; 50% N2, 50% CO2; 100% CO2) . The examined strains exhibited significant qualitative and quantitative differences in the extent and times of onset of expression of these enzymes under aerobic and modified atmospheres . No proteolytic or haemolytic activities were detected in any Aer . hydrophila cultures grown at sub-optimal temperatures under modified atmospheres containing high concentrations of CO2 (i.e . 50% CO2 or 100% CO2) . Although Aer . hydrophila can grow rapidly in modified atmospheres, the overall spoilage and pathogenic potential is grossly affected . Implications of these findings are discussed.

J Biol Chem, 2001 Jan 5, 276(1), 551 - 4
The channel-forming protein proaerolysin remains a dimer at low concentrations in solution; Barry R et al.; Proaerolysin, the proform of the channel-forming protein aerolysin, is secreted as a dimer by Aeromonas sp . The protein also exists as a dimer in the crystal, as well as in solution, at least at concentrations in the region of 500 microg/ml . Recently it has been argued that proaerolysin becomes monomeric at concentrations below 100 microg/ml and that only the monomeric form of the protoxin can bind to cell surface receptors (Fivaz, M., Velluz, M.-C., and van der Goot, F . G . (1999) J . Biol . Chem . 274, 37705-37708) . Here we show, using non-denaturing polyacrylamide electrophoresis, chemical cross-linking, and analytical ultracentrifugation, that proaerolysin remains dimeric at the lowest concentrations of the protein that we measured (less than 5 microg/ml) and that the dimeric protoxin is quite capable of receptor binding.

J Clin Microbiol, 2000 Oct, 38(10), 3785 - 90
Prevalence of enterotoxin genes in Aeromonas spp . isolated from children with diarrhea, healthy controls, and the environment; Albert MJ et al.; Aeromonads are causative agents of a number of human infections . Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear . In spite of a number of virulence factors produced by Aeromonas species, their association with diarrhea has not been clearly linked . Recently, we have characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate of Aeromonas hydrophila . Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities . We studied the distribution of the alt, ast, and act enterotoxin genes in 115 of 125 aeromonads isolated from 1, 735 children with diarrhea, in all 27 aeromonads isolated from 830 control children (P = 7 x 10(-4) for comparison of rates of isolation of aeromonads from cases versus those from controls), and in 120 randomly selected aeromonads from different components of surface water in Bangladesh . Aeromonas isolates which were positive only for the presence of the alt gene had similar distributions in the three sources; the number of isolates positive only for the presence of the ast gene was significantly higher for the environmental samples than for samples from diarrheal children; and isolates positive only for the presence of the act gene were not found in any of the three sources . Importantly, the number of isolates positive for both the alt and ast genes was significantly higher for diarrheal children than for control children and the environment . Thus, this is the first study to indicate that the products of both the alt and ast genes may synergistically act to induce severe diarrhea . In 26 patients, Aeromonas spp . were isolated as the sole enteropathogen . Analysis of clinical data from 11 of these patients suggested that isolates positive for both the alt and ast genes were associated with watery diarrhea but that isolates positive only for the alt gene were associated with loose stools . Most of the isolates from the three sources could be classified into seven phenospecies and eight hybridization groups . For the first time, Aeromonas eucrenophila was isolated from two children, one with diarrhea and another without diarrhea.

Vet Res Commun, 2000 Sep, 24(6), 371 - 7
Aeromonas hydrophila isolated from wild freshwater fish in Croatia; Topic Popovic N et al.; Aeromonas hydrophila was recovered from fish living in lake Vrana on the Croatian island of Cres . The occurrence of the bacterium in the fish was assessed and related to gross signs of disease and findings at necropsy as a potential health hazard for fish . Isolated bacteria were subjected to morphological, physiological, biochemical and antibiotic susceptibility tests . A total of 26 A . hydrophila isolates were obtained . There was a clear seasonality, as no isolates were recovered in the summer months . Most of the isolates were sensitive to all the antimicrobials used in the study except novobiocin and penicillin G . Affected fish manifested haemorrhages over the skin, in the liver, kidney and swim bladder, spleen infarcts, fatty liver, ascitic fluid and swollen haemopoietic tissues . A . hydrophila does not appear to pose a major threat for the fish in the lake at present but under unfavourable and stressful conditions it could seriously compromise fish health.

Int J Food Microbiol, 2000 Sep 15, 60(1), 65 - 74
Selective enrichment broth for the isolation of Aeromonas sp . from chicken meat; Sachan N et al.; Six selective agents (ampicillin, novobiocin, cephalothin, bile salts, brilliant green and ethanol) were tested during the development of a selective enrichment broth for the isolation of Aeromonas sp . from food . Cephalothin at 10 mg/l was found to be the best selective agent owing to its greater selectivity and efficiency in recovering stressed and lower cell concentrations of Aeromonas sp . Higher concentrations (15-25 mg/l) of cephalothin were inhibitory to some strains of A . sobria . Cephalothin (10 mg/l) was incorporated in buffered dextrin broth (BCDB-10) and alkaline peptone water (CAPW-10) and employed for the isolation of Aeromonas sp . from chicken meat naturally and artificially inoculated (with 10(9) cells/ml of A . hydrophila) . The highest isolation rate (22%) with naturally contaminated chicken was achieved with CAPW-10 in comparison to 16% with BCDB-10 and 8% with APW . Similarly, from artificially inoculated samples, 100% isolation was accomplished with CAPW-10, against 80% with BCDB-10 and 50% with APW.

Emerg Infect Dis, 2000 Sep-Oct, 6(5), 481 - 6
Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised, Taiwan; Chiu CH et al.; From 1996 to 1999, 17 culture-documented systemic infections due to novel, atypical strains of Chryseobacterium meningosepticum occurred in two newborns and 15 immunocompromised patients in a medical center in Taiwan . All clinical isolates, which were initially misidentified as Aeromonas salmonicida by an automated bacterial identification system, were resistant to a number of antimicrobial agents . The isolates were characterized as atypical strains of C . meningosepticum by complete biochemical investigation, 16S rRNA gene sequence analysis, cellular fatty acid analysis, and random amplified polymorphic DNA fingerprinting (RAPD) . This is the first report of a cluster of atypically variant strains of C . meningosepticum, which may be an emerging pathogen in newborns and the immunocompromised.

Eur J Epidemiol, 2000 May, 16(5), 447 - 53
Epidemiological relationships between Aeromonas strains isolated from symptomatic children and household environments as determined by ribotyping; Demarta A et al.; Ribotyping was used to study the epidemiology of Aeromonas associated gastro-enteritis in young children . Ribotyping patterns of 29 Aeromonas strains (16 Aeromonas caviae, 8 Aeromonas hydrophila, 3 Aeromonas eucrenophila, 1 Aeromonas veronii, and 1 Aeromonas encheleia) isolated from primary stool cultures of sick children were compared using the GelCompare software with patterns of 104 strains (39 Aeromonas eucrenophila, 29 Aeromonas caviae, 11 Aeromonas encheleia, 10 Aeromonas hydrophila, 6 Aeromonas bestiarum, 3 Aeromonas veronii, 3 Aeromonas popoffii and 3 Aeromonas media) isolated from their household environment in order to investigate the route of transmission of these bacteria . Fifteen strains (approximately 47%) isolated from stool cultures of patients showed the same riboprofile as strains found in contacts or environment . In particular, three strains isolated from patients shared the same riboprofile with strains found in their domestic environment . The wide diffusion of potentially pathogenic Aeromonas strains in our household samples, and the high rate of asymptomatic carriers among family members, suggested that predisposing factors of the host could make children prone to an Aeromonas-related intestinal disease.

Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 10691 - 6
Microbial iron transport via a siderophore shuttle: a membrane ion transport paradigm; Stintzi A et al.; A mechanism of ion transport across membranes is reported . Microbial transport of Fe(3+) generally delivers iron, a growth-limiting nutrient, to cells via highly specific siderophore-mediated transport systems . In contrast, iron transport in the fresh water bacterium Aeromonas hydrophila is found to occur by means of an indiscriminant siderophore transport system composed of a single multifunctional receptor . It is shown that (i) the siderophore and Fe(3+) enter the bacterium together, (ii) a ligand exchange step occurs in the course of the transport, and (iii) a redox process is not involved in iron exchange . To the best of our knowledge, there have been no other reports of a ligand exchange mechanism in bacterial iron transport . The ligand exchange step occurs at the cell surface and involves the exchange of iron from a ferric siderophore to an iron-free siderophore already bound to the receptor . This ligand exchange mechanism is also found in Escherichia coli and seems likely to be widely distributed among microorganisms.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 163 - 6
Pulsed-field gel electrophoriesis analyis of Aeromonas salmonicida ssp . salmonicida; Garcia JA et al.; A total of 133 strains of Aeromonas salmonicida ssp . salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns . Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish . Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.

Dev Comp Immunol, 2001 Jan, 25(1), 37 - 46
Immune responses of Nile tilapia (Oreochromis niloticus L.) clones: I . Non-specific responses; Sarder MR et al.; The importance of genetic variation in the non-specific immune responses of Nile tilapia (Oreochromis niloticus L.) clones was investigated . Fully inbred clones (IC) of Nile tilapia, produced using gynogenesis and sex reversal, and crosses between these lines (outbred clones) were used in this study . Non-specific immune responses were compared between the ICs, including serum lysozyme activity and phagocytosis, and significant differences were observed between the different groups . Their natural resistance to Aeromonas hydrophila infection was also assessed by bacterial challenge . A positive correlation was observed between the level of infection obtained and the non-specific immune parameters measured . Cumulative mortalities of fish obtained in the study showed that when a IC susceptible to A . hydrophila was crossed with a resistant IC, the resulting progeny exhibited intermediate levels of resistance to that of their parents.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 183 - 7
{Cultivation optimization of prolyl endopeptidase and high cell density fermentation}; Li M et al.; Engineered E . coli BL21/pGEM-PEP could constitutively express recombinant prolyl endopeptidase from Aeromonas punctata, which was extremely effected by culture conditions, so fermentation conditions were optimized to obtain it's high expression level . Firstly, the stability of BL21/pGEM-PEP was investigated, then, culture temperature, pH, time, medium were optimized in shake flaskd . The L9(3(4)) orthogonal experiment confirmed that shaker speed, pH, culture temperature, culture time had high degree statistical meaning . Based on these data, high cell density fermentation of E . coli BL21/pGEM-PEP on NBS BioFlo 3000 5 L fermentor was achieved, after 20 h cultivation, the final density(dwt) was 60OD600(22.5 g/L), the expressed PEP was about 28% of total cellular protein, the yield was 3.15 g per litter broth.

Diagn Microbiol Infect Dis, 2000 Aug, 37(4), 271 - 3
Spontaneous bacterial empyema caused by Aeromonas veronii biotype sobria; Wang JT et al.; Spontaneous bacterial empyema is a complication of hepatic hydrothorax in cirrhotic patients . The pathogen, clinical course and treatment strategy are different to the empyema secondary to pneumonia . A 54-year-old man, who was a cirrhotic patient with hepatic hydrothorax, was admitted to National Taiwan University Hospital for fever, dyspnea and right side pleuritic pain . The image study revealed massive right pleural effusion and no evidence of pneumonia . The culture of pleural effusion yielded Aeromonas veronii biotype sobria . The diagnosis of spontaneous bacterial empyema caused by Aeromonas veronii biotype sobria was established . To our best knowledge, Aeromonas veronii biotype sobria had never been reported in English literature as the causative pathogen of spontaneous bacterial empyema.

Appl Environ Microbiol, 2000 Sep, 66(9), 3883 - 90
Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A; Rhodes G et al.; Oxytetracycline-resistant (OT(r)) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England . The transfer of OT(r) plasmids from these isolates was investigated . Using Escherichia coli J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT(r) plasmids (designated pFBAOT1 to 17) . Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either Aeromonas hydrophila HG3 (eight isolates), A . veronii b.v . sobria HG8 (six isolates), and A . caviae HGB5 (one isolate) . One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level . Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G . Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7 tet gene determinants remaining unclassified . In all cases, Tet A was located on a 5.5-kb EcoRI restriction fragment . Hybridization with inc-rep probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids . Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT(r) plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from A . salmonicida strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital E . coli strain) . In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other . The presence of tetracycline resistance transposon Tn1721 in its entirety or in a truncated form in these plasmids was demonstrated . These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different Aeromonas species and E . coli and between the human and aquaculture environments in distinct geographical locations . Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.

Jpn J Infect Dis, 2000 Jun, 53(3), 111 - 5
Inhibition of Aeromonas caviae and A . sobria by sodium choloride, citric acid, ascorbic acid, potassium sorbate and extracts of Thymus vulgaris; Abu-Ghazaleh BM; The respective and combined effects of sodium chloride, ascorbic acid, citric acid, potassium sorbate, and Thymus vulgaris extract on the growth of Aeromonas caviae and Aeromonas sobria were investigated . Sodium chloride (3%) significantly reduced the growth and 4% NaCl inhibited growth of the tested strains . Ascorbic acid (0 . 1%), potassium sorbate (0.05%), and citric acid (0.03%) slightly inhibited growth . T . vulgaris extract (0.3%) greatly reduced the growth . Various combinations of these compounds prevented growth of the tested strains . A combination of NaCl (3%) and ascorbic acid (0 . 1%), citric acid (0.03%) and potassium sorbate (0.05%), or citric acid (0.03%) and ascorbic acid (0.1%) inhibited growth of A . caviae and A . sobria . In fish homogenates, the addition of ascorbic acid (0 . 1%) and citric acid (0.03%) was the most effective combination tested.

J Appl Microbiol, 2000 Jul, 89(1), 145 - 51
The response of Aeromonas hydrophila to oxidative stress induced by exposure to hydrogen peroxide; Landre JP et al.; Aeromonas hydrophila, an opportunist human pathogen of low virulence, was shown to display a high degree of sensitivity upon exposure to hydrogen peroxide . As with other species, Aer . hydrophila is able to develop the capacity to resist loss of viability induced by such oxidative stress . Development of stress resistance follows the archetypal profile where pre-exposure of a population to sub-lethal levels of H2O2 stimulates onset of tolerance to further exposure . Acquisition of tolerance critically requires nascent protein synthesis . Further analysis demonstrated population growth phase influences the degree of sensitivity of the organism . Late stationary phase cultures demonstrate a decreased sensitivity compared with younger populations . Significantly, it was also determined that stock culture age influenced the level of sensitivity of the derived experimental culture, where an increased stock culture age corresponded with enhanced resistance to H2O2 . These data show that Aer . hydrophila population phenotype is influenced by the phenotype of the donor stock culture.

Indian J Med Res, 2000 May, 111, 162 - 7
Production of siderophores & effect of iron restriction on the protein profiles of Aeromonas species isolated from water & patients suffering from acute diarrhoeal disease; Alavandi SV et al.; Among 48 strains of Aeromonas species, 21 isolates from patients suffering from acute diarrhoea and 27 from metropolitan water samples grown under iron-restricted conditions, 45 strains produced siderophores . Forty one of the 46 strains tested produced siderophores on chrome azurol S (CAS) agar, while 43 isolates did so when the culture supernatants of the bacterial isolates grown in minimal medium were assayed with chrome azurol S assay solution . The whole cell protein profiles of A . hydrophila strains grown under iron restricted conditions expressed new proteins that were not detected in those cultured in iron rich conditions . Five high molecular weight proteins ranging from 70 to 96 kDa were distinctly absent in cultures grown in the presence of iron, indicating their role in iron acquisition by the aeromonads.

Fish Shellfish Immunol, 2000 May, 10(4), 359 - 73
Suppression of the humoral immune response of Atlantic salmon, Salmo salar L . by the 64 kDa serine protease of Aeromonas salmonicida; Hussain I et al.; Bacteria-free supernatants of broth cultures of Aeromonas salmonicida inhibited the humoral immune response, but not the cell-mediated immune response, of Atlantic salmon to bacteriophage MS2 . The immunosuppressive factor was the 64 kDa serine protease secreted by A . salmonicida . The suppressive activity was not due to degradation of epitopes of MS2, and although serine protease degraded the heavy chain of salmon IgM in vitro there was no evidence for significant degradation in vivo . The principal lethal toxin of A . salmonicida, the glycerophospholipid: cholesterol acyltransferase did not inhibit the immune response of salmon.

Fish Shellfish Immunol, 2000 Feb, 10(2), 107 - 28
Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival; Dalmo RA et al.; Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp . salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) . The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1 . The uptake was both dependent on the LPS concentration and the time of exposure . After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration . After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS . Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument . By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer . Control specimens did not contain fluorescence or were immunohistochemically negative for LPS . In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.

J Antimicrob Chemother, 2000 Aug, 46(2), 297 - 301
Antimicrobial resistance of mesophilic Aeromonas spp . isolated from two European rivers; Goni-Urriza M et al.; The activity of 19 antibiotics and four antiseptics and/or disinfectants was studied against 138 non-redundant strains of Aeromonas spp . (104 Aeromonas caviae, 22 Aeromonas sobria and 12 Aeromonas hydrophila) isolated from two European rivers . Antibiotic resistance frequencies were: nalidixic acid, 59%; tetracycline, 14%; fosfomycin, 8%; tobramycin and cotrimoxazole, 7%; cefotaxime, 4%; chloramphenicol, 2%; gentamicin, 1% . Most of the nalidixic acid-resistant strains were susceptible to fluoroquinolones (54-98%) . Antibiotic resistance rates varied according to the source of the strains . All Aeromonas spp . strains were killed by 50 ppm of chlorine, cetylpyridinium chloride and peracetic acid, and by 1600 ppm of glutaraldehyde.

Can J Microbiol, 2000 Jul, 46(7), 674 - 8
Co-culture of Aeromonas salmonicida and host cells in intraperitoneal implants is associated with enhanced bacterial survival; Garduno RA et al.; An experimental procedure that we named "in vivo co-culture technology" allowed us to study the interactions between Aeromonas salmonicida and host cells, inside semipermeable chambers implanted in the peritoneal cavity of Atlantic salmon . Intraperitoneal implants containing bacteria and host cells, or bacteria and lysed cells, consistently yielded higher numbers of viable bacteria than implants containing bacteria only . Electron microscopy confirmed that 30 min after chamber inoculation, numerous bacteria were already internalized by exudate cells, and that at 3 h, destruction of these cells was evident . Thus, the rapid invasion and (or) the A . salmonicida-mediated lysis of host cells may constitute a survival strategy in vivo . The co-culture of bacteria with exudate peritoneal cells may be applicable to the in vivo study of other pathogens.

