Clin Infect Dis, 1998 Aug, 27(2), 332 - 44 Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions; Janda JM et al.; It has been almost 10 years since a major review on the association of Aeromonas with human disease has been published . During that period the number of valid species in the genus has grown to 14, with a new family (Aeromonadaceae) established to house this genus . Despite this explosion in the number of new genomospecies, only five (Aeromonas hydrophila, A . caviae, A . veronii, A . jandaei, and A . schubertii) are currently recognized as human pathogens . New syndromes attributed to this genus include hemolytic uremic syndrome, burn-associated sepsis, and a variety of respiratory tract infections, including epiglottitis . Convincing evidence suggests that some aeromonads do cause gastroenteritis, but it is presently unclear whether many of the strains isolated from feces are involved in diarrheal disease . Many questions regarding this genus remain unanswered. J Food Prot, 1998 Apr, 61(4), 414 - 8 Survival of Aeromonas hydrophila in fresh tomatoes (Lycopersicum esculentum Mill) stored at different temperatures and treated with chlorine; Velazquez LC et al.; This study examines the survival of two Aeromonas hydrophila strains (A . hydrophila ATCC 7965 {strain A} and A . hydrophila isolated from food {strain B} on the surface and core tissue of fresh tomatoes stored at different temperatures and the efficacy of chlorine treatment on their survival . Counts of A . hydrophila on the surface of tomatoes stored at 25 and 35 degrees C were significantly higher between days 1 and 4 for both strains as compared to results obtained at 6 degrees C . Core tissue counts of A . hydrophila cells on tomatoes dipped in a cellular suspension at 25 degrees C and stored at 25 degrees C were significantly higher (P < 0.05) than counts obtained with dip suspensions at 6 or 35 degrees C . In chopped tomatoes stored at 25 and 35 degrees C, populations of aerobic mesophiles showed significant increases after 96 and 70 h, respectively . The populations of both A . hydrophila strains in chopped tomatoes stored at 6 degrees C increased significantly after 96 h, while at 25 and 35 degrees C the counts increased in the first hours of incubation . Viable counts of A . hydrophila on the surface and central tissue of tomatoes significantly decreased (P < 0.05) when the samples were dipped for 2 min in chlorine at a concentration of 50 ppm (50 microgram/ml) . The results suggest that tomatoes should be kept at low temperatures during storage, shipping, and retail stocking and that chlorine at a concentration of 50 ppm should be used to reduce the levels of A . hydrophila. J Formos Med Assoc, 1998 Jul, 97(7), 498 - 502 Aeromonas hydrophila sepsis presenting as meningitis and necrotizing fasciitis in a man with alcoholic liver cirrhosis; Lin CS et al.; Aeromonas hydrophila is rarely reported as a causative organism of meningitis in humans . A 39-year-old man with alcoholic liver cirrhosis was admitted with a 2-day history of fever, chills, and confusion . Laboratory data revealed leukocytosis with granulocytosis, marked impairment of renal and liver function, and an elevated serum ammonia level . A . hydrophila was isolated from both blood and cerebrospinal fluid samples . Skin and soft-tissue lesions, consisting of bullae and necrotizing fasciitis, were found in the lower left leg 2 days after admission . Cultures of the bullous fluid, subcutaneous tissue, and fascia all yielded A . hydrophila . Pathologic examination revealed extensive necrosis . Although the patient was appropriately managed with antibiotics, debridement, and fasciotomies, his clinical status rapidly deteriorated, resulting in death 3 days after admission. Dis Aquat Organ, 1998 Feb 26, 32(1), 49 - 69 Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review; Wiklund T et al.; Bacterial strains of Aeromonas salmonicida included in the recognized subsp . acromogenes, subsp . masoucida, and subsp . smithia in addition to the large number of strains not included in any of the described subspecies are referred to as atypical A . salmonicida . The atypical strains form a very heterogeneous group with respect to biochemical characteristics, growth conditions, and production of extracellular proteasess . Consequently, the present taxonomy of the species A . salmonicida is rather ambiguous . Atypical A . salmonicida has been isolated from a wide range of cultivated and wild fish species, non-salmonids as well as salmonids, inhabiting fresh water, brackish water and marine environments in northern and central Europe, South Africa, North America, Japan and Australia . In non-salmonid fish species, infections with atypical strains often manifest themselves as superficial skin ulcerations . The best known diseases associated with atypical A . salmonicida are carp Cyprinus carpio erythrodermatitis, goldfish Carassius auratus ulcer disease, and ulcer disease of flounder Platichthys flesus, but atypical strains are apparently involved in more disease outbreaks than previously suspected . Macroscopical and microscopical studies of ulcerated fish indicate internal organs are infrequently invaded by atypical A . salmonicida . This view is supported by the fact that atypical strains are irregularly isolated from visceral organs of ulcerated fish . High mortality caused by atypical A . salmonicida has been observed in populations of wild non-salmonids and farmed salmonids, although the association between the mortality in the wild fish stocks and atypical A . salmonicida has not always been properly assessed . In injection experiments the pathogenicity of the atypical strains examined showed large variation . An extacellular A-layer has been detected in different atypical strains, but virulence mechanisms different from those described for (typical) A . salmonicida subsp . salmonicida, for example an extracellular metallo-protease and a different iron utilization mechanism, have been described . Limited information is available about the ecology, spread and survival of atypical strains in water . The commonly used therapeutic methods for the control of diseases in farmed fish caused by atypical A . salmonicida are generally effective against the atypical strains . Resistance to different antibiotics and transferable plasmid encoding multiple drug resistance have been observed in atpical A . salmonicida . Studies aimed at producing a vaccine against atypical strains are in progress. Burns, 1998 Jun, 24(4), 350 - 3 Aeromonas hydrophila in burn patients; Skoll PJ et al.; Burn wound infection with Aeromonas hydrophila appears to be very uncommon . This study reports on nine cases of A . hydrophila in burn patients treated over a 21 month period at the New Somerset Hospital Burn Unit . The average age of the patients was 31 years (range 24-60 years) and the average TBSA was 33% (range 16-51%) . All patients had positive wound cultures for A . hydrophila, obtained on admission or shortly thereafter . One patient also had a positive blood culture . Two patients with small partial thickness burns did not receive antibiotic therapy, and made an uneventful recovery with topical therapy alone . The other seven patients developed clinical signs of septicaemia and required parenteral antibiotics, in addition to topical therapy . One patient died of ARDS, but the other eight recovered and were discharged . No patient had evidence of myonecrosis . Small, superficial burns which culture A . hydrophila can be treated by topical therapy alone . Large and/or deep burns, require antibiotic therapy and debridement of all necrotic tissue, particularly when myonecrosis is present . The antibiotics of choice are the aminoglycosides or the quinolones. Zentralbl Hyg Umweltmed, 1998 Jun, 201(2), 199 - 203 The maximum growth temperature and human pathogenicity of psychrotrophic aeromonads; Schubert RH et al.; Determination of the maximum growth temperature of Aeromonas strains isolated from clinical material and endowed with pronounced propensity for adhesion to intestinal cells has demonstrated that all strains still grow at 39 degrees C, with this holding true for the majority of strains also at 40 degrees C . Since Aeromonas isolated obtained from water evidence to a large extent a markedly lower maximum growth temperature, the 39 degrees C growth criterion is a characteristic of Aeromonas strains isolated from clinical material. Rev Cubana Med Trop, 1997, 49(2), 84 - 5 {Comparative study of 4 methods for identifying species of the genus Aeromonas}; Longa A et al.; It is given an explanation of the results obtained with the following methods: AEROKEY II, AEROKEY II + Abbot's scheme, Api 20 NE, and the Biolog System . The study was conducted with 38 strains of Aeromonas isolated from children under 5 with acute diarrheal disease (ADD) . The AEROKEY II + Abbot's scheme proved to be the best identification method. Rev Cubana Med Trop, 1997, 49(1), 43 - 5 {Application of pyrazinamidase activity for the identification of Aeromonas species}; Bravo L et al.; 70 Aeromonas strains isolated from patients with acute diarrheal disease were classified into species by using the CAMP factor test and the Pyrazinamidase activity . It is stressed the high percent of coincidence between both tests, which are easy to make at the clinical microbiology laboratories. FEMS Immunol Med Microbiol, 1998 Jun, 21(2), 131 - 7 Possible virulence factors of Aeromonas spp . from food and water; Granum PE et al.; Thirty-one isolates of Aeromonas spp . from food and water in Norway were classified and tested for possible virulence factors including cytotoxins (tissue cultures, PCR), enterotoxins (PCR) and invasion ability (Caco-2 cells) . Five different species were recorded, A . caviae (9/31), A . hydrophila (15/31), A . schubertii (3/31), A . trota (3/31) and A . veronii biovar veronii (1/31) . One of the A . hydrophila strains was probably responsible for a small outbreak of food poisoning caused by ingestion of raw fermented fish . All the A . hydrophila strains produced and secreted cytotoxins at 37 degrees C, as well as two A . trota strains and the single A . veronii biovar veronii strain . In some cases increased cytotoxin secretion was observed under osmotic stress . The majority of the A . caviae strains which produced cytotoxins at 30 degrees C were unable to produce and/or secrete cytotoxins at 37 degrees C . One A . schubertii strain and one A . caviae strain were invasive. Dis Aquat Organ, 1998 Jun 19, 33(2), 87 - 92 Siderophore production by Aeromonas salmonicida subsp . salmonicida . Lack of strain specificity; Fernandez AI et al.; Siderophore production, presence of iron-regulated outer membrane proteins and siderophore specificity was determined among 17 isolates of Aeromonas salmonicida subsp . salmonicida obtained from Spain and Scotland . All grew in the presence of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) and siderophore production was detected using chrome azurol S (CAS) agar, confirming the presence of a high-affinity siderophore iron-uptake mechanism . The Arnow test confirmed that all isolates produced a catechol siderophore . Cross-feeding assays with indicator bacteria showed the absence of anguibactin, enterobactin, 2,3-dihydroxybenzoic acid (DHBA) and the hydroxamate siderophore, aerobactin, in the iron-restricted supernants of a representative isolate which cross fed 15/17 A . salmonicida isolates tested . Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of the same 2 major iron-regulated outer membrane proteins (IROMPs) in all isolates when grown in iron-restricted conditions and siderophore strain specificity as assessed by cross-feeding experiments was not apparent . Thus, with respect to IROMP and siderophore production A . salmonicida appears to be a homogeneous species. Lett Appl Microbiol, 1998 May, 26(5), 352 - 4 False-positive coliform reaction mediated by Aeromonas in the Colilert defined substrate technology system; Landre JP et al.; The Colilert defined substrate technology system allows specific, one-step detection of both coliforms and Escherichia coli while claiming to suppress the influence of non-coliform heterotrophs . The Colilert assay was examined in order to determine whether organisms from the genus Aeromonas could interfere and cause production of a false-positive coliform result as aquatic Aeromonas are known to constitute a fraction of the heterotrophic population found in drinking water . Results obtained clearly demonstrate that Aeromonas sp . can elicit a positive coliform type reaction at very low densities . Cell suspensions as low as 1 x 10(1) cells 10 ml-1 were observed to yield a positive reaction using Colilert reagent 4 weeks short of shelf-life expiry . Use of aged Colilert for monitoring water quality could lead to overestimation of coliforms as Aeromonas have been identified in many treated drinking water supplies. Lett Appl Microbiol, 1998 May, 26(5), 347 - 51 Bactericidal effect of chlorine on motile Aeromonas spp . in drinking water supplies and influence of temperature on disinfection efficacy; Sisti M et al.; The susceptibility of toxigenic Aeromonas spp . to free chlorine in drinking water supplies, and the influence of environmental temperature on the bactericidal activity of the oxidant, were evaluated . The results showed inactivation curves characterized by an initial phase of rapid reduction of viable cells followed by a slow inactivation of bacteria . The effect of the chlorine compound was markedly influenced by water temperature . At a summer water temperature (20 degrees C), the efficacy of the chlorine concentrations tested was found to be two to three times lower compared to that found at a winter temperature (5 degrees C) . Resistance was moderately, but significantly, greater in Aer . hydrophila vs Aer . caviae and Aer . sobria, but all Aeromonas spp . were more susceptible than Escherichia coli . Selective pressure with free chlorine did not produce Aeromonas cells with higher levels of chlorine resistance. Infect Immun, 1998 Aug, 66(8), 3825 - 31 Activation of the complement classical pathway (C1q binding) by mesophilic Aeromonas hydrophila outer membrane protein; Merino S et al.; The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied . The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D . Jeanteur, N . Gletsu, F . Pattus, and J . T . Buckley (Mol . Microbiol . 6:3355-3363, 1992), of these microorganisms . We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity . Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D) . Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated . Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein . The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing . Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups. Infect Immun, 1998 Aug, 66(8), 3501 - 9 Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants; Xu XJ et al.; Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections . The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila . Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.) . Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act) . The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis . The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector . In these mutants, the truncated act gene was integrated in place of a functionally active act gene . The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act . These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli . The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i . p., compared to 3.0 x 10(5) for the wild-type Aeromonas . Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act's biological activity and virulence in mice . The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies . Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections. Eur J Epidemiol, 1998 Apr, 14(3), 305 - 10 Clonal identification of Aeromonas hydrophila strains using randomly amplified polymorphic DNA analysis; Talon D et al.; The suitability of arbitrary primer polymerase chain reaction (RAPD) as a typing technique was evaluated by comparing it with pulsed-field gel electrophoresis (PFGE) to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections . Five isolates from patients and 10 isolates from the water supply were compared to 10 epidemiologically unrelated strains isolated from patients and rivers . Two methods were used to prepare DNA and two primers (AP3 and AP5) were selected . The discriminatory power was better with the extractive DNA preparation than the boiling method . The discrimination of closely related from less related strains by PCR using AP3 was consistent with that by PFGE: water supply of Cholet hospital contaminated with Aeromonas species was not the source of the cluster of hospital infections and only two patients were infected with clonally-related strains . RAPD using primer AP3 was simpler, cheaper, and quicker to perform than pulsed-field gel electrophoresis and is well suited for the epidemiological study of A . hydrophila isolates. Dis Aquat Organ, 1998 May 14, 33(1), 73 - 5 Generation and preliminary characterization of monoclonal antibodies directed to glycerophospholipid:cholesterol acyltransferase (GCAT) native epitopes of Aeromonas salmonicida; Lachmann I et al.; Four monoclonal antibodies (MAbs) directed to native glycerophospholipid:cholesterol acyltransferase (GCAT) epitopes of Aeromonas salmonicida were isolated using an esterase capture assay . The molecular mass of this MAb-defined antigen was estimated to be 26 kDa in SDS-PAGE . Three different epitope specificities of these MAbs were demonstrated . It was shown that all 4 MAbs recognize GCAT in culture filtrates of the strain MT004 excluding the simultaneous trapping of other components . None of the MAbs react with the denatured GCAT in Western blots. Appl Environ Microbiol, 1998 Jul, 64(7), 2473 - 8 Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota; Khan AA et al.; Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253 . Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1 . The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf(-) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109 . The nucleotide sequence of the aerA gene, located on the 1.8-kb ApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon . An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data . The deduced amino acid sequence of the aerA gene product of A . trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A . sobria 33. Sci Total Environ, 1998 Jun 18, 214, 153 - 64 Effects of mercuric chloride and sodium selenite on some immune responses of blue gourami, Trichogaster trichopterus (Pallus); Low KW et al.; The immunotoxicological effects of mercuric chloride and sodium selenite on blue gourami were studied . Some immune responses ranging from non-specific to specific were investigated . These include tissue lysozyme activity, kidney lymphocyte proliferation and plasma agglutinating antibody titre against bacteria . After 2 weeks of chronic exposure, 0.09 mg/l of Hg2+ alone induced a significant increase of kidney lysozyme activity of 4196.3 +/- 1171.0 U/g, but it decreased to 1577.4 +/- 902.4 U/g when exposed simultaneously to equiconcentration of selenium . Plasma lysozyme activity was also increased by co-administration of Hg2+ and SeO3(2-) . The level of plasma agglutinating antibody against Aeromonas hydrophila L37 was lowered in the chemical-treated fish . This indicates that the fish immunity was impaired by action of mercury and selenium . However, the in vitro lymphocyte proliferation test shows that mercury concentration lower than 0.045 mg/l Hg2+ enhanced the mitotic rate of kidney lymphocytes by approximately 30% . A high concentration of mercury caused irreversible damaging effects on con A-induced lymphoblastogenesis . In contrast, the inhibitory effect of low concentrations of mercury could be removed by washing . On the other hand, selenium showed a suppressive effect on the lymphocyte proliferation even at 0.5 mg/l. Cell Mol Life Sci, 1998 May, 54(5), 467 - 75 Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins; Robinette D et al.; Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography . The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis . Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B . The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish . These findings suggest that histones may be important defensive molecules in fish. Curr Microbiol, 1998 Jul, 37(1), 70 - 3 Purification and characterization of an arylamine N-acetyltransferase from the bacteria Aeromonas hydrophilia; Chung JG; N-acetyltransferase from Aeromonas hydrophilia was purified by ultrafiltration, DEAE-Sephacel, gel filtration chromatography on Sephadex G-100, and DEAE-5pw on high performance liquid chromatography, as judged by sodium dodecyl sulfate-polyacrylamine gel electrophoresis (SDS-PAGE) on a 12.% (wt/vol) slab gel . The enzyme had a molecular mass 44.9 kDa . The purified enzyme was thermostable at 37 degrees C for 1 h with a half-life 28 min at 37 degrees C, and displayed optimum activity at 37 degrees C and pH 7.0 . The Km and Vmax values for 2-aminofluorene were determined to be 0 . 896 mM and 2.456 nmol/min/mg protein, respectively . Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were demonstrated to be the most potent inhibitors. Ann Trop Med Parasitol, 1998 Mar, 92(2), 213 - 7 Aeromonas hydrophila soft-tissue infection as a complication of snake bite: report of three cases; Jorge MT et al.; Aeromonas hydrophila soft-tissue infection has been associated with fish and reptile bites . There have bee three recent cases from Brazil of abscesses complicating snake bites in which A . hydrophila was isolated from the purulent exudates . One of the snakes responsible for the bites was a specimen of Bothrops moojeni, and the others were most probably also lance-headed vipers . These snakes have a local necrotizing, myotoxic, oedema-inducing venom that must have favoured the multiplication in the injured tissue of A . hydrophila strains, which were probably present in the mouth, fangs or venom of the snakes . The use of a tourniquet by two of the patients probably worsened the local envenoming, and contributed to the occurrence of soft-tissue infection . The patients had a good outcome after the abscesses were incised and drained, and after being treated with chloramphenicol . Chloramphenicol appears to be a good alternative for the empirical treatment of soft-tissue infection complicating snake bite in Brazil, because: it is active against the majority of the anaerobic and aerobic bacteria found in these abscesses, including A . hydrophila; it can be administered by the oral route; and its is inexpensive . Suitable alternatives are cotrimoxazole or fluoroquinolones, to which aeromonads are usually susceptible in vitro, associated with antibiotics, such as clindamycin and metronidazole, with an anti-anaerobic spectrum. Vet Immunol Immunopathol, 1998 Feb 27, 61(2-4), 369 - 78 Immunostimulatory effects of dimerized lysozyme (KLP-602) on the nonspecific defense mechanisms and protection against furunculosis in salmonids; Siwicki AK et al.; Utilization of natural immunostimulants in fish culture offers a wide range of attractive methods for inducing and building protection against diseases . Lysozyme is an enzyme with bacteriolytic properties and is ubiquitous in its distribution among living organisms . This enzyme has antiviral, antibacterial and anti-inflammatory properties . In nature, lysozyme is found as a monomer . Lysozyme dimer is significantly less toxic than its monomer, and its high biological activity has been ascertained in cases of both viral and bacterial infections . In our study, we examined the influence of dimerized lysozyme (KLP-602) on the nonspecific cellular and humoral defense mechanisms and protection against furunculosis in rainbow trout (Oncorhynchus mykiss) . We have analyzed the immunomodulatory effects of KLP-602 after experimental infection by Aeromonas salmonicida . Application of dimerized lysozyme (KLP-602) by injection stimulated the cellular and humoral defense mechanisms and provided protection against furunculosis . By contrast, mortality rate was reduced to 45% (one injection) and 25% (three injections) using 10 or 100 microg/kg KLP-602 . Mortality in the untreated control group was 85%. Comp Immunol Microbiol Infect Dis, 1998 Jan, 21(1), 43 - 9 Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Baltic Sea: a case study; Krovacek K et al.; Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Swedish part of the Baltic Sea is reported . The pathogen was isolated from both lung and spleen specimens . All of the A . hydrophila isolates produced haemolysin and Vero active cytotoxin . The aerolysin gene was found in all tested isolates as evidenced by the polymerase chain reaction (PCR) technique . Also, all isolates tested showed identical patterns of biochemical and antibiotic resistance . As Aeromonas spp . commonly occur in aquatic environments, we suggest that organisms from this genus may also play an important role as opportunistic pathogens in morbillivirus infected seals. Can J Microbiol, 1998 Feb, 44(2), 103 - 8 Phenotypic identification of Aeromonas genomospecies from clinical and environmental sources; Borrell N et al.; A collection of 983 Aeromonas isolates from environmental and clinical sources have been identified to the genomospecies level . A phenotypic method identified 93% of the strains . The use of citrate and the production of acid from sorbitol enabled the members of the Aeromonas hydrophila complex to be separated . The most common genomospecies from intestinal sources were Aeromonas veronii biotype sobria and Aeromonas caviae . The former, together with A . hydrophila, was the most frequently isolated species of extraintestinal origin . Most pathogenic species were very prevalent in environmental samples, with A . veronii biotype sobria being the most common in lakes and reservoirs (41.5%) and in treated drinking water (25.0%), and A . caviae was the most common in sea water (26.0%) and milk products (35.5%) . Aeromonas hydrophila (18.1%) was the second most prevalent species isolated in untreated drinking water . Since Aeromonas infections are generally regarded as a water- and food-borne diseases, the high environmental prevalence of these pathogenic genomospecies should be regarded as an important threat to public health. Infect Immun, 1998 May, 66(5), 1990 - 8 Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida; Vipond R et al.; The importance of the two major extracellular enzymes of Aeromonas salmonicida, glycerophospholipid: cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid fish with furunculosis had been indicated in toxicity studies . In this study, the genes encoding GCAT (satA) and AspA (aspA) have been cloned and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic satA and aspA mutants of A . salmonicida . A pSUP202 derivative (pSUP202sac) carrying the sacRB genes was constructed to facilitate the selection of mutants . The requirement of serine protease for processing of pro-GCAT was demonstrated . Processing involved the removal of a short internal fragment . Surprisingly, pathogenicity trials revealed no major decrease in virulence of the A . salmonicida delta satA::kan or A . salmonicida delta aspA::kan mutants compared to the wild-type parent strains when Atlantic salmon (Salmo salar L.) were challenged by intraperitoneal injection . Moreover, using a cohabitation model, which more closely mimics the natural disease, there was also no significant decrease in the relative cumulative mortality following infection with either of the deletion mutants compared to the parent strain . Thus, although these two toxins may confer some competitive advantage to A . salmonicida, neither toxin is essential for the very high virulence of A . salmonicida in Atlantic salmon . This first report of defined deletion mutations within any proposed extracellular virulence factor of A . salmonicida raises crucial questions about the pathogenesis of this important fish pathogen. Infect Immun, 1998 May, 66(5), 1813 - 21 Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine; Hernanz Moral C et al.; The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined . The nucleotide sequence of the A . hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins . To obtain an effective attenuated live vaccine against A . hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A . hydrophila AG2 by means of the suicide vector pSUP202 . The A . hydrophila mutant AG2 aroA::Ka(r) was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 x 10(8) CFU . The mutants were not recoverable from the internal organs after 48 h postinoculation . Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A . hydrophila were present in the kidney and spleen 9 days postinjection . Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A . hydrophila. FEMS Immunol Med Microbiol, 1998 Mar, 20(3), 219 - 29 Analysis of the interaction of Aeromonas caviae, A . hydrophila and A . sobria with mucins; Ascencio F et al.; Aeromonas species are known to be involved in human gastrointestinal diseases . These organisms colonize the gastrointestinal tract . Aeromonas hydrophila, A . caviae, and A . sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date . Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A . caviae, A . hydrophila, and A . sobria strains . Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure . The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase . Binding of the various horseradish peroxidase-labeled mucins by A . caviae, A . hydrophila, and A . sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources . The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A . hydrophila than for A . caviae and A . sobria . Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins . The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A . caviae strain A4812 (95 and 44 kDa); A . hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A . sobria strain 48739 (95 and 43 kDa) . Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media . The culture conditions greatly influence the expression of A . hydrophila mucin-binding activity. Zentralbl Hyg Umweltmed, 1998 Feb, 200(5-6), 571 - 4 Detection of psychotrophic aeromonads in drinking water; Schubert RH; In the context of evaluating their pathogenic relevance, culture on solid media is the only approach presently suitable for culture of psychotrophic aeromonads from drinking water . In this respect, a check must be effected to ensure that the culture medium cannot engender selective culture losses for individual species . For this reason, media to which e.g . ampicillin has been added are unsuitable. Antimicrob Agents Chemother, 1998 Feb, 42(2), 436 - 9 Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv . sobria; Walsh TR et al.; The Aeromonas veronii bv . sobria metallo-beta-lactamase gene, imiS, was cloned . The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1 . To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA . Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases. Arch Microbiol, 1998 Mar, 169(3), 239 - 48 Reduction of diverse electron acceptors by aeromonas hydrophila Knight V V, Blakemore R. Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol . Final cell yields were directly proportional to the amount of terminal electron acceptor provided . Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI) . Dissimilatory Fe(III) reduction by A . hydrophila may involve cytochromes . Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm) . Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate . Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase . Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously. FEMS Microbiol Lett, 1997 Nov 15, 156(2), 199 - 204 RFLP-PCR analysis of the aroA gene as a taxonomic tool for the genus Aeromonas; Cascon Soriano A et al.; The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR . Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway . A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A . salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups . HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level. J Pak Med Assoc, 1997 Dec, 47(12), 305 - 8 Isolation and identification of Aeromonas species from human stools; Ahmed A et al.; One thousand and three diarrhoeal stool samples were processed in our laboratory during the period 1996/1997 for the presence of enteric pathogens especially Aeromonas spp., which has emerged as a new agent causing diarrhoea . Ampicillin sheep blood agar was found to be the best medium for the isolation of Aeromonas spp . from stool specimens . Enteric pathogens were found in 200 (20%) stools, of which Aeromonas spp . was the second commonest pathogen isolated amounting to 21% of isolates . This study clearly indicates that Aeromonas spp . must be looked for in every diarrhoeal stool samples, specially in children below 10 years of age . Isolation and identification is cost effective and easy, if the given protocol is observed. Haematologica, 1997 Nov-Dec, 82(6), 692 - 4 Acute rhabdomyolysis and myonecrosis complicating aeromonas bacteremia in neutropenic patients with hematologic malignancies: report of two cases; Martino R et al.; Infections by Aeromonas spp . are a rare cause of systemic infection in normal and immunocompromised hosts . We report the cases of two patients with acute non-lymphoblastic leukemia who developed septic shock by Aeromonas species with unusual soft-tissue complications . One patient who was undergoing consolidation chemotherapy developed septic shock by Aeromonas hydrophila with rhabdomyolysis and subsequent soft-tissue destruction consistent with myonecrosis . She recovered with combination antibiotic therapy and supportive care . The