Electroporation-mediated transformation of Aeromonas hydrophila

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4x10(2) CFU/microg DNA at 12.5 kV/cm and 1000 Omega, resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the first report of transformation of bacteria A. hydrophila.     

Cell-surface properties of the food- and water-borne pathogen Aeromonas hydrophila when stored in buffered saline solutions

Aeromonas hydrophila, a ubiquitous inhabitant of aquatic environments, commonly expresses several cell-surface properties that may contribute to virulence. Since many aquatic microorganisms in hostile environments can withstand starvation conditions for long periods, we examined the effect of storage under nutrient-poor conditions on the expression of cell-surface properties of this pathogen. Phenotypes studied were: (1) cell-surface hydrophobicity and charge, and (2) the ability to bind connective-tissue proteins and lactoferrin. Our results suggest that the response of A. hydrophila to nutrient-poor conditions is regimen specific. Generally, A. hydrophila cells became more hydrophobic and significantly increased their ability to bind the iron-binding glycoprotein lactoferrin when the bacterium was stored under nutrient-poor conditions; however, under these conditions, the cells seemed to lose their ability to bind connectivetissue proteins.     

Transport of glycine-betaine by Listeria monocytogenes

Uptake of [¹⁴C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4–0.5 M. The glycine-betaine transport system had a K _m of 22 M and a V _max of 11.7 nmol^-1 min^-1 mg^-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the V _max increased to 27.0 nmol^-1 min^-1 mg^-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K⁺ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.     

Hypochlorite inactivation kinetics of Listeria monocytogenes in phosphate buffer

The inactivation kinetics of Listeria monocytogenes in a phosphate buffer (PB) was determined at different hypochlorite concentrations, pH values and temperatures. D-values, using a linear regression, of L. monocytogenes in PB (pH 6.5) were 23.54, 17.40, 14.24 and 12.00s at 5, 10, 50 and 100 mg l(-1) hypochlorite, respectively, at 30 degrees C. The k-values ranged from 0.098 to 0.192s(-1) and 0.007 to 0.018s(-1) for hypochlorite concentrations (from 5 to 100 mg l(-1)) in PB (pH 6.5) and PB containing 0.1% peptone (pH 6.5), respectively, at 30 degrees C. D-values of L. monocytogenes exposed to hypochlorite were decreased with decreasing pH of PB (pH from 8.5 to 4.5). Hypochlorite showed higher antimicrobial activity at higher temperature. Not only the effect of hypochlorite concentration on the inactivation of L. monocytogenes but also other parameters like temperature, pH and suspending solutions effect the inactivation rates.     

Optional production and utilization of polysaccharide by Aeromonas hydrophila

Glucans from the fish pathogen Aeromonas hydrophila have been extracted andpurified by a method utilizing phenol/water followed by sodium deoxycholate rather than the traditional sodium hydroxide extraction. Presence of substantial amounts of these glucans was shown to be dependant on whether or not the substrate contained dextrose, a point which had import because of the low carbohydrate environment in which this species must survive and multiply. These glucans, produced in the log phase, were utilized during the later growth period.The structures of the two purified glucans were examined by methylation analysis, periodate oxidation, and enzymatic degradation. The results indicated that A. hydrophila under low-carbohydrate growth conditions produced two similar but distinguishable 14 linked glucans substituted 16 by single monosaccharide residues or short chains to give an amylopectinglycogen type of polysaccharide.     

Growth of Aeromonas hydrophila on fresh vegetables stored under a controlled atmosphere

The effects of controlled-atmosphere storage (CAS) on the survival and growth of Aeromonas hydrophila on fresh asparagus, broccoli, and cauliflower were examined. Two lots of each vegetable were inoculated with A. hydrophila 1653 or K144. A third lot served as an uninoculated control. Following inoculation, vegetables were stored at 4 or 15 degrees C under a CAS system previously shown to extend the shelf life of each commodity or under ambient air. Populations of A. hydrophila were enumerated on the initial day of inoculation and at various