Can J Microbiol, 2000 Jul, 46(7), 660 - 8
Host cell invasion and intracellular residence by Aeromonas salmonicida: role of the S-layer; Garduno RA et al.; Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages . Here we report that S+ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants . When reconstituted with exogenous S-layers, these S- mutants regained adherence . As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure . Gentamicin protection assays and electron microscopy indicated that both S+ and S- A . salmonicida invaded non-phagocytic fish cells . In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor . Lipopolysaccharide (which is partially exposed in S+ bacteria) appeared to mediate invasion . Surprisingly, A . salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts . On the contrary, bacterial viability sharply decreased after cell infection . We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines . These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A . salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.

J Infect, 2000 May, 40(3), 267 - 73
Clinical features and therapeutic implications of 104 episodes of monomicrobial Aeromonas bacteraemia; Ko WC et al.; OBJECTIVES: Aeromonas bacteraemia is not a common infectious disease, but can cause a grave outcome in infected cases . In this study, clinical presentations and prognostic factors of cases of monomicrobial Aeromonas bacteraemia were analysed . Also, the impact of beta-lactam and aminoglycoside in combination and of emerging cephalosporin-resistance during therapy was discussed . METHODS: From 1989 to 1998 in a medical centre in southern Taiwan, those cases with monomicrobial Aeromonas bacteraemia were included for study . RESULTS: A total of 104 episodes of monomicrobial Aeromonas bacteraemia, accounting for 74% of all Aeromonas bacteraemia, were encountered . The infections usually occurred in the patients with hepatic cirrhosis (54%) or malignancy (21%) and were community-acquired (74%) . Cases of community-acquired bacteraemia were more likely to have cirrhosis, a high severity score at onset, and a worse prognosis than those of nosocomial bacteraemia did and nosocomial isolates were less susceptible to cefoxitin and cefotaxime . Forty-three percent of cases had a concomitant infection focus, such as primary peritonitis, invasive cellulitis or necrotizing fasciitis, biliary tract or burn wound infections . Crude fatality rate within 2 weeks after the onset was 30% . Secondary bacteraemia and a higher severity score ( > or = 4) for illness at the first presentation were independently associated with a fatal outcome . The therapeutic superiority of beta-lactam and aminoglycoside in combination cannot be demonstrated in patients with Aeromonas bacteraemia . Cefotaxime resistance emerged in 3.4% of 58 patients treated with a cephalosporin for at least 72 h . None of the community-acquired isolates, but one-quarter of the nosocomial isolates, were resistant to cefotaxime . CONCLUSIONS: Aeromonas bacteraemia usually occurred in patients with liver cirrhosis or malignancy, and heralded a poor prognosis, especially while associated with a relevant infectious source or with a higher severity score at presentation . The superiority of aminoglycoside and beta-lactam in combination cannot be demonstrated while treating those patients, and the emergence of antimicrobial resistance to cephalosporin was a rare event during cephalosporin therapy . Thus, a broad-spectrum cephalosporin remains one of the antimicrobial alternatives for invasive community-acquired Aeromonas infections.

J Diarrhoeal Dis Res, 1999 Jun, 17(2), 75 - 80
Characterization of virulence factors of Aeromonas isolated from children with and without diarrhoea in Tripoli, Libya; Ghenghesh KS et al.; During September 1992-August 1993, stool samples from 157 children with diarrhoea and 157 matched healthy controls were examined for the presence of Aeromonas and other enteropathogens . Aeromonas strains were tested for haemolytic activity, haemagglutination patterns, and antibiotic susceptibility . In total, 62 Aeromonas were isolated, of which 27 (17.2%) were from children with diarrhoea and 35 (22.3%) from healthy controls . Only 23 (14.6%) of the diarrhoeal children and 28 (17.8%) of the healthy controls were positive for Aeromonas; of which, 4 (2.5%) of the diarrhoeal children and 6 (3.8%) of the healthy controls showed multiple species . Aeromonas hydrophila was isolated from 5 (3.2%) children with diarrhoea and from 9 (6.4%) controls, A . veronii bv sobria from 8 (5.1%) and 7 (4.5%), A . caviae from 13 (8.3%) and 17 (10.8%), and A . schubertii from 1 (0.6%) and 2 (1.3%) respectively . No significant difference in the haemolytic activity of Aeromonas was found between diarrhoeal and healthy children . However, a significant difference (p < 0.002) was observed in mannose-resistant haemagglutination (MRHA) by diarrhoeal isolates of Aeromonas (7/27, 26%) compared to the healthy controls (1/35, 3%) . Aeromonas strains were uniformly sensitive to ciprofloxacin, gentamicin, and nalidixic acid . The results of this study suggest that A . caviae strains may be associated with diarrhoea in children and MRHA may be used as one of the virulence markers for distinguishing between Aeromonas isolated from diarrhoeal children and healthy controls or environmental isolates.

J Diarrhoeal Dis Res, 1999 Mar, 17(1), 37 - 42
Virulence patterns of Aeromonas eucrenophila isolated from water and infected fish; Singh DV et al.; Six isolates of Aeromonas eucrenophila--2 from water and 4 from superficial skin ulcer of cat fish--were examined for haemagglutination, serum sensitivity, chitinase production and enterotoxicity, and correlation, if any, between them; only one strain showed haemagglutination and was inhibited by both D-mannose and L-fucose . All the strains showed resistance to normal human serum, but produced chitinase; one of them elaborated inducible chitinase . All these strains caused fluid accumulation only after 1-4 serial passages through rabbit ileal loops, of which one strain that elaborated inducible chitinase caused significantly more (p < 0.005) fluid accumulation . These observations indicate that there is no correlation between enterotoxicity and haemagglutination and/or serum resistance, and these properties did not change after animal passage . However, a correlation could be observed between elaboration of inducible chitinase and enterotoxin production.

J Diarrhoeal Dis Res, 1999 Mar, 17(1), 34 - 6
Combined infection of Norwalk-like virus and verotoxin-producing bacteria associated with a gastroenteritis outbreak; Bettelheim KA et al.; Detection of multiple pathogens, particularly a combination of viruses and bacteria, is infrequently documented in outbreaks of gastroenteritis . This paper reports the presence of Norwalk-like virus (NLV) and enterohaemorrhagic verotoxin-producing Escherichia coli in one individual, and NLV and verotoxin-producing Aeromonas sobria in another individual, both part of a large gastroenteritis outbreak . The causes of gastroenteritis in such outbreaks may be more complex than previously thought.

Rev Cubana Med Trop, 1999 Jan-Apr, 51(1), 50 - 2
Identification of Aeromonas strains of clinical origin with atypical phenotypical profiles; Bravo Farinas L et al.; A total of 47 strains of Aereomonas isolated from patients with gastroenteritis was analyzed for 40 phenotypical characters and for evaluating the numeric taxonomy based on 27 discriminatory tests . It was proved that the clinical isolates showed a relative phenotypical distance and the groups of strains that had atypical profiles were compared with the type species by the present identification schemes.

FEMS Immunol Med Microbiol, 2000 Jul, 28(3), 225 - 32
Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers; Martinez-Murcia AJ et al.; Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes . When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles . Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources . Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism . Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism . In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.

Infect Immun, 2000 Jul, 68(7), 4040 - 8
Investigation of the role of type IV Aeromonas pilus (Tap) in the pathogenesis of Aeromonas gastrointestinal infection; Kirov SM et al.; Although there is substantial evidence that type IV pili purified from diarrhea-associated Aeromonas species (designated Bfp for bundle-forming pilus) are intestinal colonization factors (S . M . Kirov, L . A . O'Donovan, and K . Sanderson, Infect . Immun . 67:5447-5454, 1999), nothing is known regarding the function of a second family of Aeromonas type IV pili (designated Tap for type IV Aeromonas pilus), identified following the cloning of a pilus biogenesis gene cluster tapABCD . Related pilus gene clusters are widely conserved among gram-negative bacteria, but their significance for virulence has been controversial . To investigate the role of Tap pili in Aeromonas pathogenesis, mutants of Aeromonas strains (a fish isolate of A . hydrophila and a human dysenteric isolate of A . veronii bv . sobria) were prepared by insertional inactivation of the tapA gene which encodes the type IV pilus subunit protein, TapA . Exotoxic activities were unaffected by the mutation in tapA . Inactivation of tapA had no effect on the bacterial adherence of these two isolates to HEp-2 cells . For the A . veronii bv . sobria isolate, adhesion to Henle 407 intestinal cells and to human intestinal tissue was also unaffected . There was no significant effect on the duration of colonization or incidence of diarrhea when the A . veronii bv . sobria strain was tested in the removable intestinal tie adult rabbit diarrhea model or on its ability to colonize infant mice . Evidence was obtained that demonstrated that TapA was expressed by both Aeromonas species and was present on the cell surface, although if assembled into pili this pilus type appears to be an uncommon one under standard bacterial growth conditions . Further studies into factors which may influence Tap expression are required, but the present study suggests that Tap pili may not be as significant as Bfp pili for Aeromonas intestinal colonization.

Shock, 2000 Jun, 13(6), 478 - 84
The cardiovascular hemodynamics and leukotriene kinetics during prostacyclin and anti-prostacyclin antibody infusions in septic shock; Tran HS et al.; This study evaluated whether or not prostacyclin (PGI2) was necessary or sufficient by itself in a pathophysiologic concentration to mediate the cardiovascular dysfunction of septic shock . Anesthetized adult swine received anesthesia only (ANESTHESIA CONTROL, n = 6); graded Aeromonas hydrophila, 10(10)/mL, infusion at 0.2 mL/kg/h that increased to 4.0 mL/kg/h over 3 h (SEPTIC SHOCK CONTROL, n = 6); pathophysiologic prostacyclin infusion to match septic shock control plasma levels without bacteremia (PGI2 INFUSION, n = 6), or graded Aeromonas hydrophila plus anti-prostacyclin antibody infusion (ANTI-PGI2-Ab INFUSION, n = 5) . This graded porcine bacteremia model was 100% lethal after 4 h . Cardiovascular hemodynamics, arterial blood gases, and plasma levels of arachidonate metabolites were measured at baseline and hourly over a 4-h period . The results showed that PGI2 was not a necessary mediator of impaired cardiovascular hemodynamics in graded bacteremia, as anti-PGI2 antibody infusion did not improve the cardiac index, systemic vascular resistance, or peripheral oxygen balance in septic animals . Also, PGI2 was not sufficient alone to cause the cardiovascular dysfunction of sepsis, as pathophysiologic infusion of PGI2 did not reproduce such changes in normal animals . PGI2 blockade during bacteremia significantly increased LTC4D4E4, and LTB4 whereas PGI2 infusion suppressed LTC4D4E4 concentration, suggesting that endogenous PGI2 may blunt leukotriene release during septic shock . These results indicate a complex dynamic equilibrium among prostacyclin and leukotrienes in septic shock.

Dis Aquat Organ, 2000 Apr 20, 40(3), 185 - 93
Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R . rugulosa; Pearson MD et al.; Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand . Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads . Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A . hydrophila), and low haemolytic activity (A . veronii biovar sobria, A . veronii biovar veronii, A . caviae, A . schubertii) were noted . DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested . Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A . hydrophila isolates and in none of the other motile aeromonads . The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays . Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species . In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A . hydrophila . Selection of suitable assay substrates is therefore important.

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1119 - 24
Aeromonas salmonicida subsp . pectinolytica subsp . nov., a new pectinase-positive subspecies isolated from a heavily polluted river; Pavan ME et al.; Aeromonas strains which phenotypically and genetically belong to the Aeromonas salmonicida species but that according to their phenotypic properties constitute a new subspecies have been isolated from the water of a heavily polluted river, the Matanza river, situated near the central district of Buenos Aires city . These strains were ascribed to the A . salmonicida species by using 65 biochemical tests and by DNA-DNA hybridization . They produce acid from -sorbitol, an unusual biochemical property found in a few members of the A . salmonicida species . They also utilize urocanic acid and do not ferment L-rhamnose or utilize LD-lactate, and are elastase- and gluconate-negative . The DNA relatedness was over 70%, the current limit accepted for the phylogenetic definition of a species, to the described A . salmonicida subspecies and nearly 100% within the new group of Aeromonas strains . Phenotypic differentiation from other A . salmonicida subspecies was readily achieved using the following characteristics: growth at 37 degrees C, melanin production, indole and Voges-Proskauer assays, growth on KCN broth, mannitol and sucrose fermentation and gas from glucose . A remarkable property of the strains of the new group was their ability to degrade polypectate, an unusual feature among Aeromonas species in general . The complete 16S rRNA gene of one strain of the new group was sequenced . Comparison with rDNA sequences of Aeromonas members available in databases revealed a close relationship between this strain and strains belonging to A . salmonicida subsp . salmonicida, masoucida and achromogenes, in agreement with the biochemical data . Since the new A . salmonicida strains constitute a tight genomic group that can be identified by phenotypic properties it was concluded that they represent a new subspecies for which the name Aeromonas salmonicida subsp . pectinolytica is proposed . The type strain of A . salmonicida subsp . pectinolytica is 34melT (= DSM 12609T).

Arch Microbiol, 2000 Apr, 173(4), 307 - 10
A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila; Wai SN et al.; Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods . Starved A . hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days . The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates . The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.

Burns, 2000 Aug, 26(5), 478 - 82
Aeromonas wound infection in burns; Kienzle N et al.; Infection of burn patients with the Aeromonas organism is an uncommon event . This paper documents four cases of Aeromonas hydrophila and one case involving both A . hydrophila and A . caviae occurring in burn patients between 1990 and 1998 at the Royal Brisbane Hospital burns unit . The organism was isolated from either skin swabs, tissue samples, blood cultures or cultured lines . In all patients there was a history of immersion in water immediately post burn . There is one case of invasion and destruction of deeper tissues and one fatality . Appropriate management requires a high index of suspicion if a history of immersion in untreated water post burn is given and the treatment involves aggressive excision and antibiotic therapy.

IUBMB Life, 1999 Aug, 48(2), 199 - 204
Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae; Lin FP et al.; Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae . Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases . Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat . However, the effect of the these mutations on the Km values were different . The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used . Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.

Syst Appl Microbiol, 1999 Dec, 22(4), 662 - 9
In vitro susceptibilities of Aeromonas genomic species to 69 antimicrobial agents; Kampfer P et al.; A total of 217 strains representing all 14 currently described genomic species in the genus Aeromonas were tested for susceptibility to 69 antimicrobial agents by a microdilution method . All species were susceptible to tetracyclines, quinolones, chloramphenicol, and most of the aminoglycosides and the cephalosporins, but were resistant to lincosamides, vancomycin, teicoplanin and some penicillins . In general, no significant differences were found that correlated with the taxonomic designation or the origin of the isolates tested . The microdilution method proved to be easy to perform allowing susceptibility testing of extensive strain collections for a large number of antimicrobial agents.

J Appl Microbiol, 2000 May, 88(5), 897 - 906
Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture; Guzman-Murillo M et al.; An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms) . Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease . Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer . veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates . The three primary-culture cells are susceptible to Aer . veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial cells . Aer . veronii adherence was dependent on bacterial load and incubation time . The effect of glycoconjugates on Aer . veronii adhesion was investigated by pre-incubating Aer . veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides . In addition, the participation of a 48-kDa Aer . veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer . veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer . veronii MCBP . Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer . veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

J Appl Microbiol, 2000 Apr, 88(4), 704 - 10
The Pseudomonas group as an indicator of potential regrowth in water distribution systems; Ribas F et al.; The regrowth of micro-organisms in the water distribution system is assisted by the presence of biodegradable organic matter (BDOC) . The low concentration of available organic carbon in this type of water favours the growth of bacteria belonging to the Pseudomonadaceae family, as this group can grow better at low concentrations of substrate than other bacteria also present in the distribution system . Although the genus Aeromonas has already been adopted as an indicator of this potential regrowth, members of the Pseudomonadaceae have not yet been proposed as indicators of potential bacterial regrowth in the water distribution system . The results are presented of a year-long study of the Barcelona distribution system in which the presence of Pseudomonas and Aeromonas was analysed and the validity of these micro-organisms as indicators of potential regrowth in the distribution system was assessed . It seems that, at least in the drinking waters of the Barcelona area, Pseudomonas is a better indicator of potential bacterial regrowth than Aeromonas.

Microbiology, 2000 Apr, 146 ( Pt 4), 999 - 1009
Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish; Zhang YL et al.; Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans . A . hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish . Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains . Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined . Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria . ORFs encoded by the remaining 46 fragments were identified as new proteins of A . hydrophila, showing no significant homology to any known proteins . Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A . hydrophila . The PPD134/91-specific genes included five known virulence factors of A . hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2) . Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads . Some of these fragments encoded virulence determinants . These included genes for the synthesis of O-antigen and type II restriction/modification system . The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A . hydrophila.

Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 551 - 8
Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 A resolution; Gilboa R et al.; SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures . It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues . It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions . The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications . Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes . The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes . The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site . A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process . These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products . These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219) . These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile . The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

Infect Immun, 2000 May, 68(5), 2808 - 18
The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages; Chopra AK et al.; An aerolysin-related cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses multiple biological activities, which include its ability to lyse red blood cells, destroy tissue culture cell lines, evoke a fluid secretory response in ligated intestinal loop models, and induce lethality in mice . The role of Act in the virulence of the organism has been demonstrated . In this study, we evaluated the potential of Act to induce production of proinflammatory cytokines associated with Act-induced tissue injury and Act's capacity to activate in macrophages arachidonic acid (AA) metabolism that leads to production of eicosanoids (e.g., prostaglandin E(2) {PGE(2)}) . Our data indicated that Act stimulated the production of tumor necrosis factor alpha and upregulated the expression of genes encoding interleukin-1beta (IL-1beta) and IL-6 in the murine macrophage cell line RAW264.7 . Act also activated transcription of the gene encoding inducible nitric oxide synthase . Act evoked the production of PGE(2) coupled to the cyclooxygenase-2 (COX-2) pathway . AA is a substrate for PGE(2), and Act produced AA from phospholipids by inducing group V secretory phospholipase A(2) . We also demonstrated that Act increased cyclic AMP (cAMP) production in macrophages . cAMP, along with PGE(2), could potentiate fluid secretion in animal models because of infiltration and activation of macrophages resulting from Act-induced tissue injury . After Act treatment of RAW cells, we detected an increased translocation of NF-kappaB and cAMP-responsive element binding protein (CREB) to the nucleus using gel shift assays . Act also upregulated production of antiapoptotic protein Bcl-2 in macrophages, suggesting a protective role for Bcl-2 against cell death induced by proinflammatory cytokines . The increased expression of genes encoding the proinflammatory cytokines, COX-2, and Bcl-2 appeared correlated with the activation of NF-kappaB and CREB . This is the first report of the detailed mechanisms of action of Act from A . hydrophila.

J Biol Inorg Chem, 2000 Feb, 5(1), 57 - 66
Amonabactin-mediated iron acquisition from transferrin and lactoferrin by Aeromonas hydrophila: direct measurement of individual microscopic rate constants; Stintzi A et al.; The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe . The ability of A . hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments . The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis . The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result . A . hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored . Addition of transferrin or lactoferrin promoted bacterial growth . Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing . Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube . Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin . The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration . Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin . Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access . Taken together, these results support an amonabactin-dependent mechanism for iron removal by A . hydrophila from transferrin and lactoferrin . The implications of these findings for an amonabactin-dependent mechanism for iron removal by A . hydrophila from transferrin and lactoferrin are discussed.

Chemotherapy, 2000 May-Jun, 46(3), 177 - 83
Interaction of cefotetan and the metallo-beta-lactamases produced in Aeromonas spp . and in vitro activity; Quiroga MI et al.; Aeromonas spp . are increasingly being recognized as human pathogens . The presence of metallo-beta-lactamases in these organisms represents a potential problem in antimicrobial therapy . Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics, but no clinical useful inhibitors of the metallo-beta-lactamases are presently known . Studying the interaction between cefotetan and Aeromonas spp . producing metallo-beta-lactamase activity, we observed that cefotetan behaved as a transient inactivator for both the crude extracts of Aeromonas strains and the purified enzymes from Aeromonas hydrophila AE036 and Aeromonas schubertii MNSA20 . The direct hydrolysis of cefotetan showed that it was a poor substrate for both purified enzymes . In view of the minimum inhibitory concentrations, cefotetan shows to be a useful antimicrobial agent against Aeromonas spp .

J Infect, 2000 Jan, 40(1), 69 - 73
Aeromonas infection in acute suppurative cholangitis: review of 30 cases; Chan FK et al.; OBJECTIVES: Aeromonads, though not common pathogens in biliary sepsis, caused substantial mortality in patients with impaired hepatobiliary function . Our aim was to study the pathogenic role of Aeromonas in acute suppurative cholangitis . METHODS: Between 1996 and 1998, the medical records of patients with a diagnosis of biliary sepsis were reviewed . Those who fulfilled the diagnostic criteria for acute suppurative cholangitis and had positive bile or blood cultures for Aeromonas species were studied . RESULTS: One thousand and forty-five patients were confirmed to have acute suppurative cholangitis . Of these, 30 patients (2.9%) had Aeromonas species isolated from bile; four were complicated by aeromonas septicaemia with simultaneous recovery of the bacteria from blood . All except two isolates were A . hydrophila . Twenty-four patients (80%) had bile duct stones, four (13%) had cholangiocarcinoma and two (7%) pancreatic cancer . Twenty-five cases (83%) had previous exploration of the biliary tract . There was substantial resistance to piperacillin (58%), ceftazidime (30%) and imipenem (15%) . Most patients improved after biliary decompression . Only three patients (10%) died, two had terminal malignancy and one had end-stage liver failure . No excess mortality was attributable to Aeromonas infection in biliary sepsis . CONCLUSIONS: Previous instrumentation facilitated ascending Aeromonas infection of the biliary tract from the gastrointestinal tract . Unlike early reports, our results showed that aeromonads did not adversely affect the clinical outcome of acute suppurative cholangitis with successful drainage of biliary obstruction.

Appl Environ Microbiol, 2000 Apr, 66(4), 1764 - 6
Identification of motile Aeromonas strains with the MicroScan WalkAway system in conjunction with the combo negative type 1S panels; Vivas J et al.; This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identifying 85 environmental, clinical, and reference strains of eight Aeromonas species.

Kansenshogaku Zasshi, 2000 Feb, 74(2), 155 - 61
{Isolation of Aeromonas species from patients with sporadic diarrhea and characterization of Aeromonas hydrophila isolates}; Okitsu T et al.; A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998 . Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A . sobria and 8 were A . caviae . The strains of A . hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups . Although all 7 strains of A . hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes . From the results of PCR assay, the tested strains of A . hydrophila were grouped into 2 hemolysin gene types of {ahh1 + ahh3 + aerA} (n = 6) and {ahh1 + aerA} (n = 1) both of which are recognized to be enteropathogenic . Five of the 7 strains of A . hydrophila belonged to serogroup O11 . These results suggest that 7 strains of A . hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A . hydrophila in humans.

Biosci Biotechnol Biochem, 2000 Feb, 64(2), 408 - 13
Purification and some properties of high-molecular-weight xylanases, the xylanases 4 and 5 of Aeromonas caviae W-61; Roy N et al.; Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 {Nguyen, V . D . et al., Biosci . Biotechnol . Biochem., 56, 1708-1712 (1993)} . Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium . The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-beta-1,4-xylanases with similar enzymatic properties except for trans-xylosidase activity . The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions . Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity.

Lett Appl Microbiol, 2000 Jan, 30(1), 57 - 60
Growth of Aeromonas species on increasing concentrations of sodium chloride; Delamare AP et al.; The growth of 16 strains of Aeromonas, representing 12 species of the genera, were examined at different salt levels (0-1.71 M NaCl) . All the strains grew on media with 0.34 M NaCl, and nine on media with 0.68 M . Two strains, Aer . enteropelogenes and Aer . trota, were able to grow on media with 0.85 M and 1.02 M NaCl, respectively . Comparison of the growth curves of Aer . hydrophila ATCC7966 and Aer . trota ATCC 49657 on four concentrations of NaCl (0.08, 0.34, 0.68 and 1.02 M) confirm the high tolerance of Aer . trota, and indicate that high concentrations of salt increase the lag time and decrease the maximum growth rate . However, both strains were able to grow, slowly, in at least 0.68 M NaCl, a sodium chloride concentration currently used as food preservative.

Infect Immun, 2000 Apr, 68(4), 1849 - 54
Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila; Nogueras MM et al.; We cloned and sequenced the structural gene for Aeromonas hydrophila porin II from strain AH-3 (serogroup O:34) . The genetic position of this gene, like that of ompF in Escherichia coli, is adjacent to aspC and transcribed in the same direction . However, upstream of the porin II gene no similarities with E . coli were found . We obtained defined insertion mutants in porin II gene either in A . hydrophila (O:34) or A . veronii sobria (serogroup O:11) serum-resistant or -sensitive strains . Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.

Immunol Rev, 2000 Feb, 173, 5 - 16
Innate immunity and the normal microflora; Boman HG; This paper discusses the following ten subtitles with the contents indicated . 1 . To meet a microbe: discusses the four alternatives in host-microbe interactions . 2 . Receptors and signal transduction giving gene activation: discusses the lipopolysaccharide receptor and the limitations of cell cultures versus use of live animals . 3 . Effector molecules--antimicrobial peptides with and without cysteines . A data base exists with over 500 sequences . This paper gives a general overview of five classes of gene-encoded effector molecules, based on the absence or presence of cysteines . These molecules are peptide antibiotics with wide spectra against different microbes . They are synthesized as propeptides and post-translational modifications are common . 4 . Effectors of innate immunity--lethal action without host damage: evaluates current opinions about the mode of action of peptide antibiotics and the fact that these effectors do not create host damage . 5 . Genes, introns and movable elements . Two cecropin genes containing movable elements and the human cathelicidin gene for proFALL-39/hCAP18 are discussed . 6 . The natural microflora . Hippos or frogs as model systems . This section includes the isolation of bacteria from the normal flora of frogs; Aeromonas hydrophila, the bacterium found on all five frog species studied; arguments and selected examples of frog-microbe interactions in vivo and in vitro; and the use of glucocorticoids as control for nuclear factor-kappa B/I kappa B alpha regulation of effector genes . 7 . The use of germ-free mice--hard facts from hard work: summarizes new findings which indicate that germ-free mice are born with a set of antibacterial peptides in their small intestine . The intestine of germ-free mice monoinfected with A . hydrophila have peptide patterns that differ depending on a pretreatment with cortisone . 8 . Looking back--an evolutionary perspective on innate immunity: arguments for an early evolutionary need for gene-encoded antibacterial factors . Caenorhabditis elegans should provide some answers . The finding of cecropin-like peptides in Helicobacter pylori and the indications that cecropins are derived from ribosomal protein L1 . 9 . What about viruses? Arguments for the lack of innate immunity against viruses . 10 . Five questions floating in the pond of immunology . The normal microflora, its size and control are too often left out from immunological thinking . Animal model systems may sometimes invite misinterpretation . Which animal species are more equal than others?

Dis Aquat Organ, 2000 Jan 14, 39(2), 109 - 19
Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses; O'hIci B et al.; The current taxonomy of Aeromonas salmonicida includes 4 subspecies . A . salmonicida subsp . salmonicida is associated with salmonid furunculosis, and A . salmonicida subsp . achromogenes, A . salmonicida subsp . masoucida, and A . salmonicida subsp . smithia are strains that show variation in some biochemical properties . This classification does not readily encompass isolates from a wide range of fish hosts currently described as atypical A . salmonicida . This study examined 17 typical strains, 39 atypical strains and 3 type A . salmonicida subspecies strains for genetic similarity using the random amplified polymophic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques . On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed . The results showed that species A . salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A . salmonicida subsp . salmonicida type strain.

J Inorg Biochem, 2000 Jan 15, 78(1), 43 - 54
Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinethiol: kinetic and spectroscopic characterization of a slow, tight-binding inhibitor-enzyme complex; Bienvenue DL et al.; The peptide inhibitor L-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP) . The overall potency (K(I)*) of LeuSH was 7 nM while the corresponding alcohol L-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (K(I) = 17 microM) . These data suggest that the free thiol is likely involved in the formation of the E x I and E x I* complexes, presumably providing a metal ligand . In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the electronic absorption and EPR spectra of {CoCo(AAP)}, {CoZn(AAP)}, and {ZnCo(AAP)} in the presence of both inhibitors . In the presence of LeuSH, all three Co(II)-substituted AAP enzymes exhibited an absorption band centered at 295 nm, characteristic of a S --> Co(II) ligand-metal charge-transfer band . In addition, absorption spectra recorded in the 450 to 700 nm region all showed changes characteristic of LeuSH and LeuOH interacting with both metal ions . EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that, in a given enzyme molecule, LeuSH interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified mu-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon binding LeuSH . EPR spectra of {CoCo(AAP)}-LeuSH, {ZnCo(AAP)}-LeuSH, and {Co_(AAP)}-LeuSH were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW) . These signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) approximately 6.5 that is characteristic of thiolate-Co(II) interactions . Combination of the electronic absorption and EPR data indicates that LeuSH perturbs the electronic structure of both metal ions in the dinuclear active site of AAP . Since the spin-spin interaction seen in resting {CoCo(AAP)} is abolished upon the addition of LeuSH, it is unlikely that a mu-S(R) bridge is established.

Dig Dis Sci, 2000 Feb, 45(2), 272 - 80
Isolation of bacteria other than Helicobacter pylori from stomachs of squirrel monkeys (Saimiri spp.) with gastritis; Khanolkar-Gaitonde SS et al.; Gastric biopsy specimens obtained from 12 squirrel monkeys (Saimiri spp.) were investigated by culture for the presence of bacteria . The stomachs of two monkeys with gastritis were colonized with gram-negative, urease-positive bacteria, identified as Ochrobactrum anthropi by the Vitek and API NFT methods (BioMerieux) . A third monkey with gastritis was positive for Aeromonas salmonicida and Pseudomonas vesicularis (both urease-negative) . No Helicobacter pylori was isolated from squirrel monkeys . Light microscopic and transmission electron microscopic examination revealed that the O . anthropi isolates were covered by extracellular material, indicating a capsule . Characterization of the O . anthropi urease revealed Michaelis-Menten constants (Km values) of 6.2 and 4.0 mM urea for the ureases of O . anthropi isolates S664 and S1835, respectively, and 3.7 for type strain 49188 . Western blot analysis using H . pylori- and H . felis-specific antibodies detected shared antigenic epitopes between the ureases of H . pylori, H . felis, and O . anthropi . The apparent molecular mass of the urease enzymes of the O . anthropi isolates was determined on 6% nondenaturing gels to be approximately 82 kDa . Antimicrobial susceptibility tests, using the MicroScan method (Dade International), revealed multidrug resistance for the O . anthropi isolates with susceptibilities for the antibiotics amikacin, ciprofloxacin, gentamicin, cefoperazone, tobramycin, imipenem, and trimethoprim/sulfamethoxazole.

An Med Interna, 1999 Dec, 16(12), 635 - 6
{Cellulitis caused by Aeromonas hydrophila}; de la Fuente Aguado J et al.; We report the case of a cirrhotic patient with leukocytoclastic vasculitis who developed a rapid and progressive cellulitis with hemorrhagic bulla and sepsis due to Aeromonas hydrophila, the portal of entry was the surgical leech of a cutaneous biopsy.

FEBS Lett, 2000 Feb 11, 467(2-3), 221 - 5
Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036; Hernandez Valladares M et al.; Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila . The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor . Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue . Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism . A possible partition of metal ions between the two binding sites is discussed.

J Med Microbiol, 2000 Feb, 49(2), 121 - 6
Production of an enterotoxin by a gastro-enteritis-associated Aeromonas strain; Trower CJ et al.; The potential of motile Aeromonas species to cause human gastrointestinal infections has been recognised recently . Considerable worldwide epidemiological, microbiological and clinical investigations have shown that some strains of the different motile aeromonads are of increasing enteropathogenic significance, especially in children, the elderly and in immunocompromised individuals . Some of the diarrhoeal symptoms of Aeromonas-associated gastro-enteritis have been attributed to enterotoxins . In this study, 15 Aeromonas isolates from clinical and non-clinical sources, representing the three motile aeromonads commonly associated with gastro-enteritis (A . caviae, A . hydrophila and A . veronii biovar sobria), were tested for their ability to cause fluid accumulation in infant mice by the suckling mouse technique . Eight isolates were found to produce enterotoxin . Of these, an A . veronii biovar sobria strain (AS15), isolated from lamb kidney, was found to produce the highest enterotoxin score . An enterotoxin of c . 40 kDa produced by A . veronii biovar sobria AS15 was purified by Sephacryl S-100 gel filtration and high-performance liquid chromatography . This enterotoxin caused marked fluid accumulation in infant mice by the suckling mouse technique . The purified enterotoxin cross-reacted with cholera toxin antibodies and was readily inactivated by heating at 56 degrees C for 10 min . The production of a 'cholera-like' enterotoxin by Aeromonas isolates from samples of animal origin suggests that these organisms could be of public health significance in food products.

J Food Prot, 2000 Dec, 63(12), 1754 - 7
Prevalence and molecular characterization of Aeromonas spp . in ready-to-eat foods in Italy; Villari P et al.; A survey was carried out in Italy to ascertain the prevalence of Aeromonas spp . in ready-to-eat foods (vegetables, cheeses, meat products, and ice creams) and the level of molecular heterogeneity of the isolates found by macrorestriction analysis of genomic DNA with pulsed-field gel electrophoresis (PFGE) . In total, 46 (14.4%) of the 320 food samples examined were found positive for Aeromonas spp . The highest percentages of isolation were discovered in vegetables, particularly lettuce (45.0%), endive (40.0%), and rucola (20.0%) . Ricotta was the only cheese type analyzed that showed a high frequency of isolation (45.0%) . Among meat products, salami and raw ham (25.0% of samples positive) and, to a lesser extent, baloney (5.0%) were found positive for Aeromonas spp . Aeromonas hydrophila was the most common isolate from foods of animal origin, whereas Aeromonas caviae was the dominant species in vegetables . No motile aeromonads were found in ice cream samples . Aeromonas isolates showed a high level of genetic heterogeneity, because 24 PFGE patterns were identified among 27 A . hydrophila strains and 20 PFGE patterns were found in 23 A . caviae isolates . In conclusion, consumers of ready-to-eat foods in Italy are regularly exposed to many genetically distinct strains of A . hydrophila and A . caviae without evident signs of malaise, and therefore, few of these strains, if any, are likely to be pathogenic.

Microb Pathog, 2000 Jan, 28(1), 25 - 36
Carboxy terminal region of haemolysin of Aeromonas sobria triggers dimerization; Nomura T et al.; Haemolysin of Aeromonas sobria is released into the culture supernatant in the form of prohaemolysin . Removal of a 42 amino acid peptide at the carboxy-terminal end converts prohaemolysin into mature haemolysin . As the role of the peptide removed from the mature haemolysin has not been studied, we mutated the haemolysin genes to delete several amino acid residues from the carboxy terminus, expressed the mutant genes in A . sobria and analysed the haemolysins produced . Deletion of more than three amino acid residues significantly reduced the efficiency of secretion of haemolysin into the culture supernatant . Mutant haemolysins with deletion of 10 amino acids were easily degraded in cells . Furthermore, cross-linking experiments indicated that the haemolysins dimerize in cells, and thus dimerized haemolysins are translocated across the outer membrane and appear in the culture supernatant . These results indicated that the carboxy-terminal end of prohaemolysin triggers dimerization of haemolysin in cells, resulting in the efficient secretion of haemolysin into the culture supernatant .