second patient developed neutropenia due to ganciclovir treatment for post-allogeneic transplant cytomegalovirus antigenemia . He developed a rapidly progressive septic shock due to Aeromonas sobria with rhabdomyolysis, multi-organ failure and bilateral lower limb myonecrosis, and died within 48 hours . The portal of entry was not identified in either case . These cases confirm the potentially aggressive nature of these bacteria in neutropenic cancer patients with an unusual tendency to produce muscular and soft-tissue destruction. Int J Food Microbiol, 1997 Sep 16, 38(2-3), 111 - 6 Effects of temperature, medium composition, pH, salt and dissolved oxygen on haemolysin and cytotoxin production by Aeromonas hydrophila isolated from oyster; Tsai GJ et al.; The effects of temperature, medium composition, pH, salt content and dissolved oxygen (DO) on the production of haemolysin and cytotoxin by one strain of Aeromonas hydrophila isolated from oyster were investigated . Four media were tested: brain heart infusion broth (BHIB), casamino acid-yeast extract broth (CAYEB), nutrient broth (NB), and trypticase soy broth (TSB) . BHIB was the best for toxin production even though the growth rates for Aeromonas hydrophila in all of these media were quite similar . Aeromonas hydrophila could produce haemolysin and cytotoxin at 37, 28 and 5 degrees C; however, the toxins were produced faster and were more stable at 28 degrees C than at 37 degrees C . Although Aeromonas hydrophila itself is tolerant to 5% (w/v) salt in BHIB and a pH range of pH 5.5 to 10.0, the production of haemolysin and cytotoxin was apparently decreased in the presence of 1-5% (w/v) NaCl or when the pH of the medium was greater or less than 7.2 . The DO values in the culture medium during the stationary growth phase also seemed to affect toxin production; greater quantities of toxins were produced when the DO values were higher. New Microbiol, 1998 Jan, 21(1), 23 - 30 Virulence factors in Aeromonas spp and their association with gastrointestinal disease; Schiavano GF et al.; Culture filtrates of eight Aeromonas strains isolated from the feces of 487 subjects (292 diarrhoeic patients and 195 asymptomatic subjects) were tested for toxin production in CHO and McCoy cells, and adhesion and invasive ability in Caco-2 cells . Among these isolates, three Aeromonas sobria and one Aeromonas caviae strains possessed virulence-associated properties . Toxin production was the most common of the three virulence properties . Two A . caviae were associated, in the absence of other diarrhoeagenic agents, with gastroenteritis; however, a virulence marker (cytotoxin) was recognized only in one strain . Two strains of A . sobria isolated from subjects with gastroenteritis were shown to be associated with one (cytotoxicity) or two (adhesion and invasive abilities) virulence factors, respectively . However, a third strain of A . sobria, although cytotoxic and invasive, was isolated from an asymptomatic subject . The results show that Aeromonas spp may act as human enteric pathogens, but also indicate that the significance of several putative virulence factors, such as production of cytotoxin and the capacity to adhere to and invade mammalian cells, remains controversial in explaining the enteropathogenesis of Aeromonads and therefore needs further studies. J Cell Biol, 1998 Feb 9, 140(3), 525 - 40 A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum; Abrami L et al.; In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells . Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases . We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation . We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization . Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes . Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments . Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited . Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics. Microbiology, 1998 Feb, 144 ( Pt 2), 299 - 307 Internalization of Aeromonas hydrophila by fish epithelial cells can be inhibited with a tyrosine kinase inhibitor; Tan E et al.; Aeromonas hydrophila is a Gram-negative bacterium that is pathogenic in fish, causing motile aeromonad septicaemia . It can enter (invade) fish cells, and survive as an intracellular parasite . The host-pathogen interaction and signal transduction pathway were studied by screening signal transduction inhibitors using carp epithelial cells and a virulent strain of the bacterium, PPD134/91 . Genistein, a tyrosine kinase inhibitor, postponed internalization of A . hydrophila into host cells, suggesting that tyrosine phosphorylation plays a role in internalization . In contrast, staurosporine, a protein kinase C inhibitor, and sodium orthovanadate, a protein tyrosine phosphatase inhibitor, accelerated internalization of PPD134/91 . Other virulent strains of A . hydrophila were also examined and it is likely that all strains, irrespective of serogroup, use the same signalling pathway to facilitate bacterial uptake. Appl Environ Microbiol, 1997 Oct, 63(10), 3770 - 5 Further characterization of Renibacterium salmoninarum extracellular products; Barton TA et al.; Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish . The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule . The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass . Separated fractions continued to produce degradation and aggregation products . One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions . Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation . Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase . The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa . Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish . Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides . The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R . salmoninarum infections. Curr Microbiol, 1998 Jan, 36(1), 19 - 23 Experimental pathogenicity of aeromonas spp . for the zebra mussel, dreissena polymorpha Maki JS, Patel G, Mitchell R. Experiments were conducted to determine whether species of Aeromonas were pathogenic to the zebra mussel Dreissena polymorpha . A . jandaei, A . veronii, and A . media, identified with Biolog, were originally isolated from dead zebra mussels . When inoculated into living mussels, these bacteria resulted in the mortality of the bivalves . Two additional species, A . salmonicida salmonicida (ATCC 33678) and A . hydrophila (ATCC 7966), were also demonstrated to be pathogenic to the mussels . In addition to the pathogenicity, the data also suggest that the zebra mussels may be an important reservoir for these bacteria in freshwater environments. Appl Environ Microbiol, 1998 Feb, 64(2), 549 - 54 Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp . strain NCIMB 10558; Liu JQ et al.; A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp . strain NCIMB 10558 was cloned and sequenced . The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues . The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized . The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position . In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine . The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms . However, lysine 207 of low-specificity L-TA from Pseudomonas sp . strain NCIMB 10558 was found to be completely conserved in these proteins . Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm . Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction. Zh Mikrobiol Epidemiol Immunobiol, 1995 Jul-Aug, (4), 9 - 12 {Bacteria of the genus Aeromonas as the causative agents of saprophytic infection}; Pogorelova NP et al.; The bacteriological study of feces from 458 patients with acute enteric diseases revealed that in 3.2% of cases (in summer in 8.1% of cases) the disease was caused by Aeromonas . In Aeromonas strains isolated from river water in the Volga delta, from fish and from raw meat the same pathogenicity factors occurred as in strains isolated from patients (hemolysin, DNA-ase, protease, lecithinase, amylase, adhesins, capacity of binding Congo red). J Bacteriol, 1998 Feb, 180(3), 667 - 73 Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae; Fukui T et al.; Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ(Ac) . Escherichia coli BL21(DE3) carrying phaJ(Ac) . under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography . The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ(Ac) except for the initial Met residue . The enoyl-CoA hydratase encoded by phaJ(Ac) exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms . These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ(Ac) is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid beta-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A . caviae. Kansenshogaku Zasshi, 1997 Nov, 71(11), 1172 - 4 {Detection of the thermostable lipase gene (lipAH) in clinical isolates of Aeromonas hydrophila belonging to the major O serogroups O11, O16 and O34}; Shibata M et al.; On the basis of DNA hybridization data and phenotypes, most pathogenic strains of Aeromonas hydrophila are grouped into hybridization group 1 (HG1) . These pathogenic strains secret the theromostable lipase, and it's gene (lipAH) has been cloned and sequenced . The present study was performed to identify the pathogenic strains of A . hydrophila by the polymerase chain reaction (PCR) technique to detect the lipAH . Synthetic oligonucleotide primers (lipAH-ns-1 and -na-1) were used in the PCR . The PCR identified 80% of lipAH-positive strains, consisting of seven of the 11 O11 strains (64%), 13 of the 15 O16 strains (87%) and 25 of the 30 O34 strains (83%) in clinical isolates of A . hydrophila used in this study. Folia Microbiol (Praha), 1997, 42(4), 385 - 9 Virulence factors-pathogenicity relationships for Aeromonas species from clinical and food isolates; Pin C et al.; The presence of virulence factors in 96 Aeromonas strains isolated from food and clinical samples was studied . Neither cytotoxic activity and hydrophobicity, not the presence of pili or an extra surface layer made it possible to establish differences between food and clinical strains . Statistical studies showed that cytotoxin production was associated with a positive Voges-Proskauer reaction, inability to ferment arabinose and a positive lysine decarboxylation . Therefore, when comparing cytotoxic clinical and food strains with lysine decarboxylation phenotype, there was a significant difference (p < 0.05) between the two groups . The association of a cytotoxin production and lysine decarboxylation character should thus be considered as a possible virulence marker. J Basic Microbiol, 1997, 37(6), 403 - 5 Effect of amino acids on the production and activities of cephalosporin C acylase and penicillin V acylase from Aeromonas species ACY 95; Dehpande BS et al.; Amino acids altered the production and activities of cephalosporin C acylase and penicillin V acylase from Aeromonas species ACY 95 to a varying degree . DL-Tryptophan enhanced the cephalosporin C acylase formation by 222% while suppressed the penicillin V acylase formation by 68%. Arch Latinoam Nutr, 1997 Mar, 47(1), 44 - 6 {Prevalence of Aeromonas spp . in surface water}; Hernandez P et al.; Some Aeromonas strains are well recognized enteropathogens according to microbiological, clinical, immunological