Microbiologia, 1994 Sep, 10(3), 257 - 62 Incidence of motile Aeromonas spp . in foods; Pin C et al.; A total of 80 food samples were purchased from local retail consumer shops and examined for the presence of motile Aeromonas spp . Of the food categories tested, poultry had the highest incidence, with 100% positive . This was followed by lamb samples, with 60% positive . Raw milk and cheese samples had very low incidence (20%) . No motile Aeromonas spp . were found in pre-prepared salads . Shellfish, fish, pork and beef samples had incidences of 40% . Most of the strains isolated were Aeromonas hydrophila, and for most of the food categories, no Aeromonas caviae isolates were obtained. Infect Immun, 1994 Sep, 62(9), 4054 - 8 Carbohydrate-reactive, pore-forming outer membrane proteins of Aeromonas hydrophila; Quinn DM et al.; Two outer membrane proteins of Aeromonas hydrophila A6, isolated in a one-step affinity chromatography process based on carbohydrate reactivity, were found to be pore-forming molecules in artificial planar bilayer membranes . These carbohydrate-reactive outer membrane proteins (CROMPs; M(r)s, 40,000 and 43,000) were subjected to amino acid analysis . The amino acid profiles for these two outer membrane proteins were almost identical . A partial protein sequence of a 14-amino-acid fragment of the 40,000-Da protein revealed homology with outer membrane porins of Escherichia coli and A . hydrophila . CROMPs were compared with carbohydrate-reactive porins also extracted from outer membranes of A . hydrophila A6 . These porins were isolated by using standard porin purification techniques (insolubility in 2% sodium dodecyl sulfate, solubility in 0.4 M NaCl, and Sephacryl S-200 gel filtration), and then Synsorb H type 2 affinity chromatography was done . The physical and functional properties of the carbohydrate-reactive porins and CROMPs were found to be identical . On the basis of pore-forming properties in planar lipid bilayers and channel inhibition with maltotriose solutions, a nonspecific, general diffusion porin and a LamB-like maltoporin were identified in both CROMP and carbohydrate-reactive porin preparations . To our knowledge, the use of carbohydrate reactivity to isolate channel-forming proteins from bacterial outer membranes has not been reported previously. Vet Immunol Immunopathol, 1994 Aug, 42(2), 199 - 208 Antigen-induced release of macrophage activating factor from rainbow trout Oncorhynchus mykiss leucocytes; Marsden MJ et al.; The production of macrophage-activating factor (MAF) by rainbow trout, Oncorhynchus mykiss, head kidney leucocytes at varying times post-immunisation, with the fish bacterial pathogen, Aeromonas salmonicida, was investigated and correlated with head kidney lymphocyte proliferation and serum antibody production . MAF production was preceded by lymphocyte proliferation and both responses were highest using whole bacterial cells as the in vitro stimulant . MAF production and antibody production increased 2-3 weeks post-immunisation, and peaked 4-5 weeks post-immunisation . The relative importance of MAF activated phagocytes in the immunological armoury of disease-resistant, vaccinated fish requires further investigation. Can J Microbiol, 1994 Aug, 40(8), 622 - 9 Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation; Garduno RA et al.; S-layers are paracrystalline protein multimers that cover the entire cell surface of many bacterial species . The presence of an S-layer in Aeromonas salmonicida (also known as A-layer) predisposed this bacterium to apparently unrelated physiological consequences: inhibition of growth at 30 degrees C, enhanced cell filamentation at 37 degrees C, and enhanced uptake of the hydrophobic antibiotics streptonigrin and chloramphenicol . Growth inhibition or enhanced filamentation was not observed when the native A-layer was missing or its arrangement altered, as in Ca(2+)-limited or Ca(2+)- and Mg(2+)-limited cells, in A-layer-negative (A-) cells with an artificially reconstituted A-layer, or in mutants unable to correctly assemble this layer . A-layer-positive cells (A+) were far more sensitive to the intracellularly acting antibiotics streptonigrin and chloramphenicol than were A- cells, and streptonigrin-resistant mutants were predominantly A- . Hemin, a compound known to specifically bind to the A-layer, alleviated streptonigrin toxicity to A+, but not A-, cells . As well, Ca(2+)- and Mg(2+)-limited cells, or mutants harboring A-layer defects had a reduced sensitivity to streptonigrin, and A- cells with reconstituted A-layers remained resistant to streptonigrin and chloramphenicol . Thus, the presence of a native A-layer arrangement on the cell surface, and not the mere presence of A-layer protein subunits, predisposed A . salmonicida toward the aforementioned physiological consequences . The A-layer is suggested to specifically effect these consequences, in particular the permeation of streptonigrin or chloramphenicol, by a specific interaction of A-layer subunits with the outer membrane. J Clin Pathol, 1994 Jul, 47(7), 642 - 6 Assessment of a chemiluminescent universal probe for taxonomical and epidemiological investigations of Aeromonas sp isolates; Carey PE et al.; AIMS--To assess a chemiluminescent universal probe for taxonomical and epidemiological investigations of Aeromonas sp isolates . METHODS--Total DNA was extracted from 69 well characterised Aeromonas sp strains and digested with the restriction endonucleases Sma I or Pst I . Following electrophoresis, the resulting fragments were transferred to a nylon membrane where they were hybridised to a commercially available universal probe of 16S + 23S rRNA . The banding patterns (ribotypes) were made visible by enhanced chemiluminescence . RESULTS--Both restriction endonucleases produced heterogeneous ribotypes so that no allocation could be made to any of the control genospecies tested . For A hydrophila and A caviae, however, groups of strains (mostly from the same patient) could be identified by indistinguishable banding patterns . A relatively high proportion (36%) of A sobria strains were non-typable . CONCLUSIONS--Although this universal chemiluminescent probe is user friendly, it is unsuitable for taxonomical investigations of Aeromonas sp . It is useful in epidemiological studies of A hydrophila and A caviae, but is of less value for A sobria. Microbiology, 1994 Jul, 140 ( Pt 7), 1731 - 6 Adaptive acid tolerance response (ATR) in Aeromonas hydrophila; Karem KL et al.; Aeromonas hydrophila, a gastrointestinal pathogen of humans, was shown to exhibit a significant adaptive acid tolerance response (ATR) capable of protecting cells from severe acid at a pH of 3.5 . The ATR was induced by exposure to a relatively mild pH level of 5.0 for 20 min . Adaptation required protein synthesis since treatment with chloramphenicol during adaptation to pH 5.0 prevented the development of acid tolerance . The adaptation to acid environment was found to be a non-transient phenomenon . Also, iron was not required for acid adaptation in A . hydrophila . Two-dimensional protein analyses revealed an increased production of 28 proteins and decreased synthesis of 10 following pH shifts from 7.2 to 5.0 . The mild pH treatment must act as a signal to A . hydrophila to adapt and survive in acid environments by producing 'protective' proteins . The adaptation and survival of this pathogen in low pH may provide valuable information about its ability to withstand acid environments in nature and in the human gastrointestinal tract. Biometals, 1994 Jul, 7(3), 227 - 36 Diversity of siderophore genes encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp; Massad G et al.; Most species of the genus Aeromonas produce the siderophore amonabactin, although two species produce enterobactin, the siderophore of many enteric bacteria . Both siderophores contain 2,3-dihydroxybenzoic acid (2,3-DHB) . Siderophore genes (designated aebC, -E, -B and -A, for aeromonad enterobactin biosynthesis) that complemented mutations in the enterobactin genes of the Escherichia coli 2,3-DHB operon, entCEBA(P15), were cloned from an enterobactin-producing isolate of the Aeromonas spp . Mapping of the aeromonad genes suggested a gene order of aebCEBA, identical to that of the E . coli 2,3-DHB operon . Gene probes for the aeromonad aebCE genes and for amoA (the entC-equivalent gene previously cloned from an amonabactin-producing Aeromonas spp.) did not cross-hybridize . Gene probes for the E . coli 2,3-DHB genes entCEBA did not hybridize with Aeromonas spp . DNA . Therefore, in the genus Aeromonas, 2,3-DHB synthesis is encoded by two distinct gene groups; one (amo) is present in the amonabactin-producers, while the other (aeb) occurs in the enterobactin-producers . Each of these systems differs from (but is functionally related to) the E . coli 2,3-DHB operon . These genes may have diverged from an ancestral group of 2,3-DHB genes. Clin Infect Dis, 1994 Jul, 19(1), 77 - 83 Aeromonas species in septicemia: laboratory characteristics and clinical observations; Janda JM et al.; We retrospectively analyzed clinical and epidemiological data on and laboratory characteristics of 53 cases of aeromonas septicemia . Only four Aeromonas genomospecies (species defined by DNA relatedness) were associated with the 53 cases, with Aeromonas hydrophila (sensu stricto) predominating (47%) . Nearly 60% of all Aeromonas isolates from blood fell into one of four somatic groups: serogroups O:11, O:16, O:18, and O:34 . Unlike Aeromonas-associated gastroenteritis, septicemia did not peak in frequency during the warmer months but rather was most common in January through March, when approximately 40% of cases occurred . In vitro tests of the pathogenicity of 20 selected blood isolates of Aeromonas indicated that resistance to complement-mediated lysis, elevated levels of protease and hemolysin activity, and the ability to elaborate siderophores correlated with higher virulence . Species and serogroup designations also correlated with the degree of virulence . Susceptibility studies of 50 strains indicated that A . hydrophila was the most drug-resistant species and that Aeromonas veronii was the most susceptible . Susceptibility to first- and second-generation cephalosporins and carbenicillin was species-associated. J Wildl Dis, 1994 Jul, 30(3), 447 - 9 Isolation of Aeromonas salmonicida from paddlefish, Polyodon spathula; Ford LA et al.; Aeromonas salmonicida was isolated from paddlefish (Polyodon spathula) mortalities collected during an epizootic of furunculosis at the Spring River State Hatchery, Arkansas (USA), in 1992 . Isolates of the bacterium were obtained from culture of gill and kidney tissue . This is the first epizootic of bacterial etiology to be reported in paddlefish. J Appl Bacteriol, 1994 Jul, 77(1), 21 - 30 Characterization of Aeromonas salmonicida subsp . salmonicida: a comparative study of strains of different geographic origin; Dalsgaard I et al.; A total of 130 strains of the fish pathogen Aeromonas salmonicida isolated in Denmark, Norway, Scotland, Canada and the USA were examined . The strains originated from farmed salmonid fish . The biochemical, physiological and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared . Aeromonas salmonicida was found to be remarkably consistent in general cultural and biochemical characteristics . It is noteworthy that the strains were positive in the fermentation of L-arabinose and were negative in the fermentation of D-arabinose . All the strains were highly proteolytic . It was observed, however, that 5% of the strains did not digest calf and trout serum and the production of haemolysin and degradation of casein by the same strains were delayed compared with the other strains . Common to all of the rough strains were auto-aggregation and ability to bind the dyes Coomassie brilliant blue and Congo red and the majority of these strains were highly hydrophobic . The strains were tested for their susceptibility to 22 antibacterial agents . Antibiotic resistance profiles of Aer . salmonicida indicated that resistance to the quinolones and oxytetracycline was increasing and that multi-resistant strains were found in several countries . The variation found in antibiograms could have potential as epidemiological markers in certain geographic areas. Can J Microbiol, 1994 Jun, 40(6), 503 - 7 Isolation and characterization of a Tn5-induced tolQ mutant of Escherichia coli; Madrid C et