J Biol Chem, 1999 Dec 31, 274(53), 37705 - 8
Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding; Fivaz M et al.; The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor . Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml . At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium . All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding . In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.

J Pediatr Hematol Oncol, 1999 Nov-Dec, 21(6), 551 - 3
Aeromonas abscess in an immunocompromised child; Halley M et al.; An 11-year-old immunocompromised child developed cellulitis and abscess due to Aeromonas hydrophila at the site of bone marrow aspiration after swimming in a freshwater lake . The patient required treatment with intravenous antibiotics and surgical debridement to eradicate the infection . Both common and unusual organisms may complicate infections at the sites of percutaneous procedures.

Syst Appl Microbiol, 1999 Sep, 22(3), 403 - 11
Polymerase chain reaction (PCR)-based typing analysis of atypical isolates of the fish pathogen Aeromonas salmonicida; Hoie S et al.; Two hundred and five isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and countries were characterized by polymerase chain reaction (PCR) targeting four genes . The chosen genes were those encoding the extracellular A-layer protein (AP), the serine protease (Sprot), the glycerophospholipid:cholestrol acetyltransferase protein (GCAT), and the 16S rRNA (16S rDNA) . All the atypical A . salmonicida isolates could be assigned to 4 PCR groups . Group 1 comprised 45 strains which tested positive for PCR amplification, using the 16S rDNA, GCAT2, Sprot2, and AP primer-sets . Group 2 comprised 88 strains with produced PCR products using the 16S rDNA, GCAT2 and AP primer-sets . Group 3 comprised 21 strains which produced PCR products using 16S rDNA, GCAT2 and Sprot2 primer-sets, and group 4 comprised 51 strains which produced PCR products using the 16S rDNA and GCAT2 primer-sets only . A . salmonicida subsp . salmonicida isolates tested, belonged to group 1 . The PCR primer-sets separated A . salmonicida from other reference strains of Aeromonas species and related bacteria with the exception of Aeromonas hydrophila . The results indicated that PCR typing is a useful framework for characterization of the increasing number of isolations of atypical A . salmonicida.

Protein Sci, 1999 Nov, 8(11), 2546 - 9
The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design; Mahadevan D et al.; The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp . (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold . A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR) . When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains . Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR . The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern . Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid . In contrast, no enzyme function has been ascribed to the TfR.

Biotechnol Bioeng, 2000 Jan 20, 67(2), 240 - 4
Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by high-cell-density cultivation of Aeromonas hydrophila; Lee SH et al.; The newly screened Aeromonas hydrophila produces copolymer consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) . The characteristics of cell growth and polymer accumulation were examined using various carbon sources . P(3HB-co-3HHx) was produced from lauric acid and oleic acid only . P(3HB-co-3HHx) content can be increased by limitation of phosphorus . A maximal P(3HB-co-3HHx) content of 28.8 wt% could be obtained in flask culture . By applying the optimally designed nutrient feeding strategy, cell dry weight, P(3HB-co-3HHx) content, and 3HHx fraction obtained over the course of 43 h were 95.7 g/L, 45.2 wt%, and 17 mol%, respectively, resulting in a productivity of 1.01 g polyhydroxyalkanoate (PHA)/L . h .

J Gastroenterol, 1999 Dec, 34(6), 700 - 1
Skip colonic ulceration in typhoid ileo-colitis; Wong SY et al.; Colonic skip lesions are typically described in Crohn's colitis, but this phenomenon has been recognized in ulcerative colitis (skipped appendiceal involvement), Behcet's colitis, cytomegaloviral colitis, and even in Aeromonas hydrophilia and Histoplasma capsulatum infection . However, skip lesions in typhoid ileo-colitis have not been reported in the English-language literature . We report herein a patient with skip ulcers due to typhoid fever.

Microbes Infect, 1999 Nov, 1(13), 1129 - 37
Enterotoxins in Aeromonas-associated gastroenteritis; Chopra AK et al.; Aeromonas species produce an array of virulence factors, and the pathogenesis of Aeromonas infections is therefore complex and multifactorial . Aeromonas-associated gastroenteritis is especially a problem in young children . The potential involvement of enterotoxins in the pathogenesis of Aeromonas infections is discussed.

Appl Environ Microbiol, 1999 Dec, 65(12), 5612 - 4
Hemolytic activity and siderophore production in different Aeromonas species isolated from fish; Santos JA et al.; The hemolytic activity and siderophore production of several strains of motile aeromonads were determined . The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples . The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure . Siderophore production was demonstrated in all but one strain of Aeromonas jandaei . No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.

Appl Environ Microbiol, 1999 Dec, 65(12), 5293 - 302
PCR detection, characterization, and distribution of virulence genes in Aeromonas spp; Kingombe CI et al.; We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no . M84709) against aerolysin genes of Aeromonas spp., suggesting the possibility of selecting common primers . Identities of 90 to 100% were found among the eight selected primers from those genes . Amplicons obtained from Aeromonas sp . reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long . Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive . PCR-restriction fragment length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns . PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11 . PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11 . PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13 . PCR-amplicon sequence analysis (PCR-ASA) revealed three main types . PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11 . PCR-ASA 2, with 82% homology, was found only in HG13 . PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11 . This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used . In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp . It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

Lett Appl Microbiol, 1999 Oct, 29(4), 211 - 5
Survival ability of cytotoxic strains of motile Aeromonas spp . in different types of water; Brandi G et al.; The ability of motile Aeromonas spp . to survive in drinking water (mineral and tap water) and in sea water was experimentally tested . Clinically isolated cytotoxic strains of A . hydrophila, A . caviae and A . sobria were selected for this study . After contamination of water samples, the survival of Aeromonas strains was studied for at least three months using viable counts . The results obtained show that the survival of the Aeromonas spp . varies considerably depending on species and water type . For all three species, the survival time was longest in mineral water, where viable bacteria of each strain were still detected after 100 d . Moreover, A hydrophila and A . caviae also re-grew on the first day . In tap water all strains showed marked survival, although to a lesser extent than in mineral water . Aeromonas cells showed a rapid decline in sea water (90% reduction in viable cells after about two d) and thus seem to be more sensitive to saline/marine stress than chlorination.

J Appl Microbiol, 1999 Oct, 87(4), 620 - 9
Monoclonal antibodies against AsaP1, a major exotoxin of the fish pathogen Aeromonas salmonicida subsp . achromogenes, and their application in ELISA; Wagner U et al.; Two monoclonal antibodies (Mabs) binding to a toxic extracellular metallo-proteinase of Aeromonas salmonicida subsp . achromogenes, AsaP1, were produced . Both reacted with common epitopes of the native enzyme and recognized this 20 kDa antigen on Western blots . One of these Mabs had an inhibitory effect on the caseinase activity of the exotoxin . A Mab-based ELISA was set up and evaluated for serological detection of AsaP1 in bacterial culture filtrates . The exotoxin was identified serologically in the extracellular products of 11 of 26 atypical Aer . salmonicida isolates, including the type strain for subsp . achromogenes NCIMB 1110 . The ELISA was approximately 100-fold more sensitive in detecting AsaP1 compared with an azocasein assay . The established serological test enables AsaP1 to be quantified reliably with a lower detection limit of about 0.12 ng ml-1 and has a potential use for the phenotypic differentiation of atypical Aer . salmonicida isolates.

Epidemiol Infect, 1999 Oct, 123(2), 299 - 307
Genetic diversity of atypical Aeromonas salmonicida studied by pulsed-field gel electrophoresis; Hanninen ML et al.; Pulsed-field gel electrophoresis (PFGE) pattern analysis with XbaI restriction enzyme was used to study the genetic heterogeneity of 88 atypical Aeromonas salmonicida strains which were earlier or during this study characterized phenotypically, by ribotyping (ClaI/PstI) and by plasmid profile analysis . The strains of certain'ribotypes were also analysed by digestion with SpeI . The strains represented different geographic locations: Finland (72 strains), Iceland (5 strains), Norway (5 strains), Sweden (4 strains) and Denmark (2 strains), and they were from 17 fish species during 1981 97 . Thirty-one PFGE genotypes found among these strains correlated well with the ribotypes, and in most cases PFGE pattern analysis subdivided ribotypes into several PFGE genotypes, and further within a PFGE genotype into subtypes . XbaI and SpeeI digests produced concordant results . In most cases, PFGE patterns of strains with the same ribotype shared many fragments, suggesting genetic relatedness . PFGE patterns of most Norwegian and Icelandic strains isolated during an approximately 10-year period had the same ribotype and their PFGE patterns shared most fragments, suggesting close genetic relatedness . Moreover, atypical strains of ribotypes B/B and H/H isolated from the same Finnish fish farms had closely related patterns suggesting genetic stability and persistence of these genotypes . Genotype 29 of Achromogenic strains was strongly associated with disease of Finnish arctic char and grayling . PFGE was shown to be a distinguishing method to study the genetic heterogeneity of atypical A . salmonicida . epidemiology of these infections.

Crit Care Med, 1999 Nov, 27(11), 2485 - 94
Platelet-activating factor and arachidonic acid metabolites mediate tumor necrosis factor and eicosanoid kinetics and cardiopulmonary dysfunction during bacteremic shock; Quinn JV et al.; OBJECTIVE: Platelet-activating factor (PAF) and eicosanoids are putative mediators of septic shock that are associated with release of tumor necrosis factor (TNF) . The purpose of this investigation was to a) examine temporal patterns of TNF and arachidonic acid metabolite release in a porcine model of bacteremic shock and b) selectively block PAF, thromboxane A2, prostacyclin, and leukotrienes to determine the relationships among these inflammatory response mediators and the alterations in cardiorespiratory dysfunction for which they are required . DESIGN: Prospective, nonrandomized, controlled trial . SETTING: Laboratory at a university medical center . SUBJECTS: Thirty-four female Yorkshire swine . INTERVENTIONS: Animals were divided into six experimental groups: five septic groups receiving an infusion of Aeromonas hydrophila at 0.2 mL/kg/hr, gradually increasing to 0.4 mL/kg/hr over 4 hrs . Each of four septic groups was pretreated with a specific mediator inhibitor (PAF receptor antagonist, n = 6; prostacyclin antibody, n = 5; leukotriene synthesis inhibitor, n = 5; and thromboxane receptor antagonist, n = 6) . One septic group (n = 6) received no mediator inhibitor and served as a septic control, and one anesthesia control group (n = 6) received no intervention . MEASUREMENTS AND MAIN RESULTS: PAF receptor blockade significantly increased systemic hypotension and mixed venous oxygen saturation and decreased pulmonary artery pressure, oxygen extraction and consumption, hemoconcentration, and levels of TNF and eicosanoids . Leukotriene inhibition increased mean arterial pressure, pulmonary and systemic vascular resistance indices, and arterial and mixed venous oxygen saturation and reduced pulmonary hypertension, oxygen delivery, oxygen extraction, oxygen consumption, and all measured mediators . Thromboxane receptor blockade lowered TNF and leukotriene levels, ameliorated systemic and pulmonary vasoconstriction, and significantly increased arterial and tissue oxygenation compared with septic controls . Prostacyclin antagonism reduced prostacyclin plasma concentrations, arterial hypoxemia, and oxygen consumption during sepsis and increased circulating leukotriene B4 . CONCLUSIONS: Elevations in plasma TNF predictably precede peak levels of eicosanoids in this model . PAF, leukotrienes, and thromboxane A2 are necessary for pulmonary hypertension during bacteremia . Systemic hypotension and increased vascular permeability are mediated by both leukotrienes and PAF . There are complex interactions among mediators during sepsis and further studies are required to define these relationships.

Indian J Med Res, 1999 Aug, 110, 50 - 5
Occurrence of haemolytic & cytotoxic Aeromonas species in domestic water supplies in Chennai; Alavandi SV et al.; A study on the occurrence of Aeromonas species in the domestic water supplies in Chennai showed that as much as 37.9 per cent of the water samples analyzed from various sources harbored Aeromonas spp . Majority of the isolates belonged to Aeromonas sobria (13.7%), A . caviae (11.6%) and A . hydrophila (9.5%) . Among the 37 metropolitan water samples analyzed, 11 samples yielded Aeromonas spp . inclusive of three isolates of A . hydrophila, four of A . sobria and two isolates each of A . caviae and A . jandaei . From a total of 28 bore well water samples analyzed, Aeromonas spp . were recovered from 15 samples, comprising five isolates of A . hydrophila, six of A . sobria and four isolates of A . caviae . Aeromonas spp . inclusive of one isolate of A . hydrophila, five of A . caviae, three of A . sobria and one isolate of A . veronii were isolated from 10 of the 30 water packets of various commercial brands sold in Chennai . Of a total of 36 isolates obtained, 32 (89%) produced beta-haemolysin with the titres ranging from 2-32 and 20 isolates (56%) were cytotoxic to vero cell monolayers . All the Aeromonas isolates were resistant to ampicillin and polymyxin B . All A . hydrophila and A . caviae isolates were also resistant to cephalothin and erythromycin and 83.3 per cent of Aeromonas isolates were resistant to erythromycin . Aeromonads resistant to tetracycline, gentamycin, co-trimoxazole and nalidixic acid appear to be emerging . The study revealed that Aeromonas spp . occur in the potable and domestic water supplies and even in the chlorinated water supplies in Chennai city, which are potentially enteropathogenic and hence may be hazardous to public health . In view of these findings drinking and domestic water quality standards need to be re-evaluated.

Biochemistry, 1999 Nov 23, 38(47), 15587 - 96
Slow-binding inhibition of the aminopeptidase from Aeromonas proteolytica by peptide thiols: synthesis and spectroscopic characterization; Huntington KM et al.; Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a-c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP) . The overall potencies (K(I)) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin . The corresponding alcohols (2a-b) are simple competitive inhibitors of much lower potencies (K(I) = 23 and 360 microM) . These data suggest that the free thiols are involved in the formation of the E . I and E.I complexes, presumably serving as a metal ligand . To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c . Both {CoZn(AAP)} and {ZnCo(AAP)}, in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S --> Co(II) ligand-metal charge-transfer band . In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion . EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified micro-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding . EPR spectra of {CoCo(AAP)}-1c, {ZnCo(AAP)}-1c, and {CoZn(AAP)}-1c were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW) . The observed signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) approximately 6.8 that is characteristic of thiolate-Co(II) interactions . These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster . Compounds 1a-c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors.

Kansenshogaku Zasshi, 1999 Oct, 73(10), 1074 - 7
{Aeromonas hydrophila septicemia with necrotizing myofascitis in a patient with hypoplastic leukemia}; Yata K et al.; A 54-year-old male was admitted to Kawasaki Medical School Hospital with the complaint of fever . His diagnosis of hypoplastic leukemia had been made one year ago . After the admission, cecal mass with pain and high fever were noted . Four days later, he suddenly lost consciousness and died . Aeromonas hydrophila was isolated from blood cultures and also from the myofascitis specimen . Autopsy specimen of the iliopsoas muscle showed necrotizing myofascitis . The specimen obtained from the cecum showed submucosal hemorrhage with edema and these findings were compatible to ischemic colitis . This pathogen is widely distributed in nature, especially in water fields . Therefore, it would be advised to consider the Aeromonas hydrophila as one of the pathological organisms pathognomonic for the septicemia, when one may see febrile and gastrointestinal symptoms in a patient with hematological malignancies.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1403 - 8
Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501; Martinez-Murcia AJ; The 16S rDNA sequences of the recently described Aeromonas encheleia and Aeromonas popoffii, were determined and compared with data from all known Aeromonas sp . Diagnostic 16S rDNA regions were also sequenced for some strains previously considered as an extension of A . encheleia and a strain of Aeromonas Group 501 (formerly Enteric Group 501) . Results indicated that A . encheleia and A . popoffii are phylogenetically separated species as originally described . A conclusion about HG11 taxonomic status is not recommended until previous discrepancies are clarified by further DNA-DNA hybridization and sequencing studies.

Microb Pathog, 1999 Oct, 27(4), 215 - 21
Identification and characterization of the Aeromonas sobria hemolysin glycoprotein receptor on intestine 407 cells; Wang A et al.; Aeromonas sobria hemolysin is important in the pathogenesis of diarrhoea caused by this enteropathogenic bacterium . By immunoprecipitation analysis using hemolysin and anti-hemolysin antibody, a 66 kDa protein (p66) was identified as a receptor for A . sobria hemolysin on Intestine 407 cells . Treatment of p66 with N-glycosidase F reduced the apparent sized of p66 to 60 kDa on SDS-polyacrylamide gels . p66, released from Intestine 407 cells following incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, bound A . sobria hemolysin . Thus treatment of Intestine 407 cells with PI-PLC resulted in the remarkable decrease of the sensitivity to A . sobria hemolysin . These results are consistent with the hypothesis that p66, the binding protein for A . sobria hemolysin, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the surface of Intestine 407 cells and probably plays a role as a receptor for A . sobria hemolysin on the intestinal cells .

Biosci Biotechnol Biochem, 1999 Aug, 63(8), 1346 - 52
XynX, a possible exo-xylanase of Aeromonas caviae ME-1 that produces exclusively xylobiose and xylotetraose from xylan; Usui K et al.; A gene, xynX, encoding a novel xylanase, was cloned from Aeromonas caviae ME-1 . This gene encoded an enzyme that was constituted of 334 amino acid residues (38,580 Da) and was similar in sequence to Family 10 (Family F) beta-1,4 endo-xylanases . XynX produced only xylobiose and xylotetraose from birch wood xylan, and xylotriose, xylopentaose, and higher oligosaccharides were not detected in the TLC analysis . We designated it as X2/X4-forming xylanase . This enzyme does not have transglycosylation activity . These data suggested that this enzyme is a possible exo-xylanase . According to homology modeling, the enzyme has a ring-shaped (alpha/beta)8 barrel (TIM barrel) structure, typical of Family 10 endo-xylanases, with the extraordinary feature of a longer bottom-loop structure.