and epidemiological evidence . The main source of infection seems to be untreated water, these microorganisms can be found in virtually all aquatic environments . Additionally, some Aeromonas, which include enterotoxigenic strains, are capable of rapid growth at 5 degrees C and even of producing toxins . Vegetable products irrigated with contaminated water may reach critical Aeromonas levels after being kept under refrigeration, this could represent a public health risk when they are consumed as uncooked salads . This study was pursued to evaluate such risk . Surface water samples were streaked on starch ampicillin and inositol-brilliant green-bile salts agar dishes . In addition, 100 ml of each sample were filtered through a 0.45 micron Millipore membrane filter . The filters were incubated on alkaline peptone water as enrichment media during 24 h at 35 degrees C . Enrichment broth was then streaked on the selective agars above mentioned . Isolates from both tests were identified using the API 20 E System . The prevalence of Aeromonas strains in the analyzed samples was 17.8% . A higher isolation rate was observed after the enrichment technique . Starch ampicillin agar showed a higher recuperation rate . A Veronii biotype sobria (formerly A . sobria) was isolated with higher frequency . Since this species has been associated with the greatest virulence, the use of contaminated water to irrigate vegetable products that are to be kept under refrigeration and consumed without ulterior cooking may represent a risk to the public health. Lett Appl Microbiol, 1997 Nov, 25(5), 363 - 6 Reappraisal of the effect of temperature on the growth kinetics of Aeromonas salmonicida; Guerin-Faublee V et al.; The effect of temperature (1-34 degrees C) on the maximum specific growth rate of Aeromonas salmonicida could not be described by the classical growth models; for some strains, two optimal temperatures at 23 degrees C and 30 degrees C were observed, as well as an unexpected increase in the pseudolag time above 27 degrees C . This could be explained by the presence of two subsets, notably S-layer+ and S-layer- sub-populations . The A- cells had higher growth parameters (Topt and mu opt) than the A+ cells and were selected by subcultures above 30 degrees C . Yet the relative proportion of A+ cells did not explain all the variation of mu max versus temperature, and the growth kinetics of an Aer . salmonicida isolate remained unpredictable. J Appl Microbiol, 1997 Nov, 83(5), 542 - 51 Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production; Gunnlaugsdottir B et al.; The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to: lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities . Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined . Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs . Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested . An extracellular metallo-caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect . Furuncular-like lesions were produced by ECPs containing AsaP1 activity . One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity . The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases. Arch Biochem Biophys, 1997 Nov 15, 347(2), 201 - 7 Determination of the amino acid sequence of the plant cytolysin enterolobin; Fontes W et al.; The cytolytic seed protein enterolobin from seeds of Enterolobium contortisiliquum was purified by using FPLC on a Mono Q column giving a single peak in capillary electrophoresis . The complete amino acid sequence of the plant cytolysin was determined by an automated method, yielding a molecular mass of 54,806 Da . Databank searches and sequence alignment demonstrated a high degree of sequence identity and similarity between enterolobin and bacterial aerolysins from Aeromonas hydrophila and A . sobria . Several key residues involved in oligomerization of A . hydrophila aerolysin are conserved in enterolobin . Circular dichroism measurements and structural predictions revealed that enterolobin is very rich in beta sheet, like aerolysin . Light-scattering studies revealed that enterolobin oligomerizes as a hexamer at pH levels below 7.0 . NaCl concentrations above 50 mM caused dimerization of enterolobin . Dithiothreitol did not cause oligomerization . Aust N Z J Ophthalmol, 1997 Nov, 25(4), 299 - 300 Aeromonas sobria endophthalmitis; Lee LR et al.; BACKGROUND: Aeromonas sobria causes a rare Gram-negative bacterial water-borne infection . It has been found in waters of North Queensland and South-east Asia . Of all Aeromonas species, A . sobria is the most virulent and invasive and has been reported to cause soft tissue infection and corneal ulcer . METHODS: A 14-year-old Caucasian male from North Queensland presented following a penetrating eye injury in which a water bird (cormorant species) had pecked his eye while he was fishing . A fulminant endophthalmitis developed despite treatment with intravenous, intravitreal and topical antibiotics and initial wound repair . Enucleation was performed . RESULTS: Aeromonas sobria was isolated from the vitreous aspirate . CONCLUSION: Aeromonas sobria infection should be suspected in water-contaminated penetrating eye injuries . The prognosis in this case was poor. J Med Microbiol, 1997 Oct, 46(10), 833 - 8 Influence of iron, growth temperature and plasmids on siderophore production in Aeromonas hydrophila; Naidu AJ et al.; Aeromonas hydrophila strains obtained from diarrhoeal samples of human patients (19 isolates) and freshwater ponds (11 isolates) were analysed for siderophore production . Both clinical and environmental isolates showed significantly increased siderophore production under iron-limiting conditions both at 28 degrees C and at 37 degrees C . Clinical isolates consistently produced higher levels of siderophores than did the environmental isolates . The role of plasmids in moderating siderophore production was studied after curing with acridine orange . Treatment with acridine orange for 24 h removed the larger plasmids but the smaller plasmids (< 5 MDa), more common in the environmental isolates, were resistant to curing . As found in the untreated isolates, the cured clinical isolates produced higher mean levels of siderophores than the cured environmental isolates . Siderophore production in A . hydrophila was significantly influenced by iron-limiting cultural conditions and the source of isolates, but plasmid content and growth temperature at 28 degrees C or 37 degrees C had little effect on production . The basis for the greater production of siderophores in clinical isolates than in environmental isolates needs further study. Am J Gastroenterol, 1997 Nov, 92(11), 2104 - 6 Aeromonas sobria-associated left-sided segmental colitis; Deutsch SF et al.; Aeromonas species, once thought pathogenic only in immunocompromised hosts, have more recently been found to be associated with many different infections in previously healthy individuals . The most common site of these infections is the gastrointestinal tract . We describe a 67-yr-old woman who presented with abdominal pain and bloody diarrhea and was diagnosed with left-sided, segmental colitis due to Aeromonas sobria, which was proven by stool culture . Extensive work-up for ischemic colitis was unremarkable, and after treatment with antibiotics, the patient's symptoms resolved, and follow-up colonoscopy failed to reveal any evidence of residual colitis or inflammatory bowel disease . Our report supports the view that Aeromonas species need to be considered in the differential diagnosis of colitis, and we believe it to be the first report of left-sided, segmental colitis secondary to Aeromonas sobria infection. Appl Environ Microbiol, 1997 Nov, 63(11), 4534 - 42 Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems; Faude UC et al.; Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990 . The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718 . These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysis of low-molecular-weight RNAs from bacterioplankton . We developed one MAb each for the bacterial isolates PX54 and PU7718 that did not show any cross-reactivity with other bacterial strains by immunofluorescence microscopy . Each MAb recognized the general lipopolysaccharide fraction of the homologous strain . These MAbs were tested successfully for their ability to be used for the in situ detection and counting of bacteria in lake water by immunofluorescence microscopy . During the spring of 1993, A . hydrophila PU7718 showed a depth distribution in Lake Plusssee with a pronounced maximum abundance at 6 m, whereas Comamonas acidovorans PX54 showed a depth distribution with a maximum abundance at the surface . The application of these MAbs to the freshwater samples enabled us to determine the cell morphologies and microhabitats of these strains within their natural environment . The presence of as many as 8,000 cells of these strains per ml in their original habitats 3 years after their initial isolation demonstrated the persistence of individual strains of heterotrophic bacteria over long time spans in pelagic habitats. Enzyme Microb Technol, 1997 Nov 15, 21(7), 472 - 8 Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae; Lin CS et al.; The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques . Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3) . The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography . The recombinant chitinase was found to be present in both the culture medium and the cytoplasm . A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining . The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively . It was stable within the pH range of 5-7 . Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed . Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity . Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods . When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides {(GlcNAc)n, n = 1-4} were detected at 24 h. Gene, 1997 Oct 15, 199(1-2), 225 - 9 Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila; Chang TM et al.; A gene (eprA1) encoding the extracellular protease of Aeromonas hydrophila AH1 has been cloned and sequenced . Nucleotide sequence analysis of eprA1 predicted a single open reading frame (ORF) of 1038 bp encoding a 346 amino acid (aa) polypeptide, with a potential 21-aa signal peptide . When the eprA1 gene was expressed in minicells, one major band of approx . 37 kDa was identified, while protease activity staining experiments identified a caseinolytic band of approx . 