al.; Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the external medium a heterologous protein, the Aeromonas hydrophila aerolysin . Genetic mapping and phenotypic characterization of E . coli CM209 showed that Tn5 is inserted in the tolQ gene . This mutant strain released a significant amount of unprocessed (proaerolysin) protein into the external medium, together with other periplasmic proteins . Similar levels of the toxin were detected in the intracellular compartments of the parental and mutant strains . Whereas inactivation of the tolQ gene itself was responsible for some of the phenotypic properties of strain CM209, such as tolerance to group A colicins or to filamentous bacteriophages, the leaky phenotype was associated with a polar effect exerted by Tn5 on distal genes of the tolQRA cluster. Vet Immunol Immunopathol, 1994 Jun, 41(3-4), 341 - 52 Genetic variation in the humoral immune response in Atlantic salmon (Salmo salar) against Aeromonas salmonicida A-layer; Stromsheim A et al.; Antibody responses to Aeromonas salmonicida A-layer were analysed in family material of Atlantic salmon (Salmo salar), consisting of 791 fish belonging to 34 full-sib groups within 12 paternal half-sib groups . The fish were immunized twice and blood samples were collected three times . Significant increase in antibody titre from first to second, and from second to third sampling, was observed . Genetic variation in antibody titres was observed at the three samplings with estimated heritabilities ranging from 0.16 to 0.20, and a significant heritability estimate was recorded in the antibody response after the second immunization (h2 = 0.16) . Moderate to high genetic (r = 0.5-0.72) and phenotypic (r = 0.29-0.57) correlations were found between the titre values at different samplings, and between selected titres and titre increases . Production parameters, such as mean slaughter weight and mean survival rate were obtained for fish which were reared commercially in the sea, and which belonged to the same full-sib groups as those analysed for A . salmonicida A-layer antibodies . No significant correlation between the mean antibody titre to A . salmonicida A-layer in this study and mean slaughter weight and survival rate in full-sib family material in the sea was observed. Biochemistry, 1994 May 3, 33(17), 5011 - 20 Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode; Jain MK et al.; A glycerophospholipid:cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila . Hydrolysis of the sn-2-ester bond of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface . This conclusion is based on the following observations . (a) When there is an excess of vesicles over enzyme, the hydrolysis of the sn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed . Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis . (b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme . (c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange . Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture . (d) Addition of DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis . Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups . Treatment of GCAT with trypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface . Alkyltrifluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT . The scooting mode analysis, which has previously been shown to provide a simplified approach for analyzing the steady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases. Vet Immunol Immunopathol, 1994 May, 41(1-2), 141 - 52 A beta-glucan inhibitable zymosan receptor on channel catfish neutrophils; Ainsworth AJ; In mice and humans zymosan binds to the complement receptor three/Mac-1 receptor; however, identification of this receptor in channel catfish (Ictalurus punctatus) has not been accomplished . Soluble fluorescein isothiocyanate (FITC) conjugated beta-glucan, a component of zymosan, was found to bind to channel catfish anterior kidney (AK) neutrophils but not to B-lymphocytes . Serum activated zymosan (SAZ)-mediated chemiluminescence responses of channel catfish AK neutrophils could be inhibited by beta-glucan but not by mannan, and inhibition of chemiluminescence responses by beta-glucan was dose dependent . Similarly, phagocytosis of FITC-SAZ could be inhibited by beta-glucan in a dose-dependent manner . Treatment of channel catfish AK neutrophils with various concentrations of trypsin resulted in inhibition of phagocytosis of FITC-SAZ but not of Aeromonas hydrophila indicating that A . hydrophila phagocytosis was mediated by a trypsin-resistant receptor . Deleting serum or using heat-inactivated serum in the mixtures for the chemiluminescence and FITC-SAZ phagocytosis assays resulted in baseline readings . These data indicate that the beta-glucan component of zymosan is responsible for zymosan phagocytosis . Furthermore, the recognition of zymosan by a specific receptor is evident based on trypsin sensitivity assays . Based on these results it is proposed that a complement receptor 3, Mac-1-like receptor, is present on channel catfish AK neutrophils. Vet Immunol Immunopathol, 1994 May, 41(1-2), 125 - 39 Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis; Siwicki AK et al.; Immunostimulant preparations Macrogard, Candida utilis, Saccharomyces cerevisiae, Evetsel, Chitosan, or FinnStim were mixed into semipurified diets and fed to groups of rainbow trout for 1 week . Fish were bled by non-lethal caudal puncture and blood samples assayed for changes in non-specific cellular immunity and humoral protein levels . In the immunostimulated fish, hematocrit levels and lymphocyte counts remained relatively stable; however, elevations were observed in oxidative radical release, myeloperoxidase activity, phagocytic indexes, and potential killing activities of phagocytic cells including neutrophils . Total plasma protein and total immunoglobulin levels were elevated by the dietary immunostimulants . A challenge with the virulent pathogen that causes furunculosis, Aeromonas salmonicida, showed that the immunostimulated groups of fish were more resistant to the disease, confirming the potential use of these substances in fish culture for the prevention of disease. Epidemiol Mikrobiol Imunol, 1994 May, 43(2), 61 - 6 {Identification of aeromonads from water sources}; Sedlacek I et al.; A total of 102 Aeromonas strains isolated from water were identified by both commercial ENTEROtest 1 & 2 kits and by several conventional tests . 83 strains (81.4%) were identified to the species level according to a differentiation table . A . hydrophila (36 strains), A . caviae (26 strains) and A . sobria (10 strains) species were isolated most frequently . Strains identified as A . allosaccharophila, A . eucrenophila, A . jandaei, A . media and A . trota were very rare . The remaining 19 strains could not be identified . The ENTEROtest kit without additional tube test was insufficient for the identification of Aeromonas strains to the species level . The arginine dihydrolase test and hydrolysis of esculin from the ENTEROtest kit were found to be the least reliable tests. Epidemiol Mikrobiol Imunol, 1994 May, 43(2), 55 - 60 {Biochemical identification of aeromonads}; Aldova E et al.; To assess the species of the genus Aeromonas in 178 strains isolated from human and animal materials and from the environment (water and hospital environment), examined along with 11 type strains of the best known species, a key was used the basis of which are in addition to biochemical tests defining the genus Aeromonas 12 tests: Voges-Proskauer, lysine decarboxylase, gas from glucose, haemolysis, gluconate oxidation, elastase, arabinose, mannose, saccharose, salicine, esculine hydrolysis, arbutine . Three tests--salicine, esculine hydrolysis, arbutine--differentiate A . hydrophila (2-3 positive) from A . sobria (0-1 positive). FEMS Microbiol Lett, 1994 May 1, 118(1-2), 163 - 6 Effect of growth temperature on complement-mediated killing of mesophilic Aeromonas spp . serotype O:34; Merino S et al.; Mesophilic Aeromonas spp . strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37 degrees C (serum-sensitive) but not when they were cultivated at 20 degrees C (serum-resistant) . These strains produced smooth lipopolysaccharide when they were grown at 20 degrees C and rough lipolysaccharide when cultivated at 37 degrees C . The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed. J Appl Bacteriol, 1994 May, 76(5), 511 - 20 Characteristics of 'atypical', cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders (Platichthys flesus (L.)); Wiklund T et al.; 'Atypical', cytochrome oxidase-negative variants of the fish pathogen Aeromonas salmonicida, isolated from ulcerated flounder (Platichthys flesus), were studied using different methods . Two of the strains possessed a protein that corresponded to the A-layer protein of Aer . salmonicida . The strains reacted with antibodies against the A-layer and monoclonal antibodies against the O-antigen of typical Aer . salmonicida . These tests confirm that the isolates from flounder should be classified as Aer . salmonicida . Analysis of the fatty acids showed that the isolates were rather homogeneous but the values of the guanine plus cytosine content of the DNA of the bacteria varied too much for any conclusion to be drawn on their taxonomic location . The strains examined exhibited several biochemical characters that differed from those of the type strains of Aer . salmonicida subsp . salmonicida and Aer . salmonicida subsp . achromogenes . The results suggest that these 'atypical', cytochrome oxidase-negative variants may form a new subspecies of Aer . salmonicida. Structure, 1994 Apr 15, 2(4), 283 - 91 Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family; Chevrier B et al.; BACKGROUND: Aminopeptidases specifically cleave the amino-terminal residue from polypeptide chains and are involved in the metabolism of biologically active peptides . The family includes zinc-dependent enzymes possessing either one or two zinc ions per active site . Structural studies providing a detailed view of the metal environment may reveal whether the one-zinc and two-zinc enzymes constitute structurally and mechanistically distinct subclasses, and what role the metal ions play in the catalytic process . RESULTS: We have solved the crystal structure of the monomeric aminopeptidase from Aeromonas proteolytica at 1.8 A resolution . The protein is folded into a single alpha/beta globular domain . The active site contains two zinc ions (3.5 A apart) with shared ligands and symmetrical coordination spheres . We have compared it with the related bovine lens leucine aminopeptidase and the cobalt-containing Escherichia coli methionine aminopeptidase . CONCLUSIONS: The environment and coordination of the two zinc ions in A . proteolytica aminopeptidase strongly support the view that the two metal ions constitute a co-catalytic unit and play equivalent roles during catalysis . This conflicts with the conclusions drawn from the related bovine leucine aminopeptidase and early biochemical studies . In addition, the known specificity of the aminopeptidase for hydrophobic amino-terminal residues is reflected in the hydrophobicity of the active site cleft. J Mol Biol, 1994 Apr 8, 237(4), 452 - 63 Mutagenesis of the paracrystalline surface protein array of Aeromonas salmonicida by endogenous insertion elements; Gustafson CE et al.; The tetragonal paracrystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor and bacteria which are unable to produce A-layer are attenuated in their ability to kill fish . Ten independent mutants of Aeromonas salmonicida which were unable to produce A-layer were isolated by growth at 30 degrees C . These mutants displayed either reduced synthesis of the A-layer subunit, synthesis of a truncated subunit, or complete loss of the ability to produce the subunit protein . Restriction mapping and analysis by polymerase chain reaction showed that the mutations had resulted from insertion of two different insertion sequence (IS) elements into different sites in the A-layer subunit gene (vapA) and its promoter . Sequence comparisons indicated that ISAS1 is unique among reported IS elements . It is 1223 bp long with imperfect terminal inverted repeats of 22 bp and insertion resulted in a duplicated 8 bp target sequence in vapA . ISAS1 expressed a 42,000 molecular weight (M(r)) protein in mini-cells . ISAS2 was 1084 bp long, expressed proteins of M(r) 38,000 and 39,000 in vitro, had imperfect 