Infect Immun, 1999 Oct, 67(10), 5192 - 9
Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila; Swift S et al.; In Aeromonas hydrophila, the ahyI gene encodes a protein responsible for the synthesis of the quorum sensing signal N-butanoyl-L-homoserine lactone (C4-HSL) . Inactivation of the ahyI gene on the A . hydrophila chromosome abolishes C4-HSL production . The exoprotease activity of A . hydrophila consists of both serine protease and metalloprotease activities; in the ahyI-negative strain, both are substantially reduced but can be restored by the addition of exogenous C4-HSL . In contrast, mutation of the LuxR homolog AhyR results in the loss of both exoprotease activities, which cannot be restored by exogenous C4-HSL . Furthermore, a substantial reduction in the production of exoprotease by the ahyI+ parent strain is obtained by the addition of N-acylhomoserine lactone analogs that have acyl side chains of 10, 12, or 14 carbons . The inclusion of N-(3-oxododecanoyl)-L-homoserine lactone or N-(3-oxotetradecanoyl)-L-homoserine lactone at 10 microM in overnight cultures of A . hydrophila abolishes exoprotease production in azocasein assays and reduces the activity of all the exoprotease species seen in zymograms.

J Food Prot, 1999 Sep, 62(9), 1045 - 9
Numbers and species of motile aeromonads during the manufacture of naturally contaminated Spanish fermented sausages (longaniza and chorizo); Encinas JP et al.; The prevalence of aeromonads in the initial mixes of 14 batches of chorizo and longaniza obtained from three small- and middle-sized factories was 78.5%, with counts ranging from >1.00 to 4.47 log10 CFU/g . Only 2 of 10 mixture samples prepared at a large and modern processing plant yielded these organisms, with levels below 1 log10 CFU/g . The hygienic status of factories significantly affected incidence and counts . Of 39 presumptive isolates from glutamate starch penicillin agar, 36 were confirmed as motile aeromonads and allocated to Aeromonas hydrophila (n = 24), A . veronii biovar sobria (n = 10), and A . caviae (n = 2) . All of them were beta-hemolytic and capable of growing at 5 degrees C . Regardless of initial contamination, aeromonads were rapidly inactivated during the early stages of manufacture.

J Clin Microbiol, 1999 Oct, 37(10), 3194 - 7
Diverse restriction fragment length polymorphism patterns of the PCR-amplified 16S rRNA genes in Aeromonas veronii strains and possible misidentification of Aeromonas species; Graf J; Restriction fragment length polymorphism analysis after PCR amplification (RFLP-PCR) of the 16S rRNA gene has been previously proposed as a rapid method to identify Aeromonas species . In the present study, the precision of RFLP-PCR was evaluated with 62 Aeromonas reference strains . The analysis revealed that Aeromonas veronii biovar sobria strains produce various patterns, possibly leading to its misidentification as an environmental species . For most other Aeromonas species little variation was noted . This study supports the usefulness of RFLP-PCR analysis to separate three clinically important species but also reveals possible misidentifications that necessitate further biochemical tests to validate the preliminary identification.

J Wildl Dis, 1999 Jul, 35(3), 536 - 41
Effects of malathion on disease susceptibility in Woodhouse's toads; Taylor SK et al.; Adult male Woodhouse's toads (Bufo woodhousi) developed clinical disease, hepatomegaly, and died at a higher rate when externally exposed once to either a high or low sublethal dose (0.011 or 0.0011 mg malathion/g toad) of field grade malathion and challenged with a sublethal dose of Aeromonas hydrophila injected intraperintoneally (1.1 x 10(4) bacteria/g toad) when compared to toads not exposed to malathion but challenged with A . hydrophila (P < 0.007) . Toads exposed to malathion (high or low dose) and challenged with A . hyydrophila had clinical disease, hepatomegaly, and died at a higher rate {9 (90%) of 10} than toads exposed to malathion alone (P < 0.002) . Toads exposed to the high and low doses of malathion had a 22% and 17% decrease in brain cholinesterase levels, respectively, when they were compared to nonmalathion exposed toads (P < 0.025, P < 0.006) . It appears that field grade malathion applied externally to adult Woodhouse's toads may cause increased disease susceptibility when challenged with a potentially pathogenic bacteria.

Vet Res, 1999 Jul-Aug, 30(4), 411 - 8
Effects of dimerized lysozyme (KLP-602) on the cellular and humoral defence mechanisms in sheatfish (Silurus glanis): in vitro and in vivo study; Morand M et al.; This study examined the effects of the dimerized lysozyme (KLP-602) on the immunocompetence cell activity in sheatfish (Silurus glanis) and its influence in vivo on the non-specific defence mechanisms and protection against motile aeromonad septicaemia (MAS) . The in vitro study showed that the lysozyme dimer (KLP-602), at concentrations between 5 and 50 micrograms/mL of medium significantly (P < 0.05) increased the respiratory burst activity and potential killing activity of pronephric macrophages, as well as the proliferative ability of pronephric lymphocytes stimulated by ConA and LPS . The in vivo study showed that injecting lysozyme dimer (Lydium-KLP) intraperitoneally at doses of 50 micrograms/kg bw stimulated cell-mediated and humoral-mediated imunity . On day 5, after application of Lydium-KLP in vivo, a statistically higher (P < 0.05) respiratory burst activity and potential killing activity of blood and pronephros phagocytes were observed . A higher proliferative ability of blood and pronephros lymphocytes stimulated by Concanavaline A (ConA) or lipopolysaccharide (LPS) was also observed . At the same time, the myeloperoxidase activity in the PMN cells and the lysozyme activity and total Ig levels in serum were significantly higher (P < 0.05), compared to the control group . A challenge test with Aeromonas hydrophila showed that dimerized lysozyme increased the protection against MAS . Dimerized lysozyme stimulates non-specific cellular and humoral mechanisms and protection against MAS in sheatfish.

Biochemistry, 1999 Aug 31, 38(35), 11433 - 9
Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols . Characterization of the hydrophobic substrate recognition site; Ustynyuk L et al.; Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP) . This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP . Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed . On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged . These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v) . All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM . The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase . Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol . The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme . On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP . Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition . Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate . Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

Res Microbiol, 1999 Jul-Aug, 150(6), 395 - 402
Two genes from the capsule of Aeromonas hydrophila (serogroup O:34) confer serum resistance to Escherichia coli K12 strains; Aguilar A et al.; The Escherichia coli DH5alpha strain as well as other K12-derived strains are unable to produce O-specific lipopolysaccharide and are thus rough and serum-sensitive . One representative recombinant clone (COS-SR1) containing Aeromonas hydrophila (serogroup O:34) chromosomal DNA conferred serum resistance to E . coli K12 strains . Genetic, biochemical, and immunological studies suggested that the two genes (orf1 and wcaJ) identified in a subclone (pAC-SR9) of COS-SR1 are necessary for the production of the colanic acid capsule at 37 degrees C on E . coli DH5alpha, rendering the strain serum-resistant . A . hydrophila strains from serogroup O:34 are able to produce capsule when they grow both in synthetic medium and in an autolysate of fish viscera . However, defined wcaJ insertion mutants of A . hydrophila 1051-88 (serogroup O:34) are unable to produce capsule on these media . This strongly suggests that both genes belong to the gene cluster responsible for capsule production (wca) of A . hydrophila 1051-88 (serogroup O:34).

Shock, 1999 Jun, 11(6), 423 - 8
Interleukin-1 mediates hemodynamic dysfunction and release of eicosanoids and tumor necrosis factor during graded bacteremia; Santos MC et al.; The pathophysiologic events of sepsis mediated by interleukin-1 (IL-1) remain ill-defined . The purpose of this study was to identify the circulatory derangements of which IL-1 was a necessary mediator and evaluate its interactions with tumor necrosis factor (TNF) and the eicosanoids during graded bacteremia . Eleven adult female swine were anesthetized, mechanically ventilated, and monitored with pulmonary artery catheters and arterial lines; they received intravenously either saline vehicle (septic control, n = 6) or human recombinant IL-1 receptor antagonist (IL-1ra, n = 5) . The animals were then infused with Aeromonas hydrophila (10(9)/mL) for 4 h at rates gradually increased from .2 mL/kg/h to 4 mL/kg/h over 3 h, then sacrificed after 4 h . Mean arterial pressure (MAP), left ventricular stroke work index (LVSWI), and systemic vascular resistance index (SVRI) were recorded at baseline and hourly thereafter, and plasma 6-keto-PGF1alpha (6-KETO), tumor necrosis factor-alpha (TNF) and leukotrienes B4(LTB4) and C4D4E4 (LTCDE), pg/mL, were measured by ELISA . MAP, LVSWI, arterial P(O2) all decreased in the septic control group to levels significantly below those of the IL-1 antagonist animals . Circulating 6-KETO, LTCDE, and TNF increased significantly in all septic animals . Plasma LTB, and TNF were reduced by IL-1 blockade, compared with septic controls . TxB2 was not affected by IL-1 inhibition . There were no intergroup differences in platelet aggregation, but the in vitro aggregation response decreased from baseline in septic controls to 54+/-27% (p < .05) . IL-1 is necessary to the development of systemic hypotension impaired LVSWI, and increased intravascular platelet aggregation during graded bacteremia . Conversely, IL-1 helps to maintain stroke volume and low SVRI in graded bacteremia, possibly through increased prostacyclin release . It may contribute to impaired pulmonary gas exchange and increased tissue oxygen demands . TNF release is stimulated in the presence of unopposed IL-1 and may be synergistic with it in the adverse hemodynamic effects of endogenous IL-1 . IL-1 is required for increased leukotriene and prostacyclin levels in this model, but it is not involved in thromboxane release . Whether the lack of survival benefit from IL-1ra in human sepsis is due to these mixed cardiopulmonary and mediator effects, to species differences, or to timing of IL-1ra administration is not clear from the data.

Dis Aquat Organ, 1999 Jun 23, 37(1), 53 - 9
Efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, for the treatment of furunculosis in Atlantic salmon Salmo salar held in seawater; Samuelsen OB et al.; This study was performed to determine the efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, in the treatment of experimental induced furunculosis in Atlantic salmon Salmo salar held in seawater . Two strains of the causative bacterium Aeromonas salmonicida subsp . salmonicida, 1 sensitive (VI-88/09/03175) and 1 resistant (3475/90) to oxolinic acid, were used . In 2 trials, cohabitational challenges were performed by introducing 8 fish challenged in advance by an intraperitoneal injection of 2.2 x 10(4) colony forming units of strain 3475/90 (Trial 1) or strain VI-88/09/03175 (Trial 2) to 10 aquaria each containing 40 healthy fish . The treatment groups in both trials consisted of 4 groups receiving either oxolinic acid (2 groups) or Vetoquinol (2 groups) and 1 control group . An unchallenged, unmedicated group was used to determine the natural mortality in the population . The recommended therapeutic dose of 25 mg oxolinic acid kg-1 fish at Days 1, 2, 4, 6, 8 and 10 following initiation of treatment was used . Oral medication initiated at Day 10 (Trial 1) or Day 11 (Trial 2) following challenge significantly (p < 0.05) lowered the specific mortality in all drug-treated groups compared to the untreated control groups . Mortality in Vetoquinol-treated groups was significantly (p < 0.05) lower than in oxolinic acid-treated groups in Trial 1 whereas no significant (p < 0.05) difference in survival rate was found between the medicated groups in Trial 2.

Protein Expr Purif, 1999 Aug, 16(3), 396 - 404
A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization; Maurice S et al.; Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease . Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein . The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3) . The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein . Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A . salmonicida bacterial cells by the glycine and the membrane extraction methods . The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography . The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques . All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage . To date, this represents the first large-scale production of biologically active recombinant A-protein .

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 183 - 90
Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain; Taguchi K et al.; The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production . Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp . 61-3 respectively were constructed and introduced into E . coli HB101 strain . On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E . coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10 . It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 67 - 72
Hemolysin of Aeromonas sobria stimulates production of cyclic AMP by cultured cells; Fujii Y et al.; Hemolysin of Aeromonas sobria possesses both cytotoxic activity against mammalian cells and enterotoxic activity . Histopathological examination revealed that hemolysin causes diarrhea without damaging the intestinal epithelial cells . And the fluid accumulated in the mouse intestinal loop by the action of the hemolysin is watery . These observations indicated that the enterotoxic activity of hemolysin is not dependent on its cytotoxic activity . To clarify the mechanism of the enterotoxic action of hemolysin, we examined cyclic nucleotide levels in cultured cells exposed to this toxin . These results showed that hemolysin stimulates the production of cyclic AMP in cultured cells and the cyclic AMPs thus produced emerge in the milieu of cells.

Mol Microbiol, 1999 Aug, 33(3), 659 - 66
The channel-forming toxin aerolysin neutralizes human immunodeficiency virus type 1; Nguyen DH et al.; Aerolysin is a channel-forming toxin secreted by Aeromonas spp . that binds to glycosyl phosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on sensitive target cells . Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier . Human immunodeficiency virus type 1 (HIV-1) produced from T cells is known to incorporate Thy-1 and other GPI-anchored proteins into its membrane . Here, we show that aerolysin is capable of neutralizing HIV-1 in a dose-dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope . Pretreatment with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins greatly reduced HIV-1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI-anchored proteins was not neutralized . Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur . These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.

Infect Immun, 1999 Aug, 67(8), 4008 - 13
Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp . serogroup O:34; Merino S et al.; Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha . Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively . Defined insertion mutants of A . hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively . Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic . A . hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp . serogroup O:34 infection process.

Int J Biol Macromol, 1999 Jun-Jul, 25(1-3), 69 - 77
Biosynthesis of polyhydroxyalkanoates (PHA) by recombinant Ralstonia eutropha and effects of PHA synthase activity on in vivo PHA biosynthesis; Kichise T et al.; Recombinant strains of Ralstonia eutropha PHB 4, which harbored Aeromonas caviae polyhydroxyalkanoates (PHA) biosynthesis genes under the control of a promoter for R . eutropha phb operon, were examined for PHA production from various alkanoic acids . The recombinants produced poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) {P(3HB-co-3HHx)} from hexanoate and octanoate, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxypentano ate) {P(3HB-co-3HV-co-3HHp)} from pentanoate and nonanoate . One of the recombinant strains, R . eutropha PHB 4/pJRDBB39d3 harboring ORF1 and PHA synthase gene of A . caviae (phaC(Ac)) accumulated copolyesters with much more 3HHx or 3HHp fraction than the other recombinant strains . To investigate the relationship between PHA synthase activity and in vivo PHA biosynthesis in R . eutropha, the PHB- 4 strains harboring pJRDBB39d13 or pJRDEE32d13 were used, in which the heterologous expression of phaC(Ac) was controlled by promoters for R . eutropha phb operon and A . caviae pha operon, respectively . The PHA contents and PHA accumulation rates were similar between the two recombinant strains in spite of the quite different levels of PHA synthase activity, indicating that the polymerization step is not the rate-determining one in PHA biosynthesis by R . eutropha . The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains were also independent of the levels of PHA synthase activity . It has been suggested that a chain-transfer agent is generated in R . eutopha cells to regulate the chain length of polymers.

Biochemistry, 1999 Jul 13, 38(28), 9048 - 53
1-Butaneboronic acid binding to Aeromonas proteolytica aminopeptidase: a case of arrested development; De Paola CC et al.; Hydrolases containing two metal ions connected by a bridging ligand catalyze reactions important in carcinogensis, tissue repair, post-translational modification, control and regulation of biochemical pathways, and protein degradation . The aminopeptidase from Aeromonas proteolytica serves as a paradigm for the study of such bridged bimetallic proteases since its three-dimensional structure is known to very high resolution and its catalytic reaction is amenable to spectroscopic examination . Herein, we report the X-ray crystal structure at 1.9 A resolution of AAP complexed with 1-butaneboronic acid (BuBA) . This structure suggests that this complex represents a snapshot of the proteolytic reaction in an arrested form between the Michaelis complex and the transition state . Comparison of the structure with spectroscopic and other data allows us to conclude that the apparently structurally symmetrical dizinc site is actually asymmetric electrostatically.

Hum Antibodies, 1999, 9(2), 111 - 24
Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle; Nishimura E et al.; Recombinant lambda light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli . Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5 . Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of lambda light chain dimers which are less reactive than the monomeric form . The lambda light chain was overexpressed in E . coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring lambda light chain in HB4C5 . Monomeric recombinant lambda light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5 . The immunostaining of lung cancer tissue sections with the recombinant lambda light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma . Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line)--xenografted nude mice by the i.p . injection of 125I-labeled recombinant lambda light chain and 125I-labeled human lambda light chain control gave tumor-specific and recombinant lambda light chain-dependent images on day 5 postinjection, and images were also detectable on day 3 . Biodistribution studies with 125I-labeled recombinant lambda light chain demonstrated that the lambda light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125I-labeled HB4C5 which could localize to the necrotic part only . These results suggest that the recombinant lambda light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects.

Indian J Med Res, 1999 Apr, 109, 136 - 40
Biochemical characteristics & secretory activity of Aeromonas species isolated from children with gastroenteritis in Chennai; Ananthan S et al.; An attempt was made to delineate the phenotypic markers for the detection of enterotoxigenic strains of Aeromonas . Eighteen Aeromonas species comprising one isolate of A . hydrophila, six isolates of A . sobria and 11 isolates of A . caviae were obtained from 379 children suffering from acute diarrhoea in Chennai . Nine of these isolates inclusive of three A . sobria and six A . caviae were found to produce secretory response in vitro in the rabbit intestinal mucosa mounted in the Ussing chambers as revealed by significant increases in the short circuit current . Eleven strains hydrolysed aesculin, 8 fermented arabinose, 6 produced acetyl methyl carbinol, 14 produced lysine decarboxylase, 3 fermented salicin, 9 produced beta-haemolysin, 9 produced CAMP-like factor and only two isolates took up congo red dye . None of these phenotypic traits were found to correlate with the in vitro secretory activity.