29 kDa determined by SDS-PAGE analysis of the minicells . The deduced C-terminal aa region (Arg-290 to Gly-313) showed sequence homology to partial C-terminal sequences of other zinc metalloproteases including Penicillium citrinum metalloprotease (PlnC), Aspergillus oryzae metalloprotease (NpII), Aspergillus flavus metalloprotease (MepA), and Aspergillus fumigatus metalloprotease (Mep20), particularly with respect to zinc-binding residues. Lett Appl Microbiol, 1997 Oct, 25(4), 269 - 73 Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapia nilotica; Khalil AH et al.; Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic and proteolytic activity were studied with respect to temperature and time of incubation as well as the lethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin product was achieved when Aer . hydrophila was grown at 35 degrees C for 30 h . Tilapia erythrocyte was found to be more susceptible than sheep erythrocyte for determining the haemolytic activity . The haemolytic activity against tilapia erythrocyte was completely inactivated after heating the ECP at 60 degrees C for 10 min or 55 degrees C for 15 min . The proteolytic activity was maximized when the bacterium was grown at 30 degrees C for 36 h . Complete inactivation of the protease enzyme was performed after heating the ECP at 80 degrees C for 10 min or 70 degrees C for 15 min . Aeromonas hydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD50 2.1 x 10(4) cell/fish), as well as heat stable unknown virulent factors that were responsible for 20% mortality . The lethality of ECP was decreased by heating and completely inactivated by boiling at 100 degrees C for 10 min. Microb Pathog, 1997 Oct, 23(4), 241 - 7 Aeromonas spp . possess at least two distinct type IV pilus families; Barnett TC et al.; Type IV pili have been purified from strains of most of the Aeromonas species associated with gastroenteritis (A . veronii biovar sobria, A . hydrophila, A . trota and A . caviae) . They appear to be a related family (molecular mass of pilin 19 to 23 kDa) with a tendency to bundle-formation . Hence, we have designated them 'bundle-forming pili' (Bfp) . A type IV pilus biogenesis gene cluster (tapABCD) recently cloned from a strain of A . hydrophila, however, encoded a 17 kDa pilin which differed significantly in its N-terminal amino acid sequence from the Bfp pilins . This paper describes the cloning of part (tapA and approximately 20% of tapB) of a homologous pilin gene cluster from a Bfp-positive strain of A . veronii biovar sobria, and presents evidence that the entire pilin gene cluster (tapABCD) is present in this strain . The predicted N-terminal amino acid sequence of the pilin encoded by the A . veronii biovar sobria tapA differed markedly from the corresponding sequence of its Bfp pilin, and those of the Bfp purified from other Aeromonas strains and species . Probing with tapA and tapD genes showed that these Bfp-positive Aeromonas strains also possessed the Tap gene cluster . TapA proteins of A . veronii biovar sobria and A . hydrophila shared 53% identity and 63% homology . We conclude that Aeromonas species are potentially able to express at least two distinct families of type IV pili (Bfp and Tap) . Shock, 1997 Oct, 8(4), 276 - 83 Differences in eicosanoid and cytokine production between injury/hemorrhage and bacteremic shock in the pig; Foex BA et al.; Plasma concentrations of the eicosanoids leukotriene (LT)B4, LTC4D4E4, thromboxane (TX)A2 and prostaglandin (PG)I2, and tumor necrosis factor (TNF) were measured during acute bacteremic shock and injury/hemorrhage in two porcine models . As TXA2 and PGI2 are rapidly metabolized, we measured their stable metabolites TXB2 and 6-keto-PGF1 alpha . Bacteremic shock was induced by a graded infusion of Aeromonas hydrophila over 4 h . Injury/hemorrhage was produced by a 30 min, 30% total blood volume hemorrhage followed by a 30 min shock period and then reinfusion of shed blood . Nociceptive afferent nerve stimulation was applied to the brachial plexi to mimic the cardiovascular responses to tissue injury . There was no increase in eicosanoid or TNF levels in the injury/hemorrhage model . In sepsis there was an early peak in TNF (at 60 min) followed by peaks in LTB4 and LTC4D4E4 at 180 min . Both TXB2 and 6-keto-PGF1 alpha showed large increases at the end of the study but there was no evidence that they had reached a peak . These results suggest that the very early inflammatory response in bacteremic shock and injury/hemorrhagic shock may be quite different . This may have implications for any therapies aimed at reducing the incidence of multiple organ failure after either of these physiological insults. Int J Syst Bacteriol, 1997 Oct, 47(4), 1157 - 64 Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended descriptions of the species Aeromonas eucrenophila and A . encheleia; Huys G et al.; The recently reported chemotaxonomic and genotypic description of two well-separated subgroups (I and II) in Aeromonas eucrenophila and their affiliation to Aeromonas encheleia and the unnamed Aeromonas DNA hybridization group (HG) 11 (G . Huys, M . Altwegg, M.-L . Hanninen, M . Vancanneyt, L . Vauterin, R . Coopman, U . Torck, J . Luthy-Hottenstein, P . Janssen, and K . Kersters, Syst . Appl . Microbiol . 19:616-623, 1996) has questioned the original species descriptions of A . eucrenophila and A . encheleia . In order to elucidate the unclear taxonomic status of these taxa in the genus Aeromonas, we have further investigated a collection of 14 reference strains and 14 related isolates encompassing the taxa A . eucrenophila subgroups I and II, A . encheleia, and HG11 by DNA-DNA hybridization (on 17 of the 28 strains) and phenotypic characterization (on all 28 strains) . Genotypically, the investigated strains could be grouped into two DNA hybridization groups that exhibited between-group homologies ranging from 42 to 52% . The members of DNA homology group I (DNA binding, 76 to 100%) were strains of A . eucrenophila subgroup I, including the type strain LMG 3774, and two A . eucrenophila-like isolates, leading to the conclusion that these strains should be considered true representatives of the species A . eucrenophila . The strains of A . eucrenophila subgroup II, HG11, and A . encheleia, on the other hand, were closely joined in DNA homology group II (DNA binding, 74 to 105%) together with two presumptive A . encheleia isolates . The fact that strain LMG 16330T of A . encheleia was the only type strain residing in DNA homology group II implies that HG11 and A . eucrenophila subgroup II should be classified in the species A . encheleia . Except for the somewhat aberrant phenotypic positions of HG11 strains LMG 13075 and LMG 13076, the establishment of DNA homology groups I and II was supported by the delineation of phena 1 and 2 (level of correlation, 90%), respectively, as revealed by numerical analysis of 136 phenotypic test results . These data indicate that A . eucrenophila and A . encheleia are phenotypically highly related but can be easily separated by testing the production of acid from D-cellobiose and lactose and the assimilation of D-cellobiose . Extended descriptions of both species are given. Infect Immun, 1997 Oct, 65(10), 4299 - 308 Hyperproduction, purification, and mechanism of action of the cytotoxic enterotoxin produced by Aeromonas hydrophila; Ferguson MR et al.; A gene encoding the cytotoxic enterotoxin (Act) from Aeromonas hydrophila was hyperexpressed with the pET, pTRX, and pGEX vector systems . Maximum toxin yield was obtained with the pTRX vector . Approximately 40 to 60% of Act was in a soluble form with the pTRX and pET vector systems . The toxin protein was purified to homogeneity by a combination of ammonium sulfate precipitation and fast protein liquid chromatography-based column chromatographies, including hydrophobic, anion-exchange, sizing, and hydroxylapatite chromatographies . Purified mature toxin migrated as a 52-kDa polypeptide on a sodium dodecyl sulfate (SDS)polyacrylamide gel that reacted with Act-specific antibodies in immunoblots . The minimal amount of toxin needed to cause fluid secretion in rat ileal loops was 200 ng, and the 50% lethal dose for mice was 27.5 ng when injected intravenously . Binding of the toxin to erythrocytes was temperature dependent, with no binding occurring at 4 degrees C . However, at 37 degrees C the toxin bound to erythrocytes within 1 to 2 min . It was determined that the mechanism of action of the toxin involved the formation of pores in erythrocyte membranes, and the diameter of the pores was estimated to be 1.14 to 2.8 nm, as determined by the use of saccharides of different sizes and by electron microscopy . Calcium chloride prevented lysis of erythrocytes by the toxin; however, it did not affect the binding and pore-forming capabilities of the toxin . A dose-dependent reduction in hemoglobin release from erythrocytes was observed when Act was preincubated with cholesterol, but not with myristylated cholesterol . With 14C-labeled cholesterol and gel filtration, the binding of cholesterol to Act was demonstrated . None of the other phospholipids and glycolipids tested reduced the hemolytic activity of Act . The toxin also appeared to undergo aggregation when preincubated with cholesterol, as determined by SDS-polyacrylamide gel electorphoresis . As a result of this aggregation, Act's capacity to form pores in the erythrocyte membrane was inhibited. Indian J Exp Biol, 1997 Feb, 35(2), 144 - 7 Enterotoxicity, haemolytic activity and antibiotic susceptibility of Aeromonas eucrenophila strains isolated from water and infected fish; Singh DV et al.; Strains of A . eucrenophila isolated from fresh water (2 strains) and infected fish (4 strains) were tested for haemolytic activity and enterotoxicity and any correlation between them . Also, the resistance patterns of A . eucrenophila were tested especially in relation to ampicillin . None of the A . eucrenophila strains caused fluid accumulation in the initial tests, however, they did so only after one to four sequential passages through the gut of a susceptible host . All the strains of A . eucrenophila showed beta-haemolytic activities . Production of beta-haemolysin could be correlated with enterotoxicity . Since all the strains of A . eucrenophila were resistant to ampicillin, media containing this antibiotic may be used for their isolation from diverse sources. EMBO J, 1997 Aug 1, 16(15), 4760 - 9 Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase; Schmitt E et al.; Peptidyl-tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl-tRNAs produced through abortion of translation . This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues . The structure of crystalline peptidyl-tRNA hydrolase could be solved at 1.2 A resolution . It indicates a single alpha/beta globular domain built around a twisted mixed beta-sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica . This similarity allowed the characterization by site-directed mutagenesis of several residues of the active site of peptidyl-tRNA hydrolase . These residues, strictly conserved among the known peptidyl-tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C-end of a neighbouring peptidyl-tRNA hydrolase molecule . Hence, several main chain atoms of three residues belonging to one peptidyl-tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl-tRNA hydrolase molecule . Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction. J Antimicrob Chemother, 1997 Aug, 40(2), 171 - 8 Distribution and expression of beta-lactamase genes among Aeromonas spp; Walsh TR et al.; Clinical and environmental isolates of Aeromonas spp . (20 Aeromonas caviae, 33 Aeromonas veronii and 21 Aeromonas hydrophila) were examined for their ability to yield mutants derepressed for beta-lactamase production and for the distribution of the three chromosomally encoded beta-lactamase genes, ampS and cepS from A . veronii bv . sobria and cphA from A . hydrophila . Of these isolates, 100% and 96% of the isolates from A . hydrophila and A . caviae, respectively, yielded beta-lactamase derepressed mutants but only 38% of A . veronii isolates yielded the resistant phenotype at 37 degrees C . However, when tested at 30 degrees C, all isolates gave rise to the derepressed mutants, indicating a temperature effect on the control mechanism . All mutants had significantly higher beta-lactamase activity against ampicillin, oxacillin, cephaloridine and imipenem . Hybridization studies with cloned aeromonas beta-lactamase genes indicated that the cephalosporinase gene, cepS, is almost ubiquitous for the three species tested . The cphA gene cross-hybridized with all isolates of A . veronii and A . hydrophila but not to A . caviae isolates . In contrast, hybridization studies using ampS revealed that only 25% of A . caviae, 45% of A . veronii and 38% of A . hydrophila tested carried the ampS gene or one closely homologous to it . Nonetheless, strains that failed to hybridize with ampS showed two serine beta-lactamases when analysed by isoelectric focusing. FEMS Microbiol Lett, 1997 Sep 1, 154(1), 1 - 7 The synthesis, secretion and role in virulence of the paracrystalline surface protein layers of Aeromonas salmonicida and A . hydrophila; Noonan B et al.; The S-layers of the Aeromonas spp . studied to date are composed of identical protein subunits which are translocated across the cytoplasmic membrane, periplasm and outer membrane to the cell surface, where they are assembled and tethered to the cell via an interaction with the O-polysaccharide side chains of the lipopolysaccharide . Aeromonas S-layers have the ability to bind a number of host factors such as fibronectin, laminin and vitronectin as well as providing resistance to serum killing and protease digestion . Aeromonas mutants unable to produce an S-layer are altered in their ability to cause disease . In the case of Aeromonas salmonicida, the loss of ability to produce an S-layer effectively abolishes virulence . However, in the case of A . hydrophila, the reduction in virulence caused by the loss of the S-layer is less significant. J Bacteriol, 1997 Sep, 179(17), 5271 - 81 Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules; Swift S et al.; Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs) . The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively . Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs . ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis . Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing . The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-L-homoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry . In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified . Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription . For A . salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A650 of 0.9 in the control to an A650 of 1.2 in the test) of protease in the culture supernatant . These data add A . hydrophila and A . salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing. Biochemistry, 1997 Aug 12, 36(32), 9837 - 46 Spectroscopically distinct cobalt(II) sites in heterodimetallic forms of the aminopeptidase from Aeromonas proteolytica: characterization of substrate binding; Bennett B et al.; The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy . EPR spectra of the high-spin S = 3/2 Co(II) ions in {CoZn(AAP)} and {ZnCo(AAP)} indicated that each metal binding site provides a spectroscopically distinct signature . For {CoZn(AAP)}, subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent . The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.29 and an E/D of 0.1 . The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT . These parameters correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion . Summation of these two species provided an excellent simulation of the observed {CoZn(AAP)} EPR spectrum . The EPR spectrum of {ZnCo(AAP)} also contained two species, at least one of which also exhibited 59Co hyperfine features . However, this signal exhibited little pH dependence, and individual species could not be isolated . The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to {CoZn(AAP)} resulted in a distinct change in the EPR spectrum; however, addition of BuBA to {ZnCo(AAP)} left the EPR spectrum completely unperturbed . These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site . On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested {Baker, J . O., & Prescott, J . M . (1983) Biochemistry 22, 5322-5331} . From difference spectroscopy and from the simulation of the {CoZn(AAP)} EPR spectrum, a third signal appearing upon BuBA binding was isolated . This signal was simulated with geff values of 2.08, 3 . 15, and 6.15 which correspond to an Ms = |+/-1/2> ground state transition with a greal of 2.41 and an E/D of 0.22 . This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT . Summation of the these three species provided an excellent simulation of the observed {CoZn(AAP)} + BuBA EPR spectrum at both pH values . This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP {Bennett, B., & Holz, R . C . (1997) J . Am . Chem . Soc . 119, 1923-1933; Chen, G., et al . (1997) Biochemistry 36, 4278-4286}. Anal Biochem, 1997 Jul 15, 250(1), 29 - 34 Continuous spectrophotometric assay of peptide deformylase; Wei Y et al.; A continuous assay for peptide deformylase has been developed using a formylated dipeptide, formyl-Met-Leu-p-nitroanilide, as substrate . Removal of the formyl group by a peptide deformylase renders the dipeptide product, which contains a free NH2 terminus, a substrate for an aminopeptidase from Aeromonas proteolytica . Sequential hydrolysis of the dipeptide by the aminopeptidase releases a p-nitroaniline, which is monitored spectrophotometrically at 405 nm . This assay was applied to determine the pH optimum and the catalytic activity of a peptide deformylase from Escherichia coli . The E . coli enzyme is most active near neutral pH (pH 7.0) and displays Michaelis-Menten kinetics toward the formylated dipeptide, with K(M) = 20.3 +/- 1.3 microM, k(cat) = 38 +/- 2 s(-1), and k(cat)/K(M) = 1.9 x 10(6) M(-1) s(-1) . It also exhibits an acylase activity, capable of deacylating N-acetyl-Met-Leu-p-nitroanilide and N-trifluoroacetyl-Met-Leu-p-nitroanilide, albeit at drastically reduced rates . These results demonstrate that the current assay is a convenient, rapid, and sensitive method for kinetic studies of peptide deformylase . The strategy employed in this work should also be generally applicable to the characterization of other acylases. Biosci Biotechnol Biochem, 1997 Jul, 61(7), 1102 - 8 Purification and properties of an aminopeptidase from a protamine-degrading marine bacterium; Obata H et al.; A protamine-degrading marine bacterium was isolated from marine soil and identified as Aeromonas salmonicida subsp . based on its taxonomical characteristics . An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized . The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12 . The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS-PAGE . The N-terminal sequence of the enzyme was H.Gly-Gln-Gln-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Ty r- Ile-Thr- . It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin . The Michaelis constant (K(m)) and the maximal rate of hydrolysis (Vmax) were, respectively, 0.28 mM and 49.4 mumol/min/mg for the L-Ala-beta-naphthylamide substrate . The optimum pH and optimum temperature were 6.5 and 45 degrees C, respectively . The purified enzyme was highly specific to L-Ala-beta-naphthylamide. Ann Clin Lab Sci, 1997 Jul-Aug, 27(4), 254 - 9 Pathogenic analysis of Aeromonas hydrophila septicemia; Chang CY et al.; Aeromonas hydrophila has emerged as a potential pathogen in the immunocompromised host . Various aeromonal infections, including septicemia, have also been reported in apparently healthy individuals . For years, researchers have disagreed over the epidemiologic roles of aeromonads in gastroenteritis . Isolation rates of aeromonads by stool culture among patients with gastroenteritis are not consistently high . Carriers of this bacterium also exist . The septicemic course is, however, often fulminant and fatal, and may lack an obvious focus . Pathogenic mechanisms are complex and largely unresolved . The objective of this study is to report the necropsy findings from a uremic patient who presented with typical aeromonal septicemia of obscure origin asking if such investigation could give insight into some of the questions mentioned previously . Western blot immunostaining for aerolysin (beta-hemolysin of aeromonads) was used to evaluate whether or not such a virulence factor is involved in the process of septic dissemination . The autopsy showed that the skin and liver contained microabscesses . The upper gastrointestinal mucosae and spleen contain patchy putrefactive lesions with adjacent focal hemorrhage . Perimortem blood cultures grew Aeromonas hydrophila . A conventional Western blot analysis of the culture supernatant failed to show aerolysin . A control Aeromonas sobia American Type Culture Collection (ATCC) strain produces readily detectable aerolysin . It is concluded that this isolate may be aerolysin-deficient or one secreting low levels of aerolysin; these would require more sensitive methods of detection . The primary focus of infection might be the upper gastrointestinal tract . Other virulence factors including the bacterial proteases and/or phospholipases might be responsible for the pathogenesis of septic dissemination. J Clin Microbiol, 1997 Jul, 35(7), 1671 - 4 Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes; Borrell N et al.; Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities . Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas . The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin . Most results from the two approaches were in agreement, but some discrepancies were discerned . Advantages over previous phenotypic and genetic methods are discussed. FEMS Microbiol Lett, 1997 Jun 15, 151(2), 245 - 8 Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39; Kataoka M et al.; L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39 . The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1) . The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction . The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5). FEMS Microbiol Lett, 1997 Jun 15, 151(2), 213 - 7 The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains; Merino S et al.; We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A . hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines . We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines . We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A . hydrophila serogroup O:34 bacterial invasion of both fish cell lines. Zentralbl Veterinarmed B, 1997 Jun, 44(4), 245 - 52 Molecular and phenotypic features of aeromonads isolated from snails (Helix aspersa) affected with a new summer disease; Kodjo A et al.; Forty-four aeromonads were isolated during the summer of 1994 from snails (Helix aspersa) affected or not by a newly described disease . Snta 1 rRNA gene restriction patterns (ribotyping) and nine selected conventional phenotypic tests were performed to retrospectively determine relationships among these isolates regarding the disease . Results from ribotyping were found highly discriminant in strain differentiation and