29 bp terminal inverted repeats and had duplicated a 3 bp target sequence . Sequence comparisons indicated that ISAS2 was also unique to A . salmonicida; however, the proteins encoded by ISAS2 showed strong homology to the putative transposases encoded by the IS30 family of IS elements . Southern analyses showed that both ISAS1 and ISAS2 were restricted to A . salmonicida strains A449 and A450 where they were present in low copy number . The ability of these two IS elements to mutate the ability of A . salmonicida to produce its paracrystalline surface array provides a novel method for the attenuation of virulence. Epidemiol Infect, 1994 Apr, 112(2), 291 - 8 Characterisation of haemolytic activity from Aeromonas caviae; Karunakaran T et al.; Aeromonas caviae, an enteropathogen associated with gastroenteritis, displays several virulence characteristics . Studies on the kinetics of growth of A . caviae and expression of beta-haemolytic toxin revealed that A . caviae produced maximum haemolytic activity extracellularly during the stationary phase . Preliminary studies on the properties of A . caviae haemolysin suggested that divalent cations (Mg2+ and Ca2+) and thiol compounds, dithiothreitol and mercaptoethanol enhanced the haemolytic activity . Addition of L-cysteine, glutathione and EDTA reduced the haemolytic activity . The iron chelator, 2-2' bipyridyl, significantly inhibited the growth of A . caviae possibly by iron limitation, with parallel enhancement of haemolysin production compared to A . caviae grown in excess of iron . These results suggest that A . caviae produces only beta-haemolysin, which resembles the haemolysins reported for several other bacteria and the activity might be regulated by environmental factors especially iron. J Clin Microbiol, 1994 Apr, 32(4), 1130 - 1 Aeromonas species exhibit aggregative adherence to HEp-2 cells; Neves MS et al.; Clinical and environmental isolates of Aeromonas species (five A . hydrophila isolates, three A . caviae isolates, and two A . sobria isolates) were tested for their adherence to HEp-2 cells . Clinical isolates of A . hydrophila and A . sobria exhibited aggregative adherence similar to that presented by enteroadherent-aggregative Escherichia coli . Bacterial aggregates adhered to cells with a typical "stacked-brick" appearance . In contrast, A . caviae strains showed a diffuse adherence pattern. Appl Environ Microbiol, 1994 Apr, 60(4), 1379 - 82 Comparison of putative virulence factors in Aeromonas hydrophila strains isolated from the marine environment and human diarrheal cases in southern Italy; Krovacek K et al.; Aeromonas hydrophila strains isolated from the same geographical region (southern Italy) but from different sources (sea sediments and human diarrhea cases) were characterized for the production of potential virulence determinants, such as production of cytotoxins, cytotonic toxins, hemolysin, and dermonecrotic factors and their capacity to adhere to human intestinal 407 cells in vitro . The results showed that isolates from both the sources produced all or some of the virulence factors which may be involved in the pathogenesis of Aeromonas-associated infections . Our study indicates that further epidemiological studies are necessary to elucidate the public health significance of infections caused by Aeromonas spp. FEMS Microbiol Lett, 1994 Mar 15, 117(1), 85 - 90 O-serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels; Esteve C et al.; The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting {SP} and precipitation after boiling {PAB}, profile of lipopolysaccharides {LPSs} and outer membrane proteins {OMPs}) was investigated in strains of the pathogenic species Aeromonas hydrophila and A . jandaei isolated from eels . Virulent strains of A . hydrophila reacted mostly with O:19 antiserum, and those of A . jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinee and Jansen system) . Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB+ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB- strains antigenically diverse that either exhibited a heterogeneous side chain or were side chain deficient . A major 50 kDa protein was only found in the PAB+ strains, whereas major OMPs detected in PAB- strains ranged from 33 to 45 kDa irrespective of the species . Epizootic eel isolates of A . hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts . In contrast, epizootic A . jandaei isolates were antigenically diverse . These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels. Changgeng Yi Xue Za Zhi, 1994 Mar, 17(1), 68 - 73 Long term survival of a patient with a regressed giant hepatocellular carcinoma after transcatheter hepatic artery embolization (TAE) complicated with liver abscess; Chuah SK et al.; A 62-year-old male patient with histologically proven hepatocellular carcinoma, received transcatheter hepatic artery embolization (TAE) therapy . Development of right pyothorax and liver abscess at the tumor region occurred 4 months later after TAE . Aeromonas hydrophila was isolated from the liver abscess . After repeated percutaneous drainage, the abscess cavity disappeared and the tumor became undetectable by ultrasonography . Nineteen months after the initial presentation, a second tumor at the dome of the right lobe liver was found . TAE was repeated . Bile stasis with stricture of left intrahepatic ducts were found by Tc-99m HIDA cholangiography and endoscopic retrograde cholangiography . The patient had a normal lifestyle until the third tumor appeared at the right lower liver 18 months after the second TAE . TAE was conducted a third time . A shunting between the hepatic artery and vein developed at the new tumor area 3 months later . The patient is surviving today which is five and a half years after the initial diagnosis . We believe that the liver abscess after TAE contributed to the complete regression of the giant tumor, in addition to the anti-tumor effect of the successful TAE. J Reconstr Microsurg, 1994 Mar, 10(2), 83 - 5 Intestinal flora of the medicinal leech Hirudinaria manillensis; Bickel KD et al.; Medicinal leeches are widely used to treat venous congestion in microvascular surgery . Aeromonas hydrophila infection, following application of the leech species Hirudo medicinalis, is a recognized complication . Administration of antibiotics directed at Aeromonas has been successful in minimizing complications of infection from this organism . A different leech species, Hirudinaria manillensis, has recently been introduced for microsurgical use . A study of the enteric content of 30 of these leeches showed that Aeromonas hydrophila was isolated in only 20 percent of animals, while the majority of remaining positive cultures were single and mixed gram-negative rods . All organisms isolated were sensitive to current recommended coverage for Aeromonas hydrophila . This study suggests that the enteric flora of different leech species may be variable and should be carefully characterized, to direct appropriate prophylactic therapy prior to release of new species for clinical use. J Med Microbiol, 1994 Mar, 40(3), 188 - 93 Cytotoxic and haemagglutinating activities of motile Aeromonas species; Majeed KN et al.; Cytotoxic and haemagglutinating properties were determined in 114 Aeromonas strains isolated from various sites in slaughtered lambs and from processed lamb meat . Cytotoxic activity on Vero cells was observed in 48 (42%) of the strains . It was more common in A . sobria and A . hydrophila isolates than with A . caviae isolates . Haemagglutination (HA) activity was found frequently in motile aeromonads irrespective of species; it was present in 50% of A . sobria strains, 51% of A . hydrophila strains and 48% of A . caviae strains . HA was inhibited by fucose, galactose and mannose at low concentration, and in most cases, two or three of these sugars were inhibitory . A significant association was found between certain HA-inhibition patterns and the production of cytotoxin by Aeromonas spp. Prikl Biokhim Mikrobiol, 1994 Mar-Apr, 30(3), 454 - 7 {Activity of lysosomal proteinases in carb tissue in aeromonas infection}; Nemova NN et al.; The activity of major lysosomal proteinases (cathepsins B and D) increased in carp tissues infested with Aeromonas hydrophila . The value and character of changes in the activity of cathepsins depended on the degree of infestation and the physiological state of fishes (normally fed or starved). Dev Comp Immunol, 1994 Mar-Apr, 18(2), 123 - 36 Immunoregulation in fish . I: Intramolecular-induced suppression of antibody responses to haptenated protein antigens studied in Atlantic salmon (Salmo salar L); Killie JE et al.; We report here evidence for intramolecular-induced suppression of the in vivo antibody response in fish, using a panel of T-dependent hapten-carrier antigens . Atlantic salmon were immunized intraperitoneally with protein antigens (Limulus polyphemus hemocyanin, chicken gamma globulin, and Aeromonas salmonicida A-layer protein) given in their native form or haptenated with either 4-hydroxy-3-iodo-5-nitrophenyl-acetic acid (NIP), 2,4,6-trinitrophenyl-acetic acid (TNP), or fluorescein-5-iso-thiocyanate (FITC) . The salmon immune system responds to these hapten-carrier antigens by eliciting high anti-hapten titers whereas the antibody titers against protein determinants were suppressed 87-99%, determined by ELISA . NIP also induced suppression of the anti-FITC response when NIP and FITC were intramolecularly conjugated to Limulus polyphemus hemocyanin (LPH) . The suppression was found to be independent of haptenation ratios and time after immunization . The possibility that haptenation interferes with or blocks the protein determinants is not likely because antisera raised against native LPH recognize LPH-specific epitopes even on heavily NIP-substituted LPH . Although the mechanism behind intramolecular-induced suppression is poorly understood, even in mammals, this study demonstrates that intramolecular-induced suppression may be one means by which antibody responses in fish are regulated . The possible impact of antigen-induced suppression on immune responses against vaccine antigens in fish is discussed. Clin Diagn Lab Immunol, 1994 Mar, 1(2), 182 - 5 Serum antibody responses of divers to waterborne pathogens; Losonsky GA et al.; To assess the significance of exposure of divers to waterborne pathogens, specific immunoglobulin G serum antibody responses to Pseudomonas and Aeromonas isolates recovered from dive sites from the respiratory tracts of nine experienced divers and seven diving trainees working in the Chesapeake Bay area over a 6- to 18-month period were measured . A significant increase in the frequency of isolation of these organisms from respiratory surfaces both groups of divers after each dive was noted, with the divers' ears being the predominant recovery site (48%; P < 10(-8), chi-square) . The acute serum responses of the majority of experienced divers (83%) showed evidence of preexisting antibody to these potential pathogens, whereas the acute serum response of only 32% of naive divers showed such evidence (P < 10(-8), chi-square) . Six months into their training, the rate of seroresponse of the trainees to organisms recovered after their first dives increased to 61% (P = 0.003, chi-square), suggesting that repeated exposure in necessary for generation of a specific systemic immunologic response . The rate of acquisition of a new seroresponse to recovered organisms was approximately 12% per dive for both groups of divers, suggesting that there is continuous exposure to, and infection with, new strains present in the water during dives . These data suggest that, in cases in which systemic antibody is important for protection, there are various levels of susceptibility to waterborne potential pathogens in both experienced and inexperienced divers. Toxicology, 1994 Feb 28, 87(1-3), 19 - 28 The cytolytic toxin aerolysin: from the soluble form to the transmembrane channel; van der Goot FG et al.; Aerolysin is a cytolytic toxin which forms channels in the plasma membranes of eucaryotic cells . The protein is secreted by Aeromonas hydrophila as an inactive protoxin . Its stability and water solubility are conferred by its ability to dimerize . Maturation of the protein occurs through proteolytic removal of a C-terminal peptide outside the secreting cell . Although the aerolysin which is so produced is still a dimer, it then has the ability to oligomerize . The oligomer is the active form of the toxin, capable of forming the transmembrane channels that disrupt cells . We review here the present knowledge about the structure of aerolysin in relation to the various steps in channel formation. Gene, 1994 Feb 11, 139(1), 87 - 91 Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila; Chopra AK et al.; A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3 . Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response . Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system . The cell lysate from this E . coli {pSL24} clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by {35S}methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) . The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C . In addition, another enterotoxin-producing clone, E . coli{pSBS32}, was isolated from cosmid and lambda bacteriophage libraries . We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment . Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells . The DNA fragments encoding these enterotoxins did not hybridize with each other . However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A . trota . Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar. Antimicrob Agents Chemother, 1994 Feb, 38(2), 353 - 5 beta-Lactam resistance of motile Aeromonas isolates from clinical and environmental sources; Morita K et al.; The MICs of various beta-lactams for 182 isolates of Aeromonas species, i.e., A . hydrophila (n = 101), A . sobria (n = 69), and A . caviae (n = 12), from clinical and environmental sources were determined by an agar dilution technique . All strains were resistant to ampicillin and susceptible to aztreonam . A . sobria and A . caviae demonstrated lower resistance rates than A . hydrophila . Penicillin-hydrolyzing beta-lactamases were detected in all strains. J Biol Chem, 1994 Jan 21, 269(3), 2146 - 50 Influence of active site and tyrosine modification on the secretion and activity of the Aeromonas hydrophila lipase/acyltransferase; Robertson DL et al.; Aeromonas sp . secrete a lipase/acyltransferase that shares several properties with the mammalian plasma enzyme lecithin:cholesterol acyltransferase . Reaction of the enzyme with tetranitromethane led to modification of 2 tyrosines and a nearly 80% decline in enzyme activity . Replacing Tyr230 with Phe altered the activity of the enzyme in the same way as did treatment with tetranitromethane . Unlike the wild type enzyme, which preferentially hydrolyzes the 2-position acyl chain of phosphatidylcholine, the Y230F mutant enzyme did not discriminate between the 1- and 2-positions of the phospholipid . Tyr230 may be necessary to correctly position phospholipid substrates at the active site . Several amino acids around the active site Ser16 of the lipase were also changed . Replacing Ser18 with Gly, bringing the enzyme's sequence into line with the "lipase consensus sequence," resulted in reduced secretion of the protein and complete loss of activity . Changing this serine to Val led to an inactive protein that was not secreted at all . Substituting Phe13 in the hydrophobic region of the consensus sequence with Ser also prevented secretion, although the mutant protein appeared to be active . The Aeromonas lipase may represent a distinct group of lipolytic enzymes which have a novel active site structure. Nature, 1994 Jan 20, 367(6460), 292 - 5 Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states; Parker MW et al.; Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections . Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers . Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution . The protoxin (M(r) 52,000) adopts a novel protein fold . Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels. J Med Microbiol, 1994 Jan, 40(1), 55 - 61 Adhesion to and invasion of human colon carcinoma Caco-2 cells by Aeromonas strains; Nishikawa Y et al.; The enteropathogenicity of Aeromonas strains that showed mannose-resistant adhesion to INT407 cells was evaluated by infecting Caco-2 cells and observing them by light and electronmicroscopy . Five of six strains adhered in large numbers to Caco-2 cells in the presence of mannose and caused cytopathic effects . Two strains of Aeromonas spp . seemed to invade Caco-2 cells, as membrane-bound bacteria were seen within the cytoplasm of these cells; however, staining by acridine orange-crystal violet appeared to show intracellular fluorescent bacteria in three strains . Fimbriae did not appear to play an important role in adhesion because fimbrial structures were not seen by transmission electronmicroscopy . Adhesion of four strains was inhibited by the addition of L-fucose . The strains were negative in the fluorescence actin staining test, which in enteropathogenic Escherichia coli strains correlates with the ability to attach and efface intestinal microvilli . The DNA of the Aeromonas strains did not hybridise with the E . coli eae and ipaB probes, associated with attaching and effacing ability and invasion, respectively . These results give support to the enteropathogenicity of adhesive strains of Aeromonas spp., although the mechanisms of adhesion, and possibly invasion, remain to be elucidated. Microbios, 1994, 77(310), 47 - 55 Adherence pattern of non-pilated Aeromonas hydrophila strains to tissue cultures; Bartkova G et al.; Adherence of non-pilated Aeromonas hydrophila strains to HEp-2, HeLa, CHO and Vero tissue culture cells was studied . The strains were isolated from intestinal and extra-intestinal human infections and from the environment . The environmental isolates revealed a diffuse type of adherence to all types of cell lines while clinical isolates showed localized adherence (50%) . Strains revealing the diffuse type of adherence showed high levels of adhesion i.e . > 20 bacteria per cell . This activity correlated with hydrophobicity of the strains tested . Haemagglutination assay with human erythrocytes was negative, showing no association with plasmid profile, Congo red binding and adherence to tissue culture cells . Thus the experiments showed that in non-pilated strains, hydrophobicity may be the major factor responsible for adherence to epithelial cells. Zentralbl Hyg Umweltmed, 1994 Jan, 195(2), 121 - 34 Assessment of some selective media for the recovery of Aeromonas hydrophila from surface waters; Bernagozzi M et al.; A comparison was made of three different growing media already proposed by some authors for the recovery of Aeromonas hydrophila in environmental water samples of various origins . The media tested were Rippey and Cabelli m Aeromonas agar, Rimler-Shotts agar and the Ryan Aeromonas Base Medium with the addition of ampicillin . The efficiency of the three media was evaluated on the basis of the following reference criteria: accuracy, selectivity and specificity . The results obtained, analyzed by calculating the Kendall concordance coefficients, demonstrated that m Aeromonas agar is the most suitable media for quantitative recovery of Aeromonas hydrophila from surface water samples even if a reduction in the total heterotrophic load of 3-logs was never obtained with this medium. Antibiot Khimioter, 1994 Jan, 39(1), 63 - 9 {Aeromonads and their sensitivity to antibacterial drugs (review of the foreign literature 1985-1992)}; Kardashova EV et al.; The review is concerned with the modern studies on the taxonomy of aeromonades and their role in the development of infectious complications of various localization, including intestinal infections . A special attention is paid to antibiotic resistance of aeromonades isolated from pathological materials and the modern notions of the factors of aeromonade pathogenicity. Clin Infect Dis, 1994 Jan, 18(1), 32 - 7 Aeromonas peritonitis; Munoz P et al.; Five new cases of peritonitis caused by Aeromonas species are reported, and 29 others described in the literature are reviewed . Males predominated (71%), and the mean age was 56.9 years . Acquisition was nosocomial in 20% of the cases . All patients except one (3%) had significant underlying diseases; 73% had chronic hepatic disease, 15% had chronic renal failure (treated with chronic ambulatory peritoneal dialysis {CAPD}), and 9% had an intestinal perforation . Symptoms were similar to those of peritonitis caused by other pathogens, with the exception of diarrhea, which occurred in 25% of cases . Blood cultures were positive in 74% of the cases . The species isolated were Aeromonas hydrophila (27), Aeromonas sobria (5), and Aeromonas caviae (2) . The overall case-fatality rate was 57% . Three strains were resistant to cotrimoxazole . Aeromonas species should be taken into account as a cause of peritonitis in patients with cirrhosis or who are undergoing CAPD. J Basic Microbiol, 1994, 34(4), 245 - 52 Factors influencing beta-galactosidase activity of Aeromonas caviae; Karunakaran T et al.; Aeromonas caviae, often reported to be associated with diarrhoeal patients, elaborates several virulence factors as well as catabolic enzymes such as xylanase and beta-galactosidase . Studies on the kinetics of growth of A . caviae and synthesis of beta-galactosidase suggested that the activity was cell associated and reached a peak during the late logarithmic phase of growth . The optimum pH for beta-galactosidase activity was 7.0 and required Ca2+ and glutathione for enhancement of its activity; IPTG also slightly improved the activity . Aerobic cultivation of A . caviae in LB containing glucose, fructose, maltose and sucrose completely inhibited the activity possibly due to acetic acid production . Addition of 100 mM cAMP to the media containing glucose (0.25%, w/v) restored the relative activity by 8.8%; however, the final pH of the media remained acidic . Aerobic growth of A . caviae with other carbon sources did not affect beta-galactosidase activity, probably as there was no acid production and thereby the final pH of the media unaltered . Arabinose, xylose and galactose induced the A . caviae beta-galactosidase activity by several folds and lactose moderately enhanced its activity. Folia Microbiol (Praha), 1994, 39(4), 331 - 6 Virulence factors in clinical and food isolates of Aeromonas species; Pin C et al.; Virulence factors were compared in 15 Aeromonas spp . isolated from faeces of patients with Aeromonas-associated gastroenteritis and in 81 strains isolated from food . Strains from food did not show differences in the distribution of virulence factors when compared with strains isolated from faeces . However, 88.8% of Aeromonas strains isolated from food were capable of producing possible virulence factors . Characterization of 28 autoagglutinating (AA+) Aeromonas spp . indicated that the human strains differed from the food strains in hemagglutinating and hemolytic capacities . These results suggest that autoagglutination associated with hemagglutinating and hemolytic capacities in food strains may be a helpful indicator of potential pathogenicity. J Biomol NMR, 1994 Jan, 4(1), 97 - 116 Assessing glycosidic linkage flexibility: conformational analysis of the repeating trisaccharide unit of Aeromonas salmonicida; Peters T et al.; A detailed conformational analysis was performed for the synthetic branched trisaccharide beta-D-ManNAc-(1-->4)-{alpha-D-Glc-(1-->3)}-L-Rha 1 which represents the repeating unit of the O-antigenic polysaccharide of Aeromonas salmonicida . The study was based on 26 experimental NOE curves from 1D transient NOE experiments, employing Gaussian-shaped inversion pulses at 600 MHz . Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations . Metropolis Monte Carlo (MMC) simulations and minimum-energy calculations with the program GEGOP were used to obtain theoretical NOE curves which were compared to the experimental ones . MMC simulations with different temperature parameters of 310, 600, 900 and 2000 K allowed identification of NOEs which are sensitive towards different conformation distributions--not only different conformations--at both glycosidic linkages in 1 . A comparison of trisaccharide 1 with the constituent disaccharides beta-D-ManNAc-(1-->4)-L-Rha 2 and alpha-D-Glc-(1-->3)-L-Rha 3 revealed effects of branching on glycosidic linkage flexibility . A quantitative evaluation was facilitated by the introduction of entropy-related flexibility parameters . Our study indicates a notable restriction of flexibility, especially at the (1-->3) linkage in 1 . Although overall flexibility in 1 is reduced as compared to the constituent disaccharides 2 and 3, it cannot be neglected altogether . In summary, combined transient NOE experiments and MMC simulations provide a simple approach to analyse glycosidic linkage flexibility. Vet Rec, 1993 Dec 18-25, 133(25-26), 617 - 21 Amoxicillin concentrations in the serum of Atlantic salmon (Salmo salar L) during furunculosis therapy; Inglis V et al.; The effectiveness of the delivery of amoxicillin to Atlantic salmon, undergoing chemotherapy in natural outbreaks of furunculosis in sea-cages, was investigated by measuring the concentration of the drug in serum samples . Five groups of 50 sera from three outbreaks were collected two hours after oral treatment with doses of 80 or 120 mg/kg bodyweight . Amoxicillin was detected in 82, 82, 92, 100 and 90 per cent of the sera in the five groups (limit of detection 0.16 microgram/ml) . Many sera contained less than the minimum inhibitory concentration of amoxicillin for the causative agent Aeromonas salmonicida (0.3 microgram/ml), but a concentration more than double the minimum inhibitory concentration was achieved in 2, 2, 56, 32 and 44 per cent of the samples . There was wide variation in the serum concentrations between individuals in the same population and between populations receiving the same treatment; this variation was associated with population factors, the severity of infection and the accuracy of medicating the feed. J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3215 - 23 Cloning and nucleotide sequence of an extracellular alpha-amylase gene from Aeromonas hydrophila MCC-1; Chang MC et al.; A gene encoding the extracellular alpha-amylase of Aeromonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101 . Subcellular fractionation of Escherichia coli JA221 carrying pCA101 revealed that approximately 60% of the amylase activity was localized in the periplasmic space . The extracellular amylase was purified to homogeneity, identified as an alpha-type and its amino-terminal sequence was determined . Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein . This 24 amino acid sequence has many of the characteristics common to known signal peptides . The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete alpha-amylases . Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several alpha-amylases were also present in A . hydrophila alpha-amylase at the corresponding positions. Clin Infect Dis, 1993 Dec, 17(6), 1058 - 60 Meningitis due to Aeromonas species: case report and review; Parras F et al.; In recent years, Aeromonas species has been reported to cause extraintestinal infections with a growing frequency . Meningitis due to Aeromonas species is, however, a rare entity . We report a case of aeromonas meningitis in a 54-year-old man with a history of chronic alcoholic liver disease who, after an episode of gastroenteritis, developed an acute clinical picture characteristic of meningitis with septic shock and ecthyma gangrenosum . Aeromonas veronii (biogroup sobria) was isolated from cultures of blood as well as from cultures of stool, peritoneal fluid, skin lesion, and CSF specimens (obtained by lumbar puncture) . Our review of seven additional cases of aeromonas meningitis in the world literature revealed that this condition is generally secondary to metastatic dissemination from primary bacteremia . Aeromonas meningitis, which may or may not be preceded by gastroenteritis, presents clinically as bacterial meningitis, although the presence of skin lesions may increase suspicion of the diagnosis . Third-generation cephalosporins are probably the therapy of choice for patients with aeromonas meningitis. Int J Food Microbiol, 1993 Dec, 20(4), 179 - 98 The public health significance of Aeromonas spp . in foods; Kirov SM; There is now evidence that some strains of Aeromonas species are enteropathogens . Such strains possess virulence properties, such as the ability to produce enterotoxins, cytotoxins, haemolysins and/or the ability to invade epithelial cells . Strains with these properties are common contaminants of drinking water and a wide range of foods . Contact or consumption of contaminated water, especially in summer, is a major risk factor in Aeromonas-associated gastroenteritis . Aeromonas-contaminated foods may also be vehicles of infection . Given the properties of strains that have been described in foods it has been suggested that food-borne illness could result not only from colonization and in vivo expression of virulence factors, but possibly also by intoxication following ingestion of foods that have been stored for a period of time, even under refrigeration . This paper reviews what is known about Aeromonas spp . in foods, their expression of virulence determinants, particularly at refrigeration temperatures, and the questions remaining to be answered to evaluate the risk they pose, so that an appropriate public health response can be determined. J Bacteriol, 1993 Dec, 175(24), 7968 - 75 Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA; Chu S et al.; The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer . Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences . Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A . salmonicida . Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A . salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures . Northern analysis of cells grown at 15 degrees C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25) . At 15 degrees C, the half-life of the vapA mRNA was 22 min, while at 20 degrees C, the half-life was significantly shorter, 11 min . The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin . Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA . vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein. Int J Food Microbiol, 1993 Nov 26, 20(3), 159 - 68 The growth and expression of virulence factors at refrigeration temperature by Aeromonas strains isolated from foods; Kirov SM et al.; A potentially significant subset (10%, 6/61) of Aeromonas strains isolated from food (milk, lamb, chicken, seafood), all A . veronii biotype sobria, were able to produce two or more exotoxins (haemolysin, enterotoxin, and cytotoxin) at 37 degrees C, and grow well at 43 degrees C . Although mesophilic organisms, they grew at 5 degrees C . In addition, they could adhere to HEp-2 cells when grown at 37 degrees C, or at 5 degrees C, and expressed flexible pili (possible colonization factors) in greater numbers at the lower temperature . These strains, as well as other exotoxin-producing strains (A . veronii biotype sobria and A . hydrophila) (33%, 20/61) lacking adhesive ability, were able to produce cytotoxins in broth cultures over a seven to 10-day period at 5 degrees C . One strain in particular, an A . hydrophila isolated from goats' milk, grew rapidly at low temperature . This psychrotrophic strain produced all three exotoxins within 3 days in broth cultures at 5 degrees C . The properties of the above strains suggest they could be of public health significance in food products that have an extended shelf-life at refrigeration temperature. Infect Immun, 1993 Nov, 61(11), 4582 - 9 Novel antigens expressed by Aeromonas salmonicida grown in vivo; Thornton JC et al.; Virulent and avirulent Aeromonas salmonicida strains grown inside intraperitoneal implants in Rainbow trout (Oncorhynchus mykiss) were examined for unique antigen expression . Western blots (immunoblots), performed with immune rabbit serum raised against in vivo-grown cells, revealed several unique antigens . With the exception of lipopolysaccharide (LPS), these novel antigens were destroyed after proteinase K treatment . The majority of these antigens were not induced in vitro in response to either iron limitation or anaerobiosis . In addition, electron microscopy demonstrated the presence of a putative capsule on in vivo-grown cells . Purification and fractionation of this carbohydrate material from cells grown in carbon-rich synthetic media resulted in the isolation and separation of an antigenically distinct LPS not seen with cells grown in standard media . Antiserum raised against in vivo-grown cells recognized both this LPS and the typical LPS of A . salmonicida apparent in in vitro-grown cells . Antiserum raised against in vitro-grown cells recognized only the LPS expressed in vitro . Antiserum directed against in vivo-grown cells was approximately 10 times more sensitive than serum directed against in vitro-grown cells in detecting A . salmonicida in infected fish kidney tissue. Can J Microbiol, 1993 Nov, 39(11), 1051 - 8 Fate of the fish pathogen Aeromonas salmonicida in the peritoneal cavity of rainbow trout; Garduno RA et al.; A model was developed to study the fate of the fish pathogen Aeromonas salmonicida in vivo, inside a specialized intraperitoneal chamber implanted in rainbow trout, Oncorhynchus mykiss . Although normally recalcitrant to lytic agents in vitro, owing to the presence of its regular surface array (S layer), A . salmonicida was rapidly killed in the peritoneal cavity by a host-derived, soluble lytic activity present in peritoneal fluid . Peritoneal fluid was also found to kill other bacteria and lyse various types of erythrocytes, but was particularly lytic to A . salmonicida . Intraperitoneal survival of injected (free) A . salmonicida cells was several orders of magnitude higher than survival of implanted (restrained) cells . Injected free cells could evade the lytic activity of peritoneal fluid because they readily spread, initiating lethal infections . One evasion strategy was envisioned to be the penetration of peritoneal and (or) tissue macrophages . In spite of the killing mechanisms of these phagocytic cells, A . salmonicida was still able to survive and even replicate inside head kidney macrophages, thereby supporting the notion of A . salmonicida as a facultatively intracellular pathogen . Intraperitoneal chambers in rainbow trout may constitute a valuable experimental tool for studying the in vivo fate of A . salmonicida, and perhaps of other fish pathogens as well. Int J Food Microbiol, 1993 Nov, 20(2), 117 - 20 Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland; Gobat PF et al.; A total of 829 poultry, meat, shellfish and fish products commonly consumed in Switzerland were qualitatively and quantitatively examined for the presence of mesophilic Aeromonas spp . Overall, aeromonads occurred in 24.1% of the samples . Raw food products were frequently contaminated (e.g . 94.1% in minced meat), with colony counts up to 6.0 x 10(6)/g . Some ready-to-eat products had a relatively high percentage of positive samples as well, such as cooked ham in slices (38.2%), mortadella (12.9%), smoked cooked sausage (15.6%), hot and cold smoked fish (10.9-14.3%) and gravad salmon (10.5%) . Colony counts, however, were somewhat lower (up to 1.7 x 10(3)/g) . The high contamination rate of cooked or hot smoked foods suggests recontamination after cooking or smoking, e.g., at the slicing and packaging stage . 