J Antibiot (Tokyo), 1999 Apr, 52(4), 398 - 406
Structure elucidation of Sch 20561, a cyclic dehydropeptide lactone--a major component of W-10 antifungal antibiotic; Afonso A et al.; Antibiotic W-10 is a fermentation complex produced by the bacterium Aeromonas sp . W-10 . The cyclic dehydropeptide lactones Sch 20562 (1) and Sch 20561 (2) are the major components of this fermentation complex and are of biological interest in view of their unique structural features and potent antifungal activity . The chemical degradation studies that were utilized in the assignment of structure 2 for Sch 20561 are described here . The structure determination of 2 made use of the ozonolytic cleavage of the dehydropeptide units to form fragments that were sequenced by mass spectrometry . The cyclic dehydropeptide lactone Sch 20561 (2) was found to be the aglycone of Sch 20562 (1) and these two natural products were correlated by a chemical transformation involving the deglucosidation of 1 to form 2.

J Antibiot (Tokyo), 1999 Apr, 52(4), 383 - 97
Structure elucidation of Sch 20562, a glucosidic cyclic dehydropeptide lactone--the major component of W-10 antifungal antibiotic; Afonso A et al.; A novel bacterium designated as Aeromonas sp . W-10 produces the antibiotic W-10 complex which comprises of two major and several minor components . The two major components from this complex, Sch 20562 (1) and Sch 20561 (1a), are of biological interest in view of their potent antifungal activity . The chemical degradation studies utilized for the assignment of structure 1 for Sch 20562 are described here . Some of the noteworthy diversity of structural features in this glucosidic cyclic dehydrononapeptide lactone 1 are: an N-terminal (D)-beta-hydroxymyristyl unit, three D-amino acid units, two (E)-alpha-aminocrotonyl units, and an O-alpha-D-glucosyl-N-methyl-L-allo-threonine unit . The structure determination of 1 utilized the selective cleavage of the dehydropeptide units by ozonolysis to form fragments that were sequenced by mass spectrometry . The stereochemistry of the amino acid units were assigned by isolation of the free amino acids from the hydrolysates of the fragments . The stereochemistry of the alpha-aminocrotonyl units and the glucosidic linkage were assigned by nmr spectroscopy and molecular rotation data.

J Hosp Infect, 1999 Apr, 41(4), 313 - 6
Pseudo-outbreak of Aeromonas hydrophila isolates related to endoscopy; Esteban J et al.; Over a two-month period there was a sudden increase in Aeromonas hydrophila isolated from colon i.e., biopsies from patients without related symptoms . Strains were studied by biotyping (API 20 NE, bioMerieux), antibiotyping, plasmid analysis, SDS-PAGE of whole cell proteins and toxicity for cell cultures . All strains gave identical results . In particular SDS-PAGE of whole cell proteins showed an identical electrophoretic pattern for all the strains, and so all were considered to be of the same clone . During the study period, endoscopic materials were disinfected with quaternary ammonia and glutaraldehyde phenate . After these disinfectants were changed to glutaraldehyde 2%, there were no more isolates of A . hydrophila from the biopsies . We conclude that SDS-PAGE can be a useful technique for epidemiological characterization of A . hydrophila.

Comp Immunol Microbiol Infect Dis, 1999 Jul, 22(3), 175 - 9
Isolation and haemolytic activity of Aeromonas species from domestic dogs and cats; Ghenghesh KS et al.; Rectal swabs from 120 domestic dogs and 15 domestic cats were examined for Aeromonas species using alkaline peptone water (pH 8.6) as the enrichment medium and blood agar containing 15 mg/l ampicillin as the plating medium . Aeromonads were isolated from 13 (10.8%) dogs and from 1 (6.7%) cat . Of the 14 aeromonads isolated in the present study only 9 were available for speciation and testing in the haemolysin assay . Of these 5 were A . sobria (including one from a cat), 2 were A . hydrophila and 2 were A . caviae . Six were positive in the haemolysin assay; 4 A . sobria (one from a cat) and 2 A . hydrophila . The presence of haemolysin producing-Aeromonas species in the faeces of domestic dogs and cats may pose a public health problem for humans who come into contact with such animals.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol, 1999 May, 123(1), 53 - 9
Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin; Ottinger CA et al.; We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A . salmonicida activity of Atlantic salmon macrophages . In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A . salmonicida failed to significantly reduce (P> 0.05) cell viability . However, binding of the lectin to A . salmonicida did result in significant (P < or = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity . Significant increases (P < or = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations . At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (P < or = 0.05) in respiratory burst was observed . The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity . The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A . salmonicida.

Biochem J, 1999 Jul 1, 341 ( Pt 1), 25 - 31
Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase; Nirasawa S et al.; An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues) . We demonstrated that a proteinase, which was isolated from the culture filtrate of A . caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide . Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged . The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain . In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate . Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not . Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all . These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E . coli.

Arch Microbiol, 1999 May-Jun, 171(6), 397 - 404
Lapstatin, a new aminopeptidase inhibitor produced by Streptomyces rimosus, inhibits autogenous aminopeptidases; Repic Lampret B et al.; Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates . The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4, AG50WX4, and HPLC RP C18 columns . By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline . Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC50 in the range of 0.3-2.4 microM . IC50 values for other enzymes tested were at least tenfold higher . Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 x 10(-7) M . Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase.

J Food Prot, 1999 May, 62(5), 463 - 6
Enumeration and confirmation of Aeromonas hydrophila, Aeromonas caviae, and Aeromonas sobria isolated from raw milk and other milk products in Northern Greece; Melas DS et al.; A total of 138 raw cow's and 57 raw ewe's milk samples; 80 pasteurized cow's milk samples; 39 Anthotyros cheese, 36 Manouri cheese, and 23 Feta cheese samples; and 15 rice pudding samples were examined for the presence and any countable population of Aeromonas species . Twenty-two (15.9%) of the 138 cow's milk samples analyzed were contaminated with A . hydrophila . In 13 of these samples, populations of 3.0x10(2) to 5.0x10(3) CFU/ml were counted in starch ampicillin agar (SAA) . Eighteen cow's milk samples (13.0%) were contaminated with A . caviae, and in eight of these samples, populations of 2.0x10(2) to 3.0x10(3) CFU/ml were counted in SAA . Five cow's milk samples (3.6%) were contaminated with A . sobria, and in two of these samples, populations of 2.5x10(3) and 5.0x10(3) CFU/ml were counted in SAA . Eleven cow's milk samples (7.9%) were contaminated with other Aeromonas spp . not classified . Eight (14.0%) of the 57 ewe's milk samples analyzed were contaminated with A . hydrophila . In these samples, populations of 5.0x10(2) to 5.0x10(3) CFU/ml were counted in SAA . Six ewe's milk samples (10.5%) were contaminated with A . caviae, and populations of 1.5x10(2) to 1.0x10(3) CFU/ ml were counted in SAA . Two ewe's milk samples (3.5%) were contaminated with A . sobria, and populations counted in SAA were 5.0x10(2) and 1.0x10(3) CFU/ml . Four samples (7.0%) were contaminated with other Aeromonas spp . not classified . A . hydrophila was recovered in 4 (10.2%) and 3 (8.3%) of the Anthotyros and Manouri cheese samples analyzed, respectively, but no countable populations were noted in SAA . None of the pasteurized milk, Feta cheese, and rice pudding samples yielded Aeromonas spp . The results of this work indicate that motile Aeromonas are common in raw milk in Greece . Also, the presence of A . hydrophila in the whey cheeses Anthotyros and Manouri indicates that postprocessing contaminations of these products with motile Aeromonas may occur during production.

Ecotoxicol Environ Saf, 1999 May, 43(1), 11 - 20
Effects of chromium on humoral and cell-mediated immune responses and host resistance to disease in a freshwater catfish, Saccobranchus fossilis (Bloch); Khangarot BS et al.; The effects of subtoxic levels of Cr on humoral and cell-mediated immune responses, blood parameters, susceptibility to bacterial (Aeromonas hydrophila) infection, and macrophage activity in the freshwater air-breathing Asian catfish, Saccobranchus fossilis, during a 28-day exposure were examined by a static bioassay test procedure . At 0.1, 1.0, and 3.2 mg / liter Cr, dose-dependent Cr accumulation in kidney, liver, and spleen was observed at the end of the experiment . Chromium exposure caused a significant change in spleen to body weight ratio . Fish exposed to Cr concentrations had lower antibody titer values, reduced numbers of splenic and kidney plaque-forming cells, and higher counts of splenic lymphocytes but reduced counts of kidney cells when compared with the control group . At 0.1, 1.0, and 3.2 mg /liter Cr, dose-dependent decreases in red blood cell counts, hemoglobin content, and packed cell volume were observed . Differential leukocyte counts revealed that Cr exposure caused a significant decrease in large and small lymphocytes, whereas neutrophils and thrombocytes increased . Effects of Cr exposure to mitogen (Con A) on proliferation of splenic and pronephric lymphocytes suggests a decrease in mitogenic response . The eye-allograft rejection time, as a parameter of cell-mediated immunity, was statistically increased at 1.0 and 3.2 mg/liter Cr . Fish exposed to Cr for 28 days exhibited higher susceptibility to A . hydrophila infection than control fish . The results suggest that Cr exposure reduced the resistance of catfish to bacterial infections . The phagocytic activity of splenic and pronephros macrophages was examined in vitro and found to be significantly decreased .

FEMS Microbiol Lett, 1999 May 1, 174(1), 131 - 6
Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model; Heuzenroeder MW et al.; Previous studies have shown that two hemolytic toxins, HlyA and AerA, contribute to the virulence of Aeromonas hydrophila . A survey was performed to gauge the distribution of hlyA and aerA genes in clinical and environmental Aeromonas isolates . For A . hydrophila, A . veronii biotype sobria and A caviae, 96%, 12% and 35% of strains, respectively, were hlyA positive, whereas, 78%, 97%, 41%, respectively, were aerA positive . All virulent A . hydrophila isolates were hlyA+ aerA+ . This genotype was most common in A . hydrophila (75.4%) followed by A . caviae (29.4%) and A . veronii biotype sobria (9.6%) . For A . hydrophila, a two-hemolytic toxin model of virulence provides the best prediction of virulence in an animal model.

FEMS Microbiol Lett, 1999 Apr 1, 173(1), 41 - 6
The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila; Dodd HN et al.; A gene encoding a deoxyribonuclease, dnsH, was cloned from Aeromonas hydrophila JMP636 . The predicted mature protein was very similar to the previously described extracellular Dns from this organism and an N-terminal region corresponding to a large putative signal sequence was predicted for the JMP636 protein . Inactivation of dnsII demonstrated that the DnsH protein was not present extracellularly in this strain . As DnsH degraded plasmid DNA and was believed to have a periplasmic location, a dnsH mutant was constructed to determine whether electroporation of A . hydrophila with plasmid DNA could be achieved . No transformants were detected . From SDS-PAGE studies, at least two additional DNases remain to be characterised from A . hydrophila JMP636.

Dev Comp Immunol, 1999 Jan-Feb, 23(1), 61 - 70
Changes in serum concentration of a serum amyloid P-like pentraxin in Atlantic salmon, Salmo salar L., during infection and inflammation; Lund V et al.; A serum amyloid P-like pentraxin has been isolated from Atlantic salmon . Salmo salar L., based on its calcium dependent binding to agarose . The subunits of approximately 37 kDa were all glycosylated and when covalently linked together formed a pentamer with disulphide bonds between all subunits . A specific rabbit antiserum raised against the pentameric form was used to follow changes in serum levels of the salmon pentraxin during infection with Aeromonas salar and inflammation induced by Escherichia coli LPS or killed A . salmonicida . The salmon pentraxin level in normal serum was in the range approximately 50-300 microg/ml . Unlike pentraxins from other species, the salmon pentraxin showed only moderate but significant increases or decreases in response to E . coli LPS or A . salmonicida infection, respectively . Although pentraxins and pentraxin-like proteins are evolutionary conserved, not all pentraxins are acute phase responders suggesting that their most ancestral function(s) are not related to acute phase induction.

Mol Cell Probes, 1999 Apr, 13(2), 93 - 8
Identification of Aeromonas trota (hybridization group 13) by amplification of the aerolysin gene using polymerase chain reaction; Khan AA et al.; Aeromonas trota is recognized as an important enteropathogen, and its haemolysin (aerolysin) is purported to be one of the virulence factors . Rapid detection and identification of A . trota is important for early and specific diagnosis of the infectious diseases that it causes . Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to amplify a species-specific sequence of the aerA gene, which encodes the aerolysin of A . trota . A DNA fragment of 622 bp was amplified from both lysed cells and isolated DNA from A . trota . The identity of the amplified 622 bp fragment was confirmed by digestion with BamH I restriction endonuclease, which produced the predicted 557 and 65 bp fragments . The lower limit for detection of the aerA gene by PCR amplification was 10 pg of total DNA or 10-15 cells ml-1 . Primer specificity for A . trota was determined by the PCR assay with cells of 55 strains of Aeromonas sppincluding all of the 14 currently recognized DNA hybridization groups . A strain of Aeromonas enteropelogenes that had been reclassified as A . trota was also PCR positive . The method described here can be used to detect aerolysin-producing A . trota (hybridization group 13) strains from environmental and clinical samples without the use of selective media or additional biochemical tests .

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 239 - 46
Signature region within the 16S rDNA sequences of Aeromonas popoffii; Demarta A et al.; To identify a group of eight Aeromonas strains of our collection showing ribotyping patterns similar to those described for the species Aeromonas popoffii, 16S rRNA gene sequence analysis was performed . Results were in agreement with the DNA binding values, and allowed the identification of a 'signature region' differentiating the A . popoffii strains from all other members of the genus Aeromonas.

J Biochem (Tokyo), 1999 Apr, 125(4), 760 - 9
Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen; Yamamura A et al.; L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp . L101, originally isolated from the organs of salmon (Oncorhynchus keta) . GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose . The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7 . Maximum activity was obtained at 55 degrees C and pH 8.5 . The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C . GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo{alpha}phenoxazin-7-ium chloride (meldola's blue), 3,3'-{3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl}-bis{2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride} (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT) . The presence of NAD(P) and oxygen gave no oxidation activity to GLDH . Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.

Microbiol Immunol, 1999, 43(1), 29 - 38
Secretion of hemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin; Nomura T et al.; The sequence at the amino terminus region of the hemolysin ofAeromonas sobria is homologous with that of aerolysin of A . hydrophila . However, there is no homology between the two toxins in the sequence at the carboxy terminal region . It has been shown that aerolysin is secreted into culture supernatant as a protoxin . This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide . However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated . In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A . sobria between Ser-446 and Ala-447, resulting in the removal of a 42 amino acid peptide . The removal of the peptide converts the prohemolysin into active hemolysin . Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin . The amounts of these mutant hemolysins accumulated in the periplasmic space of E . coli were very low compared with that of the wild-type . This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.

Ann Plast Surg, 1999 Mar, 42(3), 275 - 9
Aeromonas species isolated from medicinal leeches; Mackay DR et al.; Aeromonas hydrophila infections are a recognized complication of the use of medicinal leeches . The authors performed an experiment designed to find a safe and practical way to sterilize the leech gut of pathogenic organisms . Leeches were incubated for a 12-hour period in solutions of antibiotic effective against A . hydrophila . The incubations in the antibiotic solutions failed to eradicate pathogenic bacteria from the gut of the leeches . The authors examined cultures of bacteria isolated from the guts of the commonly used Hirudo medicinalis (European leech) and found a wide variety of pathogenic organisms . A . hydrophila is widely believed to be the most common enteric pathogen, but the authors found A . sobria more frequently in their experiment . They also cultured the guts of the leech H . michaelseni recently used clinically in South Africa . A . caviae was the most common pathogen encountered in these leeches . A . caviae and A . sobria cause a spectra of disease similar to A . hydrophila . The authors endorse the current recommendation that all patients who have leech therapy for congested flaps or replants receive broad-spectrum prophylactic antibiotics . This appears to be the safest and simplest way to prevent leech-related infections.

Microb Pathog, 1999 Feb, 26(2), 77 - 84
The type IV Aeromonas pilus (Tap) gene cluster is widely conserved in Aeromonas species; Barnett TC et al.; Nothing is known regarding the expression or function of the type IV Aeromonas pilus (Tap), which was recently identified following the cloning of a pilus biogenesis gene cluster (tapABCD) . As a first step to determine the possible significance of Tap for Aeromonas virulence, the distribution of the tapA and tapD genes in hybridization group reference strains and clinical (n=42) and environmental (n=29) isolates was determined . Homologues of tapA and tapD were present in all strains tested . Hybridization with the tapA probe enabled us to differentiate between clinical and environmental isolates of A . veronii biovar sobria .

Microb Pathog, 1999 Mar, 26(3), 149 - 58
Peroxide-inducible catalase in Aeromonas salmonicida subsp . salmonicida protects against exogenous hydrogen peroxide and killing by activated rainbow trout, Oncorhynchus mykiss L., macrophages; Barnes AC et al.; Aeromonas salmonicida subsp . salmonicida expresses a single cytoplasmically located catalase which was found to be inducible by exposure to 20 microM hydrogen peroxide in mid-exponential phase resulting in a 4 fold increase in activity . Subsequent exposure to 2 mM peroxide in late-exponential/early-stationary phase resulted in further induction of catalase activity which increased to 20 fold higher levels than those found in uninduced cultures . Exponentially induced cultures were protected against subsequent exposure to 10 mM peroxide which was lethal to non-induced cultures . Bacteria subjected to induction in mid-exponential and early-stationary phase were resistant to 100 mM peroxide, although viability was greatly reduced . Growth of the bacterium under iron-restricted conditions had no effect on the peroxide induction of catalase . As current evidence indicates, the latter is an iron-co-factored heme catalase, this result suggests that catalase induction has a high priority in the metabolism of iron . Furthermore, exposure to peroxide also induces expression of periplasmic MnSOD . A . salmonicida MT423 was resistant to normal rainbow trout macrophages, but was susceptible to killing by activated macrophages . However, if catalase was induced by prior exposure to 20 microM peroxide during mid-exponential phase, A . salmonicida was resistant to killing by activated macrophages . The ability of A . salmonicida to upregulate periplasmic MnSOD and cytoplasmic catalase production under iron restricted conditions and low level peroxide (conditions expected to exist during the early stages of an infection) may be vital for its ability to withstand attack by phagocytic cells in vivo .