suggest the possible existence of a group of Aeromonas associated with the disease . Biochemical and molecular characterizations indicated that most of these disease-associated aeromonads could belong to the phenospecies A . hydrophila . Phenospecies of aeromonads from healthy snails were distinct from those isolated from diseased snails and could be identified as A . caviae or remained undetermined . To the authors' knowledge, this is the first report of a probable pathogenic aeromonad found in snails, extending the host-pathogen spectrum of Aeromonas. Zentralbl Veterinarmed B, 1997 Jun, 44(4), 221 - 33 Computer aided assignment of motile Aeromonas strains to genospecies level by standard biochemical tests; Brunner B et al.; A procedure is given for differentiation of motile Aeromonas spp . Based on nine to 12 biochemical properties it allows an assignment of isolated Aeromonas strains first to phenotypes and, in the second step, to hybridization groups (HGs) 1-13 as well as to the genotypically defined species Aer . allosaccharophila and Aer . encheleia . The computer aided classification is carried out according to the principles of numerical taxonomy . Using this differentiation scheme 23 Aeromonas strains, which were isolated from food, and two reference strains were speciated . A total of 16 strains were assigned to Aer . hydrophila, six strains to Aer . caviae and one strain to Aer . sobria . Of the 16 Aer . hydrophile strains, 11 were attached to HG 1, one to HG 2 and four could not be classified clearly as HG 3 or HG 1 . All six Aer . caviae isolated were assigned to HG 5 and the Aer . sobria strain was ranked among HG 8 . The reference strains were assigned to the same genotypes by this method than by DNA-DNA hybridization assays. Lett Appl Microbiol, 1997 Jun, 24(6), 479 - 82 Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica); Son R et al.; Strains of Aeromonas hydrophila isolates from skin lesions of the common freshwater fish, Telapia mossambica, were screened for the presence of plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 10 antimicrobial agents . Of the 21 fish isolates examined, all were resistant to ampicillin and sensitive to gentamycin . Most isolates were resistant to streptomycin (57%), tetracycline (48%) and erythromycin (43%) . While seven of 21 isolates harboured plasmids, with sizes ranging from 3 to 63.4 kilobase pair (kb), it was only possible to associate the presence of a plasmid with antibiotic resistance (ampicillin and tetracycline) in strain AH11 . Both the plasmid and the associated antimicrobial resistance could be transferred to an Escherichia coli recipient by single-step conjugation at a frequency of 4.3 x 10(-3) transconjugants per donor cell. J Bacteriol, 1997 Jun, 179(11), 3555 - 60 L-allo-threonine aldolase from Aeromonas jandaei DK-39: gene cloning, nucleotide sequencing, and identification of the pyridoxal 5'-phosphate-binding lysine residue by site-directed mutagenesis; Liu JQ et al.; We have isolated the gene encoding L-allo-threonine aldolase (L-allo-TA) from Aeromonas jandaei DK-39, a pyridoxal 5'-phosphate (PLP)-dependent enzyme that stereospecifically catalyzes the interconversion of L-allo-threonine and glycine . The gene contains an open reading frame consisting of 1,014 nucleotides corresponding to 338 amino acid residues . The protein molecular weight was estimated to be 36,294, which is in good agreement with the subunit molecular weight of the enzyme determined by polyacrylamide gel electrophoresis . The enzyme was overexpressed in recombinant Escherichia coli cells and purified to homogeneity by one hydrophobic column chromatography step . The predicted amino acid sequence showed no significant similarity to those of the currently known PLP-dependent enzymes but displayed 40 and 41% identity with those of the hypothetical GLY1 protein of Saccharomyces cerevisiae and the GLY1-like protein of Caenorhabditis elegans, respectively . Accordingly, L-allo-TA might represent a new type of PLP-dependent enzyme . To determine the PLP-binding site of the enzyme, all of the three conserved lysine residues of L-allo-TA were replaced by alanine by site-directed mutagenesis . The purified mutant enzymes, K51A and K224A, showed properties similar to those of the wild type, while the mutant enzyme K199A was catalytically inactive, with corresponding disappearance of the absorption maximum at 420 nm . Thus, Lys199 of L-allo-TA probably functions as an essential catalytic residue forming an internal Schiff base with PLP of the enzyme to catalyze the reversible aldol reaction. J Bacteriol, 1997 Jun, 179(11), 3397 - 403 Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA; Wong CY et al.; Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C . Y . F . Wong, G . Mayrhofer, M . W . Heuzenroeder, H . M . Atkinson, D . M . Quinn, and R . L . P . Flower, FEMS Immunol . Med . Microbiol . 15:233-241, 1996) to protect mice against lethal infection by this organism . In this study, colony blot analysis of an A . hydrophila genomic library using antiserum to A . hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads . The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins . ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli . ORF1 was subsequently designated ilpA (immunophilin-like protein) . ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa . The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E . coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila . ORF3 was designated fkpA (FK506 binding protein) by analogy with the E . coli FkpA protein . Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA . hydrophila 30-kDa membrane protein . PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested . A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase . This suggests that either the fkpA gene is not essential in the virulence of A . hydrophila under these conditions or there are other genes in A . hydrophila coding for proteins with similar functions. J Biol Chem, 1997 May 2, 272(18), 12170 - 4 The glycosylphosphatidylinositol-anchored surface glycoprotein Thy-1 is a receptor for the channel-forming toxin aerolysin; Nelson KL et al.; Aerolysin is a channel-forming protein secreted by virulent Aeromonas spp . Some eucaryotic cells, including T-lymphocytes, are sensitive to very low concentrations of the toxin (<10(-9) M) . Here we show that aerolysin binds selectively and with high affinity to the glycosylphosphatidylinositol (GPI)-anchored surface protein Thy-1, which is found on T-lymphocyte populations as well as in brain . Less than 1 ng of purified Thy-1 could be detected by probing Western blots with the toxin . Mutant T-cell lines that lack the ability to add GPI anchors to Thy-1 and other surface proteins were much less sensitive to aerolysin, as were wild-type cells that were pretreated with phosphatidylinositol-specific phospholipase C to remove GPI-anchored proteins . Phosphatidylcholine/cholesterol liposomes containing purified Thy-1 in their membranes were much more sensitive to aerolysin than protein-free liposomes. Vet Microbiol, 1997 May, 56(1-2), 147 - 58 Molecular and phenotypic methods for the characterization of atypical Aeromonas salmonicida; Hanninen ML et al.; Atypical Aeromonas salmonicida form a taxonomically diverse group among the psychrophilic A . salmonicida . Characteristics of 53 atypical A . salmonicida strains originating from Finland, Denmark, Norway and Sweden were studied using 60 phenotypic tests . Ribopattern analysis and plasmid profiles were used as genetic methods . The production of brown pigment on the furunculosis agar containing L-tyrosine divided the atypical oxidase-positive strains into two groups: pigment-producing (n = 35) and achromogenic (n = 16) . PstI differentiated all the atypical A . salmonicida from A . salmonicida subsp . salmonicida . Combined ribopatterns ClaI/PstI divided pigment producing atypical strains into four major groups B/B, G/G, G/T, F/F . Most of the achromogenic oxidase-positive strains belonged to two major groups H/H or U/U . Cluster analysis of ribopatterns and plasmid profile analysis also supported the division of atypical oxidase-positive A . salmonicida into pigment-producing and achromogenic groups . The oxidase-negative strains formed a distinct group which differed from oxidase-positive atypical A . salmonicida type biochemically and in terms of ribopatterns . Our results also support the use of ribopattern analysis as a valid method to study the epidemiology of infections caused by atypical A . salmonicida on fish farms. J Comp Physiol {B}, 1997 May, 167(4), 256 - 63 Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold; Maniero GD et al.; The effect of exposure to low temperatures (5 degrees C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens . Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 degrees C compared to that of animals kept at 22 degrees C . A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 degrees C for the same length of time . Mean serum complement activity after 1 month at 5 degrees C was significantly reduced in comparison to animals held at 22 degrees C and was not detectable after 5 months in the cold . Recovery of complement levels at room temperature (22 degrees C) was also examined after cold exposure . Complement levels were significantly higher than controls (at 22 degrees C) in frogs returned to 22 degrees C for 7 and 14 days after 5 months in the cold . After frogs were held at 5 degrees C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 degrees C and continued to rise 5 and 9 days after warming . Injections with Aeromonas hydrophila following a 5-week exposure to 5 degrees C failed to cause death or observable symptoms of disease in frogs that were returned to 22 degrees C. Microb Pathog, 1997 May, 22(5), 315 - 20 Complement resistance of capsulated strains of Aeromonas salmonicida; Merino S et al.; The complement resistance of Aeromonas salmonicida strains grown under conditions promoting capsule formation was investigated using well characterized strains and their isogenic mutants . Complement resistance was previously studied using the same strains growing under non-capsulating conditions . The serum resistant strains were found to activate complement, but rapidly degrade C3b preventing productive formation of the lytic complex C5b-9 . Isogenic lipopolysaccharide rough mutants grown under non-capsulating conditions were serum sensitive, binding a large amount of C3b and leading to productive formation of C5b-9 . When grown under conditions promoting capsule formation, these mutants were partially resistant to complement because less C3b is bound to them and also partially degraded, with a concomitant reduction in lytic C5b-9. J Biotechnol, 1997 Apr 25, 54(2), 113 - 20 Purification and characterization of lipase from Aeromonas sobria LP004; Lotrakul P et al.; Lipase from Aeromonas sobria LP004, isolated from raw milk, was purified and characterized . The lipase was purified 10.29 fold to a homogeneous state by ultrafiltration and column chromatography on phenyl sepharose . The molecular weight of the lipase determined by SDS-PAGE was 97 kDa . Purified A . sobria LP004 lipase exhibited the maximum activity at pH 6.0 and 45 degrees C and was stable under alkaline conditions (pH 6.5-10.0)