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Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of South India.

A total of 319 strains of Aeromonas hydrophila were isolated from 536 fish and 278 prawns for a 2-year period. All the strains were tested for resistance to 15 antibiotics and 100% of the strains was resistant to methicillin and rifampicin followed by bacitracin and novobiocin (99%). Only 3% of the strains exhibited resistance against chloramphenicol. The multiple antibiotic resistance (MAR) indexing of A. hydrophila strains showed that all of them originated from high-risk sources.

Aeromonas hydrophila↗

Tolerance and starvation induced cross-protection against different stresses in Aeromonas hydrophila.

Aeromonas hydrophila is sometimes considered as a controversial human pathogen and reported to be susceptible to food processing procedures and environmental stresses. In this study, we have shown that early stationary phase cells of A. hydrophila were readily killed during up shifts in temperature (in the range 50-70 degrees C), the course of drying (at relative humidity, temperature and brightness of the laboratory) and after 5 min exposure to 20%, 30% and 40% v/v ethanol. However, this bacterium was found moderately susceptible to down shift to 4 degrees C in nutrient poor water, sodium chloride stresses (1.5 and 2 M) and to 12% and 15% v/v ethanol stresses. Tolerance against 1 M NaCl and 10% v/v ethanol was observed. At ambient temperature (24.5 degrees C), this microorganism exhibited a starvation survival state, which was largely independent of the initial cell concentrations (8.82, 7.71 and 6.76 log units). The cross-protection experiments showed that cells starved for short (1 day) or prolonged (50 days) periods developed increased resistance to down shift at 4 degrees C and ethanol stress. This may be of concern to the food-processing industry from the public health perspective.

Adaptation, Physiological↗

The aerobic growth of Aeromonas hydrophila and Listeria monocytogenes in broths and on pork.

Flasks of tryptic soy broth (TSB), unacidified (pH 7.2) or acidified with HCl or lactic acid to pH 6.3 or 5.5, and samples of sterile pork fat or muscle tissue, were inoculated with logarithmic phase cultures of a strain of Aeromonas hydrophila or a strain of Listeria monocytogenes. The broth cultures were incubated at temperatures between 0 and 25 degrees C, and growth rates were determined from optical density increases. The tissue samples were incubated at temperatures between -2.4 and 25.2 degrees C, and growth rates were determined from viable count increases. Both organisms grew without lag in all broths at temperatures greater than 10 degrees C. A hydrophila did not grow at 5 degrees C in TSB acidified with lactic acid to pH 5.5, and grew in other broths at that temperature after a lag of about 10 h. The organism did not grow in either broth of pH 5.5 at 2 degrees C, but grew in other broths at that temperature after a lag of about 40 h. A hydrophila did not grow in any broth at 0 degrees C. L. monocytogenes grew in all broths at 5 degrees C only after a lag of about 60 h, and did not grow in any broth at 2 degrees C. For both organisms, the rates of growth, at any temperature, were lower in broths of pH 6.3, and lower again in broths of pH 5.5, than in the unacidified broth. Growth rates in the broths of pH 6.3 were similar, but growth rates were lower in lactic acid acidified broth of pH 5.5 than in HCl acidified broth of that pH. The data for the growth of each organism in each medium were well described by the regression line of the plot of the square roots of growth rates against temperature. A hydrophila grew on fat tissue of pH 6.3 +/- 0.3, without lag, at 1.8 degrees C and higher temperatures at rates greater than the rates of growth in unacidified TSB. Numbers of the organism declined on muscle tissue of pH 5.6 +/0 0.2 at any temperature. L. monocytogenes grew on fat tissue without lag at -0.3 degrees C and higher temperatures, at rates which at lower temperatures were greater than the rates of growth in unacidified TSB. The organism grew on muscle tissue only at temperatures greater than or equal to 15.4 degrees C, at rates which were less than the rates of growth in lactic acid acidified broth of pH 5.5. Models derived from the cultivation of A. hydrophila and L. monocytogenes in commercial broths appear to be highly unreliable guides to the behaviours of those organisms on pork.

Aerobiosis↗

Purification and characterization of lipase from Aeromonas sobria LP004.

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) and at temperatures lower than 40 degrees C. This lipase could be classified as a 1,3-position specific enzyme and its catalytic activity was calcium dependent. PMSF, a serine enzyme inhibitor and 2-mercaptoethanol, a reducing agent, did not affect the enzyme activity.

Aeromonas↗

Aeromonas hydrophila in chlorinated water supplies.

Methods for the isolation and enumeration of Aeromonas hydrophila in water supplies are described. Examination of 286 chlorinated samples collected between July and October showed that 19% of otherwise uncontaminated waters contained Aerom. hydrophila, rising to 71% of those from which Escherichia coli was isolated. The isolation rate during January and February was only 7% for all samples. On the basis of these results, tap water is a likely source of the aeromonads found in human intestinal flora.