J Wildl Dis, 1999 Jan, 35(1), 64 - 9
Mortality of captive Canadian toads from Basidiobolus ranarum mycotic dermatitis; Taylor SK et al.; Twenty-six adult free-ranging Canadian toads (Bufo hemiophrys) were collected from northeastern North Dakota (USA) during the last week of August 1994 and placed in captivity . During late December and January 1995, 21 Canadian toads died . Clinical signs included increased time sitting in water bowls, darkened dorsal skin, constant arching of their backs, and hyperemia and sloughing of ventral epidermis . The condition progressively worsened until death occurred within 5 to 7 days after onset of clinical disease . Mycotic dermatitis due to Basidiobolus ranarum was diagnosed in all toads and the fungus was isolated from 11 (52%) of 21 toads . Histology of the ventral skin and digits revealed numerous fungal spherules and occasional hyphae without significant inflammatory reaction . This condition clinically resembled red leg associated with Aeromonas hydrophila and many other bacterial organisms, and the diseases could be confused without appropriate diagnostic tests . This also is the first report of B . ranarum causing clinical disease in a toad species.

J Appl Microbiol, 1999 Feb, 86(2), 187 - 93
DNA probes specific for Aeromonas hydrophila (HG1); Oakey HJ et al.; Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes . From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested . Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences . Three of these individual amplicons had a sequence specific to Aer . hydrophila (HG1) isolates . The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR) . The specificity of the PCR was achieved using a modified hot-start procedure . The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.

Blood, 1999 Mar 1, 93(5), 1749 - 56
Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin; Brodsky RA et al.; Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene . The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins . Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors . We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin . The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59(+) cells in the samples measured by flow cytometry . The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes . PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity . Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry . Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific . Aerolysin should also be useful in studying PNH biology.

Fundam Clin Pharmacol, 1999, 13(1), 102 - 6
Destabilase complexes--natural liposome produced by medicinal leeches Hirudo medicinalis; Nikonov GI et al.; Electrophoretic analysis of destabilase preparation demonstrates the presence of protein combinations with MW 12.3, 25 and 50 kD . Fraction (MW 12.3 D) is a monomer of destabilase aggregation having properties of micellar proteins and represents a stable lipid-protein complex, where the role of lipid component is played by the stable analogue of prostacyclin (MW 391 D) . The synthesis of a low molecular fraction of destabilase is fulfilled with bacteria--symbiont of leeches Aeromonas hydrophila . When the destabilase (MW 12.3 kD) contacts with blood a process of complexe formation is triggered with hirudin and blood plasma kallikrein inhibitor, forming a stable 'destabilase complex' (DC; MW 25 kD), possessing also a high aggregation capacity . Polymer forms of the destabilase complex form a liposome changing its spatial orientation depending on the nature of the solvent . Such structural organization provides a high stability of DC components and a rapid penetration through cellular membranes (transmembrane transfer) and it also provides prophylactic antithrombotic action in the case of peroral application to animals, due to the blockade of vascular platelets (inhibition of platelet aggregation by prostacyclin analogue) and plasmic (inhibition of thrombin activity and blood plasma kallikrein) links of the hemostasis process . Destabilase fraction with MW 50 kD is a dimer of the destabilase complex . As a result of DC destruction (liposome), hirudin, prostacycline analogue and blood plasma kallikrein inhibitor are released.

J Clin Microbiol, 1999 Mar, 37(3), 706 - 8
Antimicrobial susceptibility patterns of Aeromonas jandaei, A . schubertii, A . trota, and A . veronii biotype veronii; Overman TL et al.; Fifty-six isolates of four Aeromonas species, which have been documented as causative agents of human infections or isolated from human clinical specimens, were subjected to antimicrobial susceptibility testing using a MicroScan WalkAway conventional (overnight incubation) gram-negative panel . The four species tested and the number of isolates of each were as follows: Aeromonas jandaei, 17; A . schubertii, 12; A . trota, 15; and A . veronii biotype veronii, 12 . All isolates of A . trota were susceptible to all antimicrobial agents tested, except cefazolin (20% of isolates were resistant) and cefoxitin (13% of isolates were resistant) . All isolates of A . schubertii and A . veronii biotype veronii, as well as 88% of A . jandaei isolates, were resistant to ampicillin . Resistance to ampicillin-sulbactam ranged from 25% of A . schubertii strains to 100% of A . veronii biotype veronii strains . Cefazolin resistance ranged from 17% of A . veronii biotype veronii isolates to 59% of A . jandaei isolates . Imipenem resistance was detected in 65% of A . jandaei strains and 67% of A . veronii biotype veronii strains . A . jandaei displayed resistance to piperacillin and ticarcillin in 53 and 71% of the isolates, respectively . A . veronii biotype veronii strains were 100% susceptible to piperacillin and 100% resistant to ticarcillin . These antibiogram data may be useful in establishing the identification of these four species when members of the genus Aeromonas are isolated from human clinical sources.

J Hosp Infect, 1999 Jan, 41(1), 45 - 9
An in-vitro evaluation of the activity of chlorine against environmental and nosocomial isolates of Aeromonas hydrophila; Chamorey E et al.; A chlorine preparation was tested against 34 strains of Aeromonas hydrophila isolated from a hospital water facility and patients with nosocomial infections in a French medical school hospital . In-vitro bactericidal activity was determined by a macro- and micromethod, using specific interfering substances over a range of dilutions and temperatures, after 1 and 5 minute exposure times . A 10(5)-fold reduction in the challenge inoculum was taken as the criterion of efficacy . Results obtained by both methods agreed and indicated a median minimal bacterial concentration of chlorine of from 0.95 ppm without interfering substances to 297 ppm in the presence of interfering substances . These data indicate that Aeromonas hydrophila isolates are relatively resistant to chlorine, thus precluding the use of chlorine alone as a disinfectant for water supplies contaminated with Aeromonas spp.

J Food Prot, 1999 Jan, 62(1), 30 - 4
Prevalence and characteristics of Aeromonas species isolated from processed channel catfish; Wang C et al.; From August 1994 to May 1995, 238 channel catfish fillets collected from three processing plants in the Mississippi Delta at four time periods were tested for the presence of Aeromonas species . Identification of Aeromonas spp . was accomplished using an automated Vitek bioassay system with gram-negative and nonfermenter cards . Approximately 36.1% were positive for A . hydrophila, 35.7% for A . sobria, and 10.9% for A . caviae . All three Aeromonas spp . were found in all three processing plants, and the incidence of A . hydrophila contamination appeared to be higher in summer than other seasons . Eighty-six percent of the Aeromonas isolates were hemolytic on 5% sheep blood agar plates . Most isolates were susceptible to chloramphenicol, neomycin, streptomycin, and trimethoprim-sulfamethoxazole and resistant to ampicillin and bacitracin . Results suggest that Aeromonas spp . are prevalent in processed channel catfish, and most isolates are hemolytic and resistant to ampicillin and bacitracin.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 69 - 75
Co-expression of polyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonas caviae establishes copolyester biosynthesis pathway in Escherichia coli; Fukui T et al.; Polyhydroxyalkanoate biosynthesis genes of Aeromonas caviae were expressed in Escherichia coli LS5218 (fadR atoC(Con)), and the polyhydroxyalkanoate-producing ability of the recombinants was investigated . A LS5218 strain harboring only phaCAc (polyhydroxyalkanoate synthase gene) did not accumulate any polyhydroxyalkanoate from dodecanoate in spite of the existence of translated polyhydroxyalkanoate synthase protein, whereas co-expression phaCAc and phaJAc ((R)-specific enoyl-CoA hydratase gene) resulted in the accumulation of P(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer up to 7-11 wt% of dry cell weight from octanoate and dodecanoate . These results indicated that both phaCAc and phaJAc are essential for E . coli LS5218 to establish the polyhydroxyalkanoate biosynthesis pathway from alkanoic acids . The copolyester content in the strain expressing both the genes under the lac promoter control reached to 38 wt% from dodecanoate . Enzyme assays suggest that efficient monomer formation via beta-oxidation by a high level expression of phaJAc was important to achieve a high polyhydroxyalkanoate content in the recombinant E . coli.

Zentralbl Hyg Umweltmed, 1998 Dec, 201(4-5), 423 - 30
Aeromonas hydrophila associated with a severe outbreak of infection in farmed rabbits; Paniagua C et al.; An outbreak of infection with a high rate of mortality has been detected in an industrial rabbitry in Spain . Aeromonas hydrophila was isolated in pure culture from liver, lung, heart and spleen of ill and moribund animals . All the isolates belonged to O:11 serogroup, autogglutinated after boiling, were resistant to the bactericidal action of the fresh rabbit serum and did not agglutinate in acriflavine . They were producers of hemolysins and proteases but were not enterotoxigenic.

J Commun Dis, 1998 Jun, 30(2), 71 - 8
Virulence factors of aeromonads--an emerging food borne pathogen problem; Agarwal RK et al.; Aeromonas spp . possess a number of virulence properties which are considered responsible for intestinal and extra-intestinal infections in human beings and also for a wide variety of infections in animals . The paper discusses current status of colonization and toxic factors of Aeromonas spp., especially in relation to food and biochemical markers . Future research needs are also identified.

J Med Microbiol, 1998 Jun, 47(6), 527 - 31
Characterisation of a type IV pilus produced by Aeromonas caviae; Kirov SM et al.; A pilus produced by a clinical isolate of Aeromonas caviae (strain CA195) was purified and partially characterised . The Mr of the pilin was estimated to be 23 kDa by SDS-PAGE . Its N-terminal amino-acid sequence showed that it was closely related to 'bundle-forming' type IV pili purified from other Aeromonas spp . associated with gastro-enteritis and considered to be important intestinal colonisation factors . Bundle-forming pili, often with a polar location, were seen on the surface of strain CA195 which was highly adherent to HEp-2 cells . Removal of surface structures by mechanical means decreased adhesion (by > or = 50%) suggesting that these pili played some role in HEp-2 cell binding . This pilus type could prove an important marker for enteropathogenic A . caviae which appear to lack other putative virulence factors.

Eur J Biochem, 1998 Dec 1, 258(2), 313 - 9
Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding--comparisons with the homologous enzyme Aeromonas proteolytica aminopeptidase; Papir G et al.; Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase . In addition, a unique Ca2+-binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme . Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure-dependent manner . Among the three hydrophobic 4-nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in k(cat)/Km) . Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine-like) residues at their N-termini and smaller enhancements for inhibitors with rigid (phenylalanine-like) residues.

Lett Appl Microbiol, 1998 Dec, 27(6), 349 - 51
Isolation of Aeromonas salmonicida in association with purple-pigmented bacteria in sediment from a Scottish loch; Austin DA et al.; Aeromonas salmonicida was recovered in close association with an unidentified purple-pigmented organism, which was isolated from sediment in a Scottish loch during November (1997) and February (1998) . However, there has not been any evidence of A . salmonicida infections, specifically furunculosis, associated with the fish in this loch.

Microbiol Immunol, 1998, 42(10), 703 - 14
Purification and characterization of enterotoxin produced by Aeromonas sobria; Fujii Y et al.; We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay . The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay . Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities . These results showed that the enterotoxin of A . sobria possesses hemolytic activity . Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin . As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A . sobria but also of A . hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms . The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH) . The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation . However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.

Commun Dis Public Health, 1998 Dec, 1(4), 267 - 70
The risk to public health of aeromonas in ready-to-eat salad products; Mattick KL et al.; Mesophilic Aeromonas spp . are isolated regularly from cases of gastrointestinal infection and have markers indicative of enteropathogenicity . Is aeromonas, which is present in a large proportion of ready-to-eat salads, actually a gastrointestinal pathogen? Isolates of mesophilic aeromonas from salads were characterised in terms of their ability to grow at refrigeration temperatures over the given shelf life and by the presence of markers of potential virulence . The major phenospecies present in salads, A.caviae, showed little enteropathogenic potential . Thirty-five per cent of aeromonas salad isolates are A.hydrophila or A.sobria, however, and all isolates tested had at least one marker of enteropathogenicity, including cytotoxin and haemolysin production, adherence to epithelial cells, and resistance to certain antibiotics Despite the presence of markers of enteropathogencity, the lack of epidemiological evidence of a link between infectious intestinal disease and the consumption of salads suggests that their contamination with aeromonas does not pose a significant risk to health in immunocompetent adults.

Commun Dis Public Health, 1998 Dec, 1(4), 263 - 6
Optimisation of the protocol for detection of Aeromonas species in ready-to-eat salads, and its use to speciate isolates and establish their prevalence; Mattick KL et al.; Aeromonas spp . are detected in more than 500 cases of gastrointestinal infection each year in England and Wales . This study aimed to identify their prevalence in ready-to-eat salads, which are a potential source of aeromonas infection . The protocol for isolation of mesophilic Aeromonas spp . from salads was optimised . Using the improved method, Aeromonas spp were isolated from 19 of 25 samples (25 g) of ready-to-eat salad products . Aeromonas organisms were counted, isolates were identified to species level, and the effect of pH on colonisation of salads was assessed . Aeromonas was present at high levels in six salads (> or = 100 cfu/g) . The major species present in salads was Aeromonas caviae, but A.hydrophila and A.sobria, which have more pathogenic potential, were also isolated . It is hoped that this study will help to assess the risk to public health of aeromonas in salads.

Microbiology, 1998 Nov, 144 ( Pt 11), 3087 - 96
Construction of a physical and preliminary genetic map of Aeromonas hydrophila JMP636; Dodd HN et al.; A physical and preliminary genetic map of the Aeromonas hydrophila JMP636 chromosome has been constructed . The topology of the genome was predicted to be circular as chromosomal DNA did not migrate from the origin during PFGE unless linearized by S1 nuclease . Cleavage of the chromosome with PacI and PmeI produced 23 and 14 fragments, respectively, and enabled calculation of the genome size at 4.5 Mb . Digestion of the chromosome with I-CeuI produced 10 fragments, indicating that 10 rrl (23S) genes were likely to be present . Hybridizations between DNA fragments generated with PacI, PmeI and I-CeuI were used to initially determine the relationship between these segments . To accurately map genes previously characterized from JMP636, the suicide vector pJP5603 was modified to introduce restriction sites for PacI and PmeI, producing pJP9540 . Following cloning of genes into this vector and recombinational insertion into the JMP636 chromosome, PacI and PmeI cleavage determined the location of genes within macrorestriction fragments with the additional bands produced forming hybridization probes . From the data generated, it was possible to form a physical map comprising all the fragments produced by PacI and PmeI, and assign the contig of I-CeuI fragments on this map . The preliminary genetic map defines the location of six loci for degradative enzymes previously characterized from JMP636, while the locations of the 10 sets of ribosomal genes were assigned with less accuracy from hybridization data.

J Biol Chem, 1998 Dec 4, 273(49), 32656 - 61
The pore-forming toxin proaerolysin is activated by furin; Abrami L et al.; Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila . Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation . This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A . Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin . We also investigated the potential role of these enzymes in the in vivo activation of the protoxin . We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin . The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells . The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin . Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.

Infect Immun, 1998 Dec, 66(12), 5711 - 24
Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes; Pizarro-Cerda J et al.; Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment . In this study, we have examined the intracellular pathway of the virulent B . abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells . At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1 . At approximately 1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61beta but negative for the mannose 6-phosphate receptors and cathepsin D . Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles . At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61beta- and protein disulfide isomerase-positive compartment . Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment . These results are compatible with the hypothesis that pathogenic B . abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.

J Pak Med Assoc, 1998 Jun, 48(6), 158 - 61
Sensitivity pattern and beta-lactamase production in clinical isolates of Aeromonas strains; Ahmed A et al.; Of the 43 Aeromonas spp . isolated from various clinical samples 94% isolates were Beta-lactamase producers . The isolates were tested for sensitivity by disc diffusion method which is commonly used in Pakistan . MIC was determined by using Epsilometer test (E-test) method . More than 80% isolates were sensitive to cephalosporins and quinolones . However, resistance to commonly used antibiotics was very high, 94% isolates were resistant to ampicillin which corresponds to the betalactamase production . More than 60% of the isolates were resistant to cotrimoxazole and 40% to chloramphenicol, hence quinolones and cephalosporins appear to be the drugs of choice for treating serious Aeromonas infections . The MIC range of Aeromonas was best for cefotaxime < 0.06 - 1.0 ug/ml . MIC 90 for cefotaxime was 0.50 ug/ml, for imipenem 0.25 ug/ml and for ciprofloxacin 2.0 ug/ml.

Rev Cubana Med Trop, 1995, 47(3), 215 - 6
{New species of Aeromonas isolated in Cuba}; Bravo L et al.; One hundred and fifty-five strains of Aeromonas isolated in the stools of children under 5 years presenting with acute diarrheal disease were studied . Using the Aerokey II system for the identification of species, 47 strains were identified as Aeromonas caviae, 58 as Aeromonas hydrophila, 23 as Aeromonas veronii biovar sobria, 14 as Aeromonas trota, 9 as Aeromonas veronii biovar veronii, 2 as Aeromonas jandaei and 2 as Aeromonas shubertii, Emphasis is placed on the advantages of this method which allowed for the classification of new species not identified previously in our country.

Rev Cubana Med Trop, 1995, 47(2), 114 - 7
{Phenotype markers in strains of Aeromonas isolated in Cuba from children with an acute diarrheic disease}; Bravo Farinas L et al.; Twenty-seven cases of Aeromonas isolated from 300 five-years children with acute diarrheal disease (EDA) were studied, with the aim for demonstrating the occurrence of some phenotypical markers associated with enteropathogenicity; among them were lysine decarboxylation, acetil methyl carbinol production, enterotoxigenicity, cytotoxicity and hemolysis . The percentage of analysed strains had two or more of the investigated markers, and 13 (48.1%) lysed the rabbit erythrocytes with titres higher than than 1:16 . The presence of markers in Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae was demonstrated.

