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 kno