Aeromonas↗

Aeromonas hydrophila infection of a puncture wound.

Aeromonas hydrophila is a gram-negative organism generally considered a pathogen of low virulence, rarely reported as causing infection in man. Recently it has been recognized as causing infection in wounds. This article reports the case of a patient who suffered a puncture wound while standing in a fresh water lake. He subsequently developed a rapidly progressing cellulitis requiring hospitalization. This organism was cultured from the wound and responded dramatically to appropriate antimicrobial therapy.

Adult↗

Fatal Aeromonas hydrophila bacteremia in a hemodialysis patient treated with deferoxamine.

A 49-year-old woman undergoing long-term hemodialysis and treated with deferoxamine (DFO) 1.5 g twice weekly for aluminum bone disease developed fever and bilateral calf pain caused by myonecrosis with gas gangrene. She had a rapidly fatal outcome. The cultures of blood and aspirates from both calf muscles demonstrated Aeromonas hydrophila. No obvious entry point could be traced. The in vitro growth of the patient's strain was found to be stimulated by the deferoxamine-iron complex in an iron-deprived medium. It is suggested that high-dose DFO therapy in this patient was responsible for promoting a bacterial infection by this microorganism.

Aeromonas hydrophila↗

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

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

Acute-Phase Proteins↗

Aeromonas hydrophila in burn patients.

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.

Adult↗

Hand and forearm infections caused by Aeromonas hydrophila.

This is a report of two cases of hand and forearm infections caused by Aeromonas hydrophila. This organism is resistant to penicillin and most cephalosporins and is found in most freshwater environments. It is recommended that broad-spectrum coverage such as a combination of cephalosporin and aminoglycoside antibiotic be used.

Adolescent↗

Sequence analysis and biochemical characterisation of chromosomal CAV-1 (Aeromonas caviae), the parental cephalosporinase of plasmid-mediated AmpC 'FOX' cluster.

Aeromonas caviae CIP 74.32 was resistant to amoxicillin, ticarcillin and cephalothin, and susceptible to cefoxitin, cefotaxime, ceftazidime, aztreonam and imipenem. This strain produced a cephalosporinase (pI 7.2) and an oxacillinase (pI 8.5). The cephalosporinase gene cav-1 was cloned and sequenced. Unlike A. caviae donor, Escherichia coli pNCE50 transformant producing CAV-1 beta-lactamase was resistant to cefoxitin. The deduced protein sequence CAV-1 contained 382 amino acids, and shared >96% homology with FOX-1 to FOX-5 cephalosporinase. CAV-1 presented only two amino acid substitutions (Thr270Ser and Arg271Ala) with FOX-1. CAV-1 is the chromosomal putative ancestor of the FOX family, a cluster of class C/group 1 plasmidic cephalosporinases spreading in Klebsiella and E. coli clinical isolates via conjugative plasmids.

Aeromonas↗

A novel electrochemically active and Fe(III)-reducing bacterium phylogenetically related to Aeromonas hydrophila, isolated from a microbial fuel cell.

A facultative anaerobic bacterium was isolated from a mediator-less microbial fuel cell fed with artificial wastewater containing acetate and designated as PA3. The isolate was identified as a strain of Aeromonas hydrophila based on its biochemical, physiological and morphological characteristics as well as 16S rDNA sequence analysis and DNA-DNA hybridization. PA3 used glucose, glycerol, pyruvate and hydrogen to reduce Fe(III), nitrate and sulfate. Cyclic voltammetry showed that PA3 was electrochemically active and was the culture collection strain A. hydrophila KCTC 2358. Electricity was generated from a fuel cell-type reactor, the anode compartment of which was inoculated with cell suspensions of the isolate or A. hydrophila KCTC 2358. The electrochemical activities are novel characteristics of A. hydrophila.

Aeromonas hydrophila↗

Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101.

Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII). Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII). In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.

Adsorption↗

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila.

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.

Aeromonas↗

Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis.

Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium. The results indicated that three isolates could degrade chitin at high pH. Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No. 4.1 could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki (Bt-k) toward diamondback moth larvae. Thus, it might be useful to increase the toxicity of B. thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into Bt. To investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5 alpha using 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate. The four plasmids were then introduced into B. thuringiensis subsp. israelensis (Bt-i) strain c4Q272 by electroporation. Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.

Aeromonas hydrophila↗

Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila.

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.

Aeromonas hydrophila↗

Molecular and phenotypic methods for the characterization of atypical Aeromonas salmonicida.

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.

Aeromonas↗

Characterization of atypical Aeromonas salmonicida by different methods.

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.

Aeromonas↗