J Food Prot, 1998 Oct, 61(10), 1321 - 9
Adhesion of Aeromonas hydrophila to water distribution system pipes after different contact times; Assanta MA et al.; Scanning electron microscopy observation was used to investigate the ability of Aeromonas hydrophila to attach to various water distribution pipe surfaces, such as stainless steel, copper, and polybutylene, after different contact times at ambient and storage temperatures . Surface energy value of each surface was estimated by contact angle measurements using water, alpha-bromonaphthalene, and dimethyl sulfoxide . Our results indicated that Aeromonas cells could easily attach to all surface types after exposures as short as 1 or 4 h at both temperatures (4 and 20 degrees C) . Polybutylene, a low-energy surface (41.2 mJ.m-2), followed by stainless steel (65.7 mJ.m-2), was most colonized by Aeromonas cells, whereas few cells were observed on copper, which has a surface energy of 45.8 mJ.m-2 . Extracellular materials could also be observed on polybutylene surfaces, especially after 1 and 4 h of exposure at the refrigeration temperature.

Appl Environ Microbiol, 1998 Nov, 64(11), 4194 - 201
Molecular characterization of plasmid-mediated oxytetracycline resistance in Aeromonas salmonicida; Adams CA et al.; Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli . The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp . The molecular basis for antibiotic resistance in OT-resistant isolates of A . salmonicida was determined . The OT resistance determinant from one plasmid (pASOT) of A . salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe . The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A . salmonicida . Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A . salmonicida isolates carried the class A resistance determinant . Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.

Mol Microbiol, 1998 Oct, 30(2), 341 - 52
Secretion and properties of the large and small lobes of the channel-forming toxin aerolysin; Diep DB et al.; Aerolysin is a dimeric protein secreted by Aeromonas spp . that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing . The protein comprises two lobes joined by a short arm . The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site . We cloned and expressed the DNA for both lobes of the toxin separately and together in A . salmonicida . The large lobe produced alone was secreted, although more poorly than native protein . The small lobe with the arm produced by itself was not secreted . When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex . Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer . The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25 degreesC . The large lobe with the arm was also purified . It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization . The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers . Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Scand J Immunol, 1998 Oct, 48(4), 357 - 63
Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora; Simmaco M et al.; Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora . Their skin glands produce a secretion containing 20-30 different peptides, some antimicrobial some neurotrophic . As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short periods of time . The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans . From an alternative frog strain of A . hydrophila, Bo-3, we isolated a spontaneous and stable mutant (Bo-3N), resistant to nalidixic acid, here used to follow the host-microbe interactions in experimental infection of mouth and skin of Rana esculenta . The skin peptides had been previously isolated, sequenced and cloned . We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IkappaBalpha was up-regulated . Experimental mouth infections with 20 million cells of A . hydrophila Bo-3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 microl 'saliva' . An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min . Using different enzyme-linked immunosorbent assays (ELISAs) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A . hydrophila . An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A . hydrophila only corresponding to a few per cent of the positive control . For skin infections we bathed the frogs for 10 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml . Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one-third of that of the non-bathed control frog . HPLC analysis of the peptide pattern confirmed these findings . The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila . The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate . One such function is involved in the control of the natural flora.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Jul, 120(3), 559 - 69
Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida; Hoover GJ et al.; In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A . salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M) . A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated . Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE . Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein . Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms . A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV) . A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout . A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37) . This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences . These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A . salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Zentralbl Veterinarmed B, 1998 Sep, 45(7), 443 - 5
Haemorrhagic septicaemia by Aeromonas salmonicida subsp . salmonicida in a black-tip reef shark (Carcharhinus melanopterus); Briones V et al.; One black-tip reef shark (Carcharhinus melanopterus) was found dead without previous signs of disease . Major lesions consisted in cutaneous erythema, mainly at the base of the fins, focal to diffuse inflammatory lesions in gills and intestinal wall, and discrete haemorrhages in the same organs, liver and kidneys . Microcolonies of Gram-negative rods were observed in the lamina propia of gills, underneath the intestinal mucosa and randomly distributed in the renal and hepatic parenchymas . Also, emboli containing Gram-negative rods were observed in capillaries of these organs . Aeromonas salmonicida subsp . salmonicida was isolated in pure culture from gills, liver and intestine . Specific immunostaining confirmed the relationship between the isolate and lesion-associated bacteria . No previous reports on this infection in sharks have been found in the literature.

J Clin Microbiol, 1998 Nov, 36(11), 3188 - 92
Inducible beta-lactam resistance in Aeromonas hydrophila: therapeutic challenge for antimicrobial therapy; Ko WC et al.; Despite the abundant amount of knowledge about inducible chromosomally mediated beta-lactamases among Aeromonas species, extended-spectrum beta-lactam-resistant A . hydrophila strains selected in clinical practice were rarely reported . In the present study, two strains of A . hydrophila, A136 and A139, with markedly different susceptibilities to extended-spectrum cephalosporins were isolated from blood and the tip segment of an arterial catheter of a burn patient . Another strain (A136m) was selected in vitro by culturing A136 in a subinhibitory concentration of cefotaxime, the beta-lactam agent administered for the treatment of Aeromonas bacteremia in this patient . Typing studies by arbitrarily primed PCR and pulsed-field gel electrophoresis indicated a clonal relationship among strains A136, A136m, and A139 . These strains were identified to be of DNA hybridization group 1 . Wild-type strain A136 was resistant only to ampicillin and cephamycins, but A136m and A139 were highly resistant to the expanded- and broad-spectrum cephalosporins . The presence of increased beta-lactamase activity in A139 suggests that A139 is a derepressed mutant which overexpresses beta-lactamases . These results call attention to the use of beta-lactam agents for the treatment of invasive Aeromonas infections.

Rev Cubana Med Trop, 1996, 48(1), 50 - 2
{Aeromonas hydrophila pneumonia associated with a traffic accident . Report of a case}; Vazquez Piloto A et al.; The case of a patient who was driving a car after getting drunk is presented . His car turned over and he fell into an irrigation canal, and, as a result, he suffered from an incomplete drowning syndrome . He was admitted in the Intensive Care Unit with acute inflammatory pneumonia and a strain of Aeromonas hydrophila was isolated in blood . The patient's evolution was favorable . It is the first report on a case like this in our country.

Rev Cubana Med Trop, 1992, 44(2), 134 - 6
{CAMP factor for the differentiation among Aeromonas species}; Bravo Farinas L et al.; The CAMP factor technique, described by Natale Figura as a presumptive method for the differentiation of the species hydrophila, sobria and caviae, was applied to 80 Aeromonas strains isolated from children under 5 years with acute diarrheic disease . The typical phenomenon was seen in aerobiosis and anaerobiosis conditions in 10 strains, which were classified as Aeromonas hydrophila . In aerobiosis conditions/alone, it was observed in 20 strains, which were identified as Aeromonas sobria . It was not observed in either of the incubation conditions above mentioned in 50 of the remaining strains, which were identified as Aeromonas caviae . The advantages of applying this new technique is emphasized.

Mol Microbiol, 1998 Sep, 29(5), 1237 - 47
An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP-binding cassette mutations on complex formation and function; Schoenhofen IC et al.; The energy-dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins . An 85 kDa complex containing the two proteins was identified in wild-type cells but not in cells producing either protein alone . Radiolabelling followed by cross-linking, immunoprecipitation and then reduction of the cross-links confirmed the presence of the two proteins in the same complex . The complex could also be extracted intact from cell membranes with non-ionic detergents . A G229D substitution in the kinase-3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase-1a motif (K56) with Arg . The G229D mutant contained very little of the ExeA-ExeB complex, but overexpression of the mutant complex until wild-type levels were achieved allowed normal secretion . In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present . These results are consistent with a model in which binding of ATP by ExeA is required for ExeA-ExeB complex formation, while hydrolysis is required for its function in secretion once established.

Res Microbiol, 1998 Jun, 149(6), 407 - 16
Mesophilic Aeromonas strains from different serogroups: the influence of growth temperature and osmolarity on lipopolysaccharide and virulence; Merino S et al.; Growth of mesophilic Aeromonas sp . strains from serogroups O:13, O:33 and O:44 at different temperatures and osmolarity resulted in changes in the lipopolysaccharide (LPS) and virulence of the strains tested, as we had previously reported for strains from serogroup O:34 . The effect of osmolarity could be observed when the cells grew at 37 degrees C but not at 20 degrees C . Purified LPS from cells cultivated at 20 degrees C (high or low osmolarity) or at 37 degrees C at high osmolarity was smooth, whereas the LPS extracted from the cells cultivated on low osmolarity was rough . The smooth strains were resistant to the bactericidal activity of non-immune serum, while the rough strains were sensitive and showed better adhesion to Hep-2 cells than the rough strains . Furthermore, the smooth strains were more virulent for fish and mice than the rough strains . For mesophilic Aeromonas sp . strains from serogroups O:1 to O:44, these changes were not observed, except for serogroups O:13, O:33, O:34 and O:44.

Res Microbiol, 1997 Sep-Oct, 148(7), 625 - 31
The role of the capsular polysaccharide of Aeromonas hydrophila serogroup O:34 in the adherence to and invasion of fish cell lines; Merino S et al.; The ability of Aeromonas hydrophila serogroup O:34 strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared . The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation . However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade the fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation . Isogenic unencapsulated mutants grown under conditions promoting capsule formation showed a lower ability to invade the fish cell lines than the parental capsulated strains . From these results, we concluded that the capsular polysaccharide is an important factor in intracellular invasion.

Appl Environ Microbiol, 1998 Oct, 64(10), 3776 - 83
Distribution and life strategies of two bacterial populations in a eutrophic lake
Weinbauer MG, Hofle MG.
Monoclonal antibodies and epifluorescence microscopy were used to determine the depth distribution of two indigenous bacterial populations in the stratified Lake Plusssee and characterize their life strategies . Populations of Comamonas acidovorans PX54 showed a depth distribution with maximum abundances in the oxic epilimnion, whereas Aeromonas hydrophila PU7718 showed a depth distribution with maximum abundances in the anoxic thermocline layer (metalimnion), i . e., in the water layer with the highest microbial activity . Resistance of PX54 to protist grazing and high metabolic versatility and growth rate of PU7718 were the most important life strategy traits for explaining the depth distribution of the two bacterial populations . Maximum abundance of PX54 was 16,000 cells per ml, and maximum abundance of PU7718 was 20,000 cells per ml . Determination of bacterial productivity in dilution cultures with different-size fractions of dissolved organic matter (DOM) from lake water indicates that low-molecular-weight (LMW) DOM is less bioreactive than total DOM (TDOM) . The abundance and growth rate of PU7718 were highest in the TDOM fractions, whereas those of PX54 were highest in the LMW DOM fraction, demonstrating that PX54 can grow well on the less bioreactive DOM fraction . We estimated that 13 to 24% of the entire bacterial community and 14% of PU7718 were removed by viral lysis, whereas no significant effect of viral lysis on PX54 could be detected . Growth rates of PX54 (0.11 to 0.13 h-1) were higher than those of the entire bacterial community (0.04 to 0.08 h-1) but lower than those of PU7718 (0.26 to 0.31 h-1) . In undiluted cultures, the growth rates were significantly lower, pointing to density effects such as resource limitation or antibiosis, and the effects were stronger for PU7718 and the entire bacterial community than for PX54 . Life strategy characterizations based on data from literature and this study revealed that the fast-growing and metabolically versatile A . hydrophila PU7718 is an r-strategist or opportunistic population in Lake Plusssee, whereas the grazing-resistant C . acidovorans PX54 is rather a K-strategist or equilibrium population.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1560 - 7
Molecular properties and activity of a carboxyl-terminal truncated form of xylanase 3 from Aeromonas caviae W-61; Okai N et al.; Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 {Nguyen, V.D . et al., Biosci . Biotechnol . Biochem., 56, 1708-1712 (1993) and Appl . Environ . Microbiol., 57, 445-449 (1991)} . While preserving a purified xylanase 3 preparation from A . caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one . It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease . We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing . By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr . Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan . In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products . Xylanase 3 hydrolysed X4 through X6 . However, Xyn3tr had no activity towards X4 and X5 . The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E . coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured . The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr . Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.

J Appl Microbiol, 1998 Sep, 85(3), 501 - 11
Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea; Fiorentini C et al.; A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995 . Biotyping at the species level allowed the identification of 96 strains (46%) as Aer . caviae, 46 (22%) as Aer . sobria, 33 (16%) as Aer . hydrophila and 25 (12%) as Aer . veronii . Eight strains (4%) were regarded as unnamed aeromonads . Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer . hydrophila strains were more commonly isolated from cleaner water . Aeromonas sobria and Aer . veronii were equally distributed in both estuaries . There was no correlation between temperature and numbers of aeromonads in either estuary . Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes . Several different PhP-types were found in each estuary, yielding a high diversity for these strains . However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions . The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.

J Appl Microbiol, 1998 Mar, 84(3), 423 - 30
Identification of Aeromonas strains of different origin to the genomic species level; Kaznowski A; A total of 71 Aeromonas strains was identified with established genomic species by DNA-DNA hybridization . The strains were isolated from diarrhoeal stools, dead and live fish, drinking, lake, river and sea water, municipal sewage and aluminium rolling emulsion . The strains were allocated to seven hybridization groups (HGs) but the majority belonged to HG 4 (42%), HG 8/10 (30%) and HG 3 (18%) . All strains were examined by 136 phenotypic tests . Useful phenotypic characters for separation of Aeromonas HG 1-3 genospecies were: utilization of DL-lactate, urocanic acid and growth at 40.5 degrees C . Few phenotypic differences were detected between strains of HG 4, HG 5 and HG 6 . Most isolates of the Aer . veronii biotype sobria (HG 8) showed a characteristic biochemical profile: positive V.P . (Voges-Proskauer) reaction, oxidation of gluconate, production of gas from glucose, susceptibility to cephalotin, no hydrolysis of elastin, arbutin and aesculin, and no acid production from L-arabinose, arbutin and salicin.

J Appl Microbiol, 1998 Mar, 84(3), 383 - 92
Incidence of mesophilic Aeromonas within a public drinking water supply in north-east Scotland; Gavriel AA et al.; The motile mesophilic Aeromonas are ubiquitous to a wide variety of aquatic environments including drinking water distribution systems . Concern over the presence of mesophilic Aeromonas in public drinking water supplies has been expressed in recent years as it has been regarded as a pathogenic organism of importance in gastroenteritis . A major drinking water distribution system in north-east Scotland was monitored over a 12 month period to determine the prevalence of mesophilic Aeromonas . These data were examined in relation to chlorine concentration, pH, temperature, rainfall and the standard bacteriological indicators of water quality . Aeromonas were isolated to varying degrees from 21 of the 31 reservoirs investigated . The maximum recovery observed during the study was 605 cfu in 300 ml . The probability of isolation generally decreased with increasing levels of chlorination, although this oxidant was found to be ineffective in many reservoirs . Certain reservoirs with poor chlorination profiles yielded very few isolates, whereas some highly chlorinated sites liberated Aeromonas frequently and in relatively high numbers . A seasonal pattern in the incidence of Aeromonas emerged with infrequent isolation during the winter period increasing to a peak during the summer, with most isolates recovered when water temperature was > 12 degrees C . An association was demonstrated between the pattern of Aeromonas isolations and that of rainfall . No relationship was apparent between incidence of Aeromonas and total heterotrophic plate counts.

Microbiology, 1998 Aug, 144 ( Pt 8), 2141 - 9
Physical map of the chromosome of Aeromonas salmonicida and genomic comparisons between Aeromonas strains; Umelo E et al.; I-Ceul and Pmel physical maps of the Aeromonas salmonicida A449 chromosome were constructed using PFGE . The circular chromosome of A . salmonicida A449 was estimated to be 4658 +/- 30 kb . The approximate location of several genes, including those encoding proteins implicated in virulence, were identified . The map showed that the known virulence-factor-encoding genes were not clustered . The I-Ceul genomic digestion fingerprints of several typical and atypical strains of A . salmonicida were compared . The results confirmed the homogeneity of typical strains, which provided further support for the clonality of the population structure of this group . Extensive diversity was observed in the I-Ceul digestion fingerprint of atypical strains, although a clonality was observed in the strains isolated from diseased goldfish . The results suggest that comparison of I-Ceul digestion fingerprints could be used as a powerful taxonomic tool to subdivide the atypical strains and also help clarify some of the current confusion associated with the taxonomy of the genus Aeromonas.

J Appl Microbiol, 1998 Jun, 84(6), 999 - 1006
Identification of atypical Aeromonas salmonicida: inter-laboratory evaluation and harmonization of methods; Dalsgaard I et al.; The atypical isolates of Aeromonas salmonicida are becoming increasingly important as the frequency of isolation of bacteria belonging to this group continues to rise . The primary object of this study was to compare and evaluate the results obtained in various laboratories concerning the biochemical identification of atypical Aer . salmonicida before and after standardization of media and methods . Five laboratories examined 25 isolates of Aer . salmonicida from diverse fish species and geographical locations including the reference strains of Aer . salmonicida subsp . salmonicida (NCMB 1102) and Aer . salmonicida subsp . achromogenes (NCMB 1110) . Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase . The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical reactions . After standardization, improvement was obtained with the identification; however, disagreement was still observed between the different laboratories . These findings demonstrate the difficulties involved in a proper identification of atypical Aer . salmonicida and also that data presented in the literature on various strains of Aer . salmonicida are not readily comparable . This paper seems to be the first on standardization of microbiological tests for identification of fish pathogens and the results obtained show the need for standardization of methods both within and between laboratories.

Clin Infect Dis, 1998 Aug, 27 Suppl 1, S48 - 53
Metallo-beta-lactamases: a class apart; Bush K; Metallo-beta-lactamases have recently become more prominent among the beta-lactam-hydrolyzing enzymes . Two major functional groups of enzymes have been identified, with little structural similarity among the groups . One group is a set of enzymes with broad substrate specificities capable of hydrolyzing most beta-lactams except monobactams . A second group is composed of the "true" carbapenemases, enzymes that exhibit poor hydrolysis of penicillins and cephalosporins . This latter group has been found primarily in Aeromonas species . To date, only a small number of carbapenem-resistant isolates have been reported to produce metallo-beta-lactamases, in part because of the ease with which this resistance can be acquired by other means: permeability changes and an increase in chromosomal cephalosporinase production . However, the appearance of these enzymes on plasmids in Japan poses a worrisome problem . It is anticipated that plasmid-mediated resistance to carbapenems will continue to increase, perhaps compromising the use of these agentsPublication Types:

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