61.2% of the identified strains were Aeromonas hydrophila, followed by 22.5% Aeromonas caviae and 16.3% Aeromonas sobria. J Med Microbiol, 1993 Nov, 39(5), 325 - 33 Typing of Aeromonas spp . by numerical analysis of immunoblotted SDS-PAGE gels; Mulla R et al.; One hundred and three isolates of Aeromonas spp., collected from both environmental sources and patients, were examined by SDS-PAGE of whole cells followed by immunoblotting with polyclonal rabbit antiserum raised against whole cells of A . sobria . All isolates were typable, yielding 15-20 well separated bands . Reproducibility of the technique was good and discrimination excellent, yielding 30 types amongst 103 isolates . Immunoblot type was not related to biochemical phenotype . Attempts to correlate immunoblot type with serotype were unsuccessful because only 42% of the strains tested could be serotyped. FEBS Lett, 1993 Nov 1, 333(3), 296 - 300 Physical and chemical characterization of the oligomerization state of the Aeromonas hydrophila lipase/acyltransferase; Ausio J et al.; Aeromonas glycerophospholipid:cholesterol acyl transferase undergoes a conformational transition upon activation by treatment with trypsin . Chemical cross-linking and sedimentation velocity analysis showed that the lipase dimerizes due to removal of a region near its C-terminus . The lipase monomer has a sedimentation coefficient s20.w = 2.83 S, whereas the dimer has s20.w = 3.65 +/- 0.22 S . Hydrodynamic analysis using these sedimentation values and the masses determined by mass spectrometry indicated that the monomers are aligned side-by-side in the dimer . An important change occurs in the apparent partial specific volume of the molecule upon activation. J Mol Biol, 1993 Oct 20, 233(4), 753 - 65 Distribution of surface-exposed and non-accessible amino acid sequences among the two major structural domains of the S-layer protein of Aeromonas salmonicida; Doig P et al.; The tetragonally arranged crystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor . Circular dichroism studies in the presence or absence of 0.1% sodium dodecyl sulfate showed that the secondary structure of A-protein, and its 39,439 molecular weight amino-terminal trypsin-resistant peptide, were altered . In both cases alpha-helix was increased significantly at the expense of beta-structure when SDS was added . Western and dot immunoblotting, immuno-microscopy and enzyme-linked immunosorbent assay with monospecific polyclonal antiserum and eight monoclonal antibodies specific for epitopes exposed on the surface of native A-layer showed that the 481 residue A-protein subunit and the surface of A-layer were conserved antigenically . Mimeotope analysis of nonapeptides representing the sequence of A-protein allowed identification of 146 residues in presumed linear epitopes accessible on the surface of A-layer . Inaccessible or non-epitopic residues accounted for 70% of the protein . The majority of inaccessible residues were in the N-terminal 301 residues of A-protein . Dispersed among these were 65 surface-accessible residues in five linear epitope clusters illustrating the complex folding of this major structural domain of A-protein . The C-terminal 180 residues carried fewer linear epitopes but contained the major region of A-layers surface-accessible sequence, including four linear epitopes in predominantly hydrophobic sequence . Four A-layer surface-binding monoclonal antibodies also bound to this minor structural domain, although the epitopes of only two were identified by mimeotope analysis . The epitopes of six A-layer surface-binding monoclonals could not be identified, suggesting that A-layer may also contain conformation dependent surface epitopes. Vet Rec, 1993 Oct 16, 133(16), 389 - 91 Assessment of the antimicrobial sensitivity of Aeromonas salmonicida isolates from farmed Atlantic salmon in Scotland; Grant AN et al.; Sixty-five isolates of Aeromonas salmonicida were assessed for antimicrobial sensitivity by disc diffusion tests and the results compared with the minimum inhibitory concentration for each isolate . The results demonstrated that disc diffusion, using a standard technique, may be used to categorize isolates as sensitive or resistant provided that the corresponding minimum inhibitory concentrations are known. Microb Pathog, 1993 Oct, 15(4), 313 - 7 The role of a 40-megadalton plasmid in the adherence and hemolytic properties of Aeromonas hydrophila; Hanes DE et al.; A cured strain of Aeromonas hydrophila, MS-2PC, was examined for phenotypic changes in antibiotic resistance, adherence, and hemolysis . Parental strain MS-2 was resistant to ampicillin, novobiocin, and carbenicillin; MS-2PC, which lacked a 40-MDa plasmid, was also resistant to ampicillin but was sensitive to novobiocin and carbenicillin . The adherence of these isolates to CaCo-2 and HeLa cells was examined . MS-2PC demonstrated greater attachment to both cell lines than did strain MS-2 (p < 0.05) . MS-2PC also demonstrated greater hemolysis activity than did MS-2 (p < 0.01) . The 40-Mda plasmid was isolated and reintroduced into MS-2PC . The resulting transformant, 20T, regained resistance to carbenicillin and novobiocin . The attachment ability of 20T was equal to that of MS-2, and both strains demonstrated significantly lower attachment ability than that of MS-2PC (p < 0.01) . Strains MS-2 and 20T exhibited the same hemolysis pattern, which was markedly less than that of strain MS-2PC . These results indicate that the 40-Mda plasmid which codes for antibiotic resistance also controls other functions of A . hydrophila MS-2. Comp Immunol Microbiol Infect Dis, 1993 Oct, 16(4), 267 - 72 Fatal septicaemia caused by Aeromonas hydrophila in a patient with cirrhosis; Krovacek K et al.; In this case report from Italy we describe a fatal infection caused by A . hydrophila in a 39 yr old cirrhotic patient . This pathogen was isolated as a pure single culture from the patient's blood sample . The patient died on the second day of hospitalization from overwhelming sepsis . The A . hydrophila isolate was tested for different potential virulence properties, such as invasiveness, adherence, exotoxins production, presence of fimbriae and for the patterns of resistance to a variety of antimicrobial agents . Although, the Aeromonas species are infrequently reported as a cause of human infections, the present case study confirms the capability of these pathogens to induce serious human infections. New Microbiol, 1993 Oct, 16(4), 333 - 42 Characterization of mesophilic Aeromonas from clinical specimens by computerized analysis of SDS-PAGE protein profiles and by enzymatic activity; Arzese A et al.; Mesophilic Aeromonas (Aeromonas hydrophila, Aeromonas sobria, Aeromonas caviae) have recently been considered important aetiological agents of human diseases, mainly gastrointestinal infections . Although several findings have pointed out the significance of this group of microorganisms as enteric pathogens and suggested the presence of virulence factors, epidemiological and clinical studies are limited by the difficulty of correctly identifying mesophilic Aeromonas at the species level . SDS-PAGE of radiolabelled total protein profiles and bacterial enzymatic activities were used to type 31 clinical isolates (6 A . hydrophila, 7 A . sobria and 18 A . caviae) from patients with gastroenteritis and from healthy controls . Analysis of SDS-PAGE protein patterns, reinforced by the UPGMA-grouping system (AMBIS software) provided a good characterization of A . caviae strains as a homogeneous group of microorganisms, possessing significant differences from the other two species of mesophilic Aeromonas, in good agreement with biochemical and enzymatic tests . Data obtained in analyzing A . sobria protein profiles clearly showed two groups, with a correlation coefficient (CC) = 0.70, which in our experience is a doubtful value for assigning two strains to the same species . Strains biochemically identified as A . hydrophila showed a CC = 0.64, which is equally not acceptable for species assignment . Inter-species comparison highlighted this heterogeneity, showing two mixed subgroups, both containing strains that were assigned to A . sobria and A . hydrophila species on the basis of biochemical features.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Syst Bacteriol, 1993 Oct, 43(4), 855 - 6 Aeromonas enteropelogenes and Aeromonas ichthiosmia are identical to Aeromonas trota and Aeromonas veronii, respectively, as revealed by small-subunit rRNA sequence analysis; Collins MD et al.; The 16S rRNA gene sequences of the type strains of Aeromonas enteropelogenes and Aeromonas ichthiosmia were determined by polymerase chain reaction direct sequencing in order to clarify their interrelationships with other aeromonad species . On the basis of 16S rRNA gene sequence analysis, A . enteropelogenes and A . ichthiosmia were found to be identical to Aeromonas trota and Aeromonas veronii, respectively. Antibiot Khimioter, 1993 Oct-Nov, 38(10-11), 20 - 2 {The comparative characteristics of the antibiotic sensitivity of psychrophilic and mesophilic aeromonads}; Shenderovich VA et al.; The general regularities of the antibiotic susceptibility of psychrophilic and mesophilic aeromonads were determined . The antibioticograms were in general similar . Still, there was observed a higher susceptibility of Aeromonas salmonicida to tetracycline, chloramphenicol and rifampicin, as well as a larger number of strains susceptible to semisynthetic broad spectrum penicillins (ampicillin and carbenicillin) and cephazoline . The susceptibility to aminoglycosides was lower. FEMS Microbiol Lett, 1993 Sep 1, 112(2), 191 - 7 Molecular cloning and nucleotide sequence analysis of the maltose-inducible porin gene of Aeromonas salmonicida; Dodsworth SJ et al.; The gene for the Aeromonas salmonicida maltose-inducible porin (maltoporin) was cloned into phagemid pTZ18R in two restriction fragments, 0.6-kb PstI/KpnI and 1.7-kb SphI, of genomic DNA and their nucleotide sequences were determined . Open reading frames of 1329 and 1335 bp translated into sequences of 443 and 445 amino acids, with a 23 or 25 amino acid signal sequence and a 420 amino acid mature protein of molecular mass 46424 Da . Putative ribosome binding sites, AGGA and GGGAA, occurred 9 bp upstream of two possible ATG initiation codons . The A . salmonicida gene product showed a high degree of similarity with Escherichia coli LamB, and codon usage was very similar to that of another A . salmonicida outer membrane protein but markedly different from those of extracellular proteins. Am J Clin Pathol, 1993 Sep, 100(3), 308 - 10 Failure of the Vitek AutoMicrobic system to detect beta-lactam resistance in Aeromonas species; Schadow KH et al.; The ability of the Vitek AutoMicrobic system (AMS; Vitek, Inc., Hazelwood, MO) and disk-diffusion method to detect beta-lactam resistance was assessed with 25 strains from four species of Aeromonas . A very major error was indicated when a strain was shown to be susceptible by the AMS or disk-diffusion method but resistant by the agar dilution method . The rates for very major errors for disk diffusion and the AMS were 0% and 43%, respectively . The beta-lactam agents and numbers of very major errors for the AMS were as follows: ticarcillin, 17; mezlocillin, 3; piperacillin, 4; cephalothin, 9; cefazolin, 3; cefoxitin, 1; cefotetan, 2; and cefuroxime, 1 . Thus, these data suggest that the AMS currently is not reliable for testing the resistance of Aeromonas to beta-lactam agents. Infect Immun, 1993 Sep, 61(9), 3854 - 62 Aeromonas salmonicida grown in vivo; Garduno RA et al.; The virulent fish pathogen Aeromonas salmonicida was rapidly killed in vivo when restricted inside a diffusion chamber implanted intraperitoneally in rainbow trout . After a period of regrowth, the survivors had acquired resistance to host-mediated bacteriolysis, phagocytosis, and oxidative killing, properties which were subsequently lost by growth in vitro . Resistance to bacteriolysis and phagocytosis was associated with a newly acquired capsular layer revealed by acidic polysaccharide staining and electron microscopy . This capsular layer shielded the underlying, regular surface array (S-layer) from immunogold labeling with a primary antibody to the S-layer protein . Resistance to oxidative killing was mediated by a mechanism not associated with the presence of the capsular layer . An attenuated vaccine strain of A . salmonicida grown in vivo failed to express the capsular layer . Consequently, the in vivo-grown cells of this attenuated strain remained as sensitive to bacteriolysis, and as avidly adherent to macrophages, as the in vitro-grown cells . The importance of these new virulence determinants and their relation to the known virulence factors of A . salmonicida are discussed. J Diarrhoeal Dis Res, 1993 Sep, 11(3), 157 - 60 Adherence of haemagglutinating and non-haemagglutinating clinical and environmental isolates of Aeromonas; Singh DV et al.; Twelve haemagglutinating and non-haemagglutinating isolates of Aeromonas spp., comprising 6 each of clinical and environmental origin, were examined for their ability to adhere to rabbit intestinal epithelium, for inhibition of adhesion with sugars, and for delineation of the portion of intestine, jejunum, or ileum that is most susceptible to adhesion . Although the environmental isolates of Aeromonas haemagglutinated human erythrocytes that were inhibited by D-mannose and/or L-fucose, the majority of the clinical isolates of Aeromonas adhered to rabbit intestinal epithelium in almost equal proportions regardless of their haemagglutination (HA) properties, species designation, and source of isolation . Adhesion of both haemagglutinating and non-haemagglutinating isolates of Aeromonas was inhibited by sugars; however, the ability of sugar inhibition to adhere was similar to that observed with HA . This study suggests that adhesion is probably mediated by a variety of pilus or non-pilus colonisation factors which may or may not be a haemagglutinin . The jejunum was found to be more susceptible to adhesion than the ileum . However, no appreciable difference was observed in the number of adhered bacteria to adjacent loops. Immunology, 1993 Sep, 80(1), 68 - 72 Biological markers of macrophage activation: applications for fish phagocytes; Enane NA et al.; The immune defence mechanisms of fish seem to be related and similarly competent to those of mammals . Because of this, there is an increased interest in the immune responses of fish as models for higher vertebrates in immunological/immunotoxicological studies . Macrophages (M phi), phagocytic cells of the mammalian and teleost immune system which reside in tissues, represent a quiescent population of cells . However, upon stimulation, alterations in the physiology of these resident M phi occur which can be defined in terms of activation . This study was undertaken to determine whether biological markers used to assess mammalian M phi activation are applicable for use with fish M phi . Cells were recovered from the peritoneal cavity of non-injected and Aeromonas salmonicida-injected fish, and differences between resident and elicited M phi were evaluated with respect to protein content, phagocytic competence, enzyme activities and hydrogen peroxide production . Results demonstrate that biological markers used to assess mammalian M phi activation, with the exception of acid phosphatase activity, can be used to characterize the activation state of trout M phi, and that the activation process in both fish and mammals may occur by similar mechanism(s). Zentralbl Mikrobiol, 1993 Sep, 148(6), 441 - 7 Aeromonas-associated gastroenteritis in Egypt; Ghanem EH et al.; Aeromonas spp . including A . hydrophila, A . sobria, and A . caviae, were recovered from the feces of 88% of diarrheic Egyptian children . In contrast, only 45% of nondiarrheic children contained Aeromonas spp . A probable source of Aeromonas spp . is from drinking water inasmuch as nine out of ten samples analysed from the district of Cairo in which the children resided tested positive for Aeromonas spp . Enterotoxigenicity of the isolates from various sources was tested . 33% of the diarrheic samples produced enterotoxin whereas 47% of the nondiarrheic and 56% of the tap water strains produced enterotoxin. J Biol Chem, 1993 Aug 25, 268(24), 18272 - 9 Dimerization stabilizes the pore-forming toxin aerolysin in solution; van der Goot FG et al.; Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila . Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate . Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism . This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation . However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected . Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer . Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin . These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur. Appl Environ Microbiol, 1993 Aug, 59(8), 2437 - 41 Effects of nutrients on exopolysaccharide production and surface properties of Aeromonas salmonicida; Bonet R et al.; Extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Aeromonas salmonicida A450 and the influence of the capsule on cell surface properties were studied . A . salmonicida did not produce CPS or EPS when glucose, phosphate, magnesium chloride, or trace mineral components were absent from the medium . The addition of yeast extract improved capsule production . Neither EPS nor CPS formation depended on the C/N ratio, although it appeared to be influenced by the level of carbon and nitrogen in the culture . Both EPS and CPS production started at the end of the logarithmic growth phase . The amounts of EPS and CPS produced were not influenced by temperature changes between 15 and 20 degrees C and was maximal from pH 7 to 7.5 . Cell surface properties were strongly influenced by capsule production; high CPS production was associated with enhanced cell hydrophilicity and autoagglutination . The effect of CPS on cell surface properties was independent of the presence of the surface protein array (A-layer). Appl Environ Microbiol, 1993 Aug, 59(8), 2411 - 7 Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate; Anguita J et al.; A structural gene which codes for an extracellular lipase (EC 3.1.1.3) in Aeromonas hydrophila H3, which was isolated from a female hospitalized patient, was cloned in Escherichia coli by using pBR322 as a vector . Lipase purified from both A . hydrophila culture supernatant and the periplasmic fluids of E . coli containing the lip determinant in the original clone (plasmid pLA2) showed an M(r) of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the M(r) determined by Sephacryl S-200 chromatography . Regarding substrate specificity, the optimum chain lengths for the acyl moiety were C6 for ester hydrolysis and C6 and C8 for triacylglycerol hydrolysis . Sequence analysis showed a major open reading frame of 2,052 bp, which predicts a polypeptide with an M(r) of 71,804 . The polypeptide was found to contain an amino acid sequence (V-H-F-L-G-H-S-L-G-A) which is highly preserved among lipases. J Med Microbiol, 1993 Aug, 39(2), 107 - 13 Characterisation of strains of Aeromonas spp . by phenotype and whole-cell protein fingerprint; Millership SE et al.; Sixty-eight isolates of Aeromonas spp . were examined biochemically and their cell proteins were analysed by silver-stained SDS-PAGE . Protein fingerprints did not correlate with phenotype . However, consideration of both phenotype and fingerprint showed clustering of epidemiologically related isolates . There was also evidence that similar strains could be found in infected people and water or other environmental samples. J Infect Dis, 1993 Jul, 168(1), 215 - 8 Diarrhea associated with Aeromonas species in children in day care centers; de la Morena ML et al.; Outbreaks of diarrhea caused by enteropathogens have been reported in day care centers (DCC), but Aeromonas species have not been implicated . This study evaluated 381 children involved in 51 outbreaks in four DCC to determine the association of Aeromonas species with diarrhea and to characterize the isolates . The organism was identified in two outbreaks of diarrhea . In one, Aeromonas species were isolated from 6 (24%) of 25 children and in the other from 5 (21%) of 24 children . Seven other Aeromonas strains from children in DCC were studied . Fourteen (78%) of 18 were Aeromonas caviae and 15 were from children with diarrhea . Of the isolates, 75% did not have plasmids detected; all others had unique plasmid patterns . All strains had different DNA content . Twenty-two control isolates of Aeromonas from children with diarrhea in Mexico and Dallas had different chromosomal DNA patterns . Most Aeromonas infections were associated with symptoms . Chromosomal DNA patterns differentiated Aeromonas strains better than did plasmid DNA patterns . The outbreaks of diarrhea were unusual in that several different Aeromonas genospecies were involved in each outbreak. Comp Biochem Physiol Comp Physiol, 1993 Jul, 105(3), 479 - 84 The effect of bacteria infection on mean selected body temperature in the common Agama, agama agama: a dose-response study; Ramos AB et al.; 1 . The fever response was studied in 43 common agamas using a self-pairing experiment in which animals received an intraperitoneal injection of sterile saline and an injection of one of six dosages of dead Aeromonas sobria (1 x 10(6), 1 x 10(7), 1 x 10(8), 1 x 10(9), 1 x 10(10), and 1 x 10(11) total organisms) . 2 . The results demonstrated a significant increase in Tb (1.6-3.1 degrees C) above the mean selected body temperature (MSBT) of the saline injection animals over a bacteria infection range of three orders of magnitude . At 1 x 10(8) organisms, an increase was observed on bacteria day 1 while at dosages of 1 x 10(9) and 1 x 10(10) an increase was observed on bacteria days 1 and 2 . 3 . At dosages of 1 x 10(6) and 1 x 10(7) there was no difference between saline MSBT and bacteria MSBT . 4 . At a dosage of 1 x 10(11), MSBTs on bacteria days 1 and 2 were below saline MSBT . 5 . The average duration of the fever response is related to the level of infection; however, the magnitude of the fever is relatively independent of the level of infection. Int J Food Microbiol, 1993 Jun 1, 18(4), 339 - 42 Occurrence of Aeromonas spp . in samples of ground meat and chicken; Hanninen ML; Aeromonas spp . was commonly isolated from ground meat and chicken samples at the retail level . The dominant species in ground meat were A . hydrophila and A . caviae . In chicken, A . sobria was common while A . caviae was isolated infrequently . Although A . hydrophila was isolated from 75% of ground meat samples and 62% of chicken samples, DL-lactate-positive A . hydrophila (genospecies 1) was isolated from only 25 or 37% or respective samples . Sorbitol-positive A . hydrophila (genospecies 3) was common in both ground meat and chicken. Antimicrob Agents Chemother, 1993 Jun, 37(6), 1324 - 8 High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance; Segatore B et al.; The Aeromonas hydrophila AE036 chromosome contains a cphA gene encoding a metallo-beta-lactamase highly active against carbapenem antibiotics . This enzyme was induced in strain AE036 to the same extent by both benzylpenicillin and imipenem . When the cphA gene was inserted into plasmid pACYC184, used to transform Escherichia coli DH5 alpha, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DH5 alpha(pAA20R), expressing the Aeromonas metallo-beta-lactamase, were significantly increased, but those of penicillins and cephalosporins were not . When the metallo-beta-lactamase purified from E . coli DH5 alpha(pAA20R) was assayed with several beta-lactam substrates, it hydrolyzed carbapenems but not penicillins or cephalosporins efficiently . These results demonstrate that this metallo-beta-lactamase possesses an unusual spectrum of activity compared with all the other class B enzymes identified so far, being active on penems and carbapenems only . This enzyme may thus contribute to the development of resistance to penems and carbapenems but not other beta-lactams. J Bacteriol, 1993 May, 175(10), 3105 - 14 An Aeromonas salmonicida gene which influences a-protein expression in Escherichia coli encodes a protein containing an ATP-binding cassette and maps beside the surface array protein gene; Chu S et al.; A conserved Aeromonas salmonicida gene (abcA) affecting expression of the surface array protein gene (vapA) in Escherichia coli was identified . The 924-bp gene starts 205 bp after vapA and codes for a protein with a deduced molecular weight (M(r)) of 34,015 containing an N-terminal P-loop and significant homology to the ATP-binding cassette transport protein superfamily . AbcA was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by using T7 polymerase expression and DNA-directed translation and was copurified with the sarkosyl-soluble cytoplasmic membrane fraction . The protein displayed aberrant migration during SDS-PAGE . A lacZ fusion containing 128 bp of upstream sequence and 387 bases in the 5' end of abcA was constructed, and the beta-galactosidase activity of the abcA-lacZ fusion gene was shown to be similar in E . coli and A . salmonicida . The 130,000-M(r) AbcA-LacZ fusion protein was purified, and by using an ATP affinity column, the 129 AbcA N-terminal P-loop-containing residues were shown to bind ATP. Infect Immun, 1993 May, 61(5), 2172 - 81 An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis; Vaughan LM et al.; Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish . The disease is responsible for severe economic losses in intensively cultured salmon and trout . Bacterin vaccines provide inadequate protection against infection . We have constructed an aromatic-dependent mutant of A . salmonicida in order to investigate the possibility of an effective live-attenuated vaccine . The aroA gene of A . salmonicida was cloned in Escherichia coli, and the nucleotide sequence was determined . The codon usage pattern of aroA was found to be quite distinct from that of the vapA gene coding for the surface array protein layer (A layer) . The aroA gene was inactivated by inserting a fragment expressing kanamycin resistance within the coding sequence . The aroA::Kar mutation was introduced into the chromosome of virulent A . salmonicida 644Rb and 640V2 by allele replacement by using a suicide plasmid delivery system . The aroA mutation did not revert at a detectable frequency (< 10(-11) . The mutation resulted in attenuation when bacteria were injected intramuscularly into Atlantic salmon (Salmo salar L.) . Introduction of the wild-type aroA gene into the A . salmonicida mutants on a broad-host-range plasmid restored virulence . A . salmonicida mutant 644Rb aroA::Kar persisted in the kidney of brown trout (Salmo trutta L.) for 12 days at 10 degrees C . Vaccination of brown trout with 10(7) CFU of A . salmonicida 644Rb aroA by intraperitoneal injection resulted in a 253-fold increase in the 50% lethal dose (LD50) compared with unvaccinated controls challenged with a virulent clinical isolate 9 weeks later . A second vaccination after 6 weeks increased the LD50 by a further 16-fold. Microb Pathog, 1993 May, 14(5), 411 - 5 Effects of the acetylcholinesterase toxin of Aeromonas hydrophila on the central nervous system of fish; Rodriguez LA et al.; The purified acetylcholinesterase (AcChE) toxin, crude extracellular products (ECP) or viable virulent Aeromonas hydrophila were injected intraperitoneally into rainbow trout in different sublethal and lethal doses . When fish showed signs of morbidity, brain tissue was excised and assayed for acetylcholinesterase activity . In all cases there was a large increase in AcChE activity (about 40-fold for purified AcChE-toxin) . This was shown to be due to an accumulation of the fish's own AcChE and not the bacterial toxin . Nevertheless, the latter was detected in brain homogenates from fish in all treatment groups using a rabbit antiserum to the purified toxin to probe Western blots of brain homogenates, demonstrating that the toxin does gain access to brain tissue and is produced during in vivo infection . The results strongly suggest that this toxin plays a central role in the pathogenesis of A . hydrophila infection. Can J Microbiol, 1993 May, 39(5), 513 - 23 Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila; Chopra AK et al.; The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined . A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3 . Cell lysates of Escherichia coli (lambda CH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously . All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies . Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E . coli (lambda CH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin . DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide . Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent . Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently . Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A . hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1993 May, 26(2), 78 - 85 Isolation and characterization of Aeromonas from seafoods in Taipei; Yaun SS et al.; A total of 124 fresh seafoods and 158 processed seafoods collected from the retail markets and supermarkets in Taipei were tested for the contamination with motile Aeromonas spp . Of the fresh seafoods analyzed, 88% displayed the presence of Aeromonas . The isolation rates of various samples were as follows: 100%, freshwater fish; 95%, seawater fish; 78%, fish fillets; 84%, shrimp and crab of the crustacea group; 83%, bivalve shellfish and 84%, non-bivalve shellfish of the mollusca group, and 100%, seaweed . Of the 158 processed seafoods, 11% were contaminated by Aeromonas . The isolation rates were as follows: 0%, canned, dried, or frozen fresh seafood; 18%, salted seafood; 30%, fish cake; 7% vacuum-packaged fish cakes; 14%, frozen seafood dumplings; 8%, cooked seafoods . One hundred and eighty-three Aeromonas strains isolated in this survey were characterized to species level and tested for their ability to produce beta-hemolysin . Ninety-eight percent (98%) of the A . hydrophila produced beta-hemolysin on 5% blood agar, 94% of the A . sobria and 33% of the A . caviae produced beta-hemolysin . Thus it is likely that fresh seafoods are potentially significant sources of the virulent Aeromonas species and may play an important role in the epidemiology of Aeromonas-associated gastroenteritis. Antimicrob Agents Chemother, 1993 Apr, 37(4), 905 - 7 In vitro susceptibilities of tropical strains of Aeromonas species from Queensland, Australia, to 22 antimicrobial agents; Koehler JM et al.; Greater than 90% of 131 strains of Aeromonas species were susceptible to the aminoglycosides, ureidopenicillins, extended-spectrum cephalosporins, aztreonam, quinolones, tetracycline, and chloramphenicol, and all were uniformly resistant to ampicillin . Except for amoxicillin-clavulanate, sulfonamide, trimethoprim, and trimethoprim-sulfamethoxazole, there was good correlation between the results obtained by the agar dilution and disk diffusion techniques. FEMS Microbiol Lett, 1993 Apr 1, 108(2), 151 - 5 Identification of Aeromonas schubertii and Aeromonas jandaei by using a polymerase chain reaction-probe test; Ash C et al.; Two oligonucleotide primers were used in a polymerase chain reaction-protocol to amplify a region (approx . 850 bp) of the 16S rRNA gene of Aeromonas schubertii and Aeromonas jandaei . Hybridization of the polymerase chain reaction products to specific internal probes provided a highly specific method for the identification of these two species. Epidemiol Infect, 1993 Apr, 110(2), 279 - 87 Adherence to HEp-2 cells and enteropathogenic potential of Aeromonas spp; Grey PA et al.; Aeromonas strains (total = 60) of clinical, water and food origin were tested for adherence to HEp-2 cells . Environmental strains were selected (except for A . caviae) to include primarily those expressing other virulence-associated properties . Adhesion was markedly species-dependent (A . veronii biotype sobria, 15 of 26 {58%} . A caviae, 4 of 12 {33%} and A . hydrophila, 2 of 8 {11%}) . A . veronii biotype sobria were adhesive, irrespective of source (62 and 54% for clinical and environmental strains, respectively) . Adherent strains of this species were enterotoxin-positive and most (13 of 15) grew at 43 degrees C . A . caviae isolated from clinical specimens contained a higher proportion (75%) of adherent strains than environmental strains (13%) . Virulent subsets of A . veronii biotype sobria and A . caviae are adherent to HEp-2 cells . The HEp-2 assay is a useful model for investigating mechanisms of adherence and enteropathogenicity of virulent Aeromonas species. Appl Environ Microbiol, 1993 Mar, 59(3), 874 - 80 Survival of nonculturable Aeromonas salmonicida in lake water; Morgan JA et al.; The survival of Aeromonas salmonicida subsp . salmonicida was investigated in sterile and untreated lake water . In sterile lake water (filtered and autoclaved), it was found that cells of A . salmonicida entered a nonculturable but viable condition . Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential . For survival studies in untreated lake water, A . salmonicida was marked with the xylE gene by using the plasmid pLV1013 . Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin . Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A . salmonicida (pLV1013) . The results indicated that A . salmonicida entered a nonculturable condition in untreated lake water over a 21-day study . The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed. Dev Comp Immunol, 1993 Mar-Apr, 17(2), 129 - 40 Activation of rainbow trout (Oncorhynchus mykiss) phagocytes by muramyl dipeptide; Kodama H et al.; Immunoenhancing activity of synthetic muramyl dipeptide (MDP) was investigated in relation to the activation of rainbow trout (Oncorhynchus mykiss) phagocytes and to nonspecific protection against challenge with the fish pathogen Aeromonas salmonicida . Head kidney phagocytes collected from MDP-injected fish showed significant chemotactic activity against zymosan-activated normal rainbow trout serum, and phagocytic activities against both A . salmonicida and plastic beads . The MDP-activated phagocytes also showed enhanced superoxide generation . When normal phagocytes were exposed to supernatants of phagocyte or peripheral blood lymphocyte cultures of MDP-injected fish, the cells showed significant chemotactic activity, indicating that the MDP-activated cells produced phagocyte-activating factor . Injection of MDP to fish provided protection against challenge with virulent A . salmonicida. J Med Microbiol, 1993 Mar, 38(3), 227 - 34 Isolation and purification of Aeromonas sobria cytotonic enterotoxin and beta-haemolysin; Gosling PJ et al.; Aeromonas sp., grown in tryptone soya broth supplemented with yeast extract, 0.6%, pH 7.5, and incubated with agitation at 100 oscillations/min for 15 h at 37 degrees C produced optimal amounts of beta-haemolysin and cytotonic enterotoxin . More prolonged incubation resulted in the loss of enterotoxic activity and anion exchange chromatographic analysis indicated the presence of a moiety capable of breaking down the toxin . Anion exchange fast protein liquid chromatography resulted in a single peak of haemolytic activity and two peaks with enterotoxic activity . The cytotonic enterotoxin was purified from the fraction most active in the infant mouse assay; the second peak, which did not cross-react immunologically, may represent a second cytotonic enterotoxin . Neither peak was observed in the chromatographic fractions of filtrates from strains devoid of activity in the infant mouse assay . Purified enterotoxin, estimated to have a mol . wt of 15 kDa by SDS-PAGE, caused fluid accumulation in the infant mouse assay, was non-haemolytic to rabbit erythrocytes, caused an increase in cAMP activity in tissue culture cells and did not cross-react immunologically with components of cholera toxin or the whole toxin . Purified beta-haemolysin had an estimated mol . wt of 55 kDa, lysed rabbit erythrocytes and did not cause fluid accumulation in the infant mouse test. J Diarrhoeal Dis Res, 1993 Mar, 11(1), 30 - 4 Detection of Aeromonas hydrophila serogroup 0:34 in faeces using an enzyme-linked immunosorbent assay; Merino S et al.; A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was developed for detection of Aeromonas hydrophila serogroup 0:34 . The assay uses a detector antibody which shows no cross-reactions with Aeromonas strains not belonging to serogroup 0:34 or non-Aeromonas competing organisms . The detector antibody is mixed with the sample and incubated for 1 h; it is then microcentrifuged and the supernatant (unabsorbed antibody) titered on a microtiter plate coated with A . hydrophila cells from serogroup 0:34 . All A . hydrophila strains from serogroup 0:34 that we tested in this manner reacted strongly with the detector antibody . Also, by culturing and performing the immunoassay with the detector antibody we established and quantified the presence of A . hydrophila 0:34 on different samples. J Hosp Infect, 1993 Mar, 23(3), 223 - 8 Bacteriological investigation of the occurrence and antibiotic sensitivities of the gut-flora of the potential southern African medicinal leech, Asiaticobdella buntonensis (Hirudinidae); Wilken GB et al.; The lack of availability of medicinal leeches is a major impediment to the widespread use of leech therapy for treatment of congested flaps and replants in southern Africa . An investigation into the suitability of an alternative leech, the indigenous southern African leech, Asiaticobdella buntonensis, was therefore started . The risk of hospital-acquired infection related to the use of leeches and the antibiotic sensitivities of bacteria isolated from the gastro-intestinal tract of wild-caught leeches were investigated . Eleven bacterial genera were isolated but Aeromonas were most frequently isolated, occurring in 82% of microbiological samples . All were sensitive to cefotaxime and amikacin . The gut-flora and their sensitivities to 19 antibiotics were similar to those reported for the traditional medicinal leech, Hirudo medicinalis . These results emphasize the need to anticipate unusual infections when prescribing prophylactic or curative antibiotics in the course of leech therapy. Microbiologia, 1993 Feb, 9 Spec No, 49 - 56 {Incidence, behavior and control of Aeromonas hydrophila in meat and dairy products}; Garcia-Lopez ML et al.; This review deals with several aspects of Aeromonas hydrophila and other motile Aeromonas species associated with foodborne illness . Although it is mainly dedicated to the factors affecting growth and survival of this species in foods of animal origin, information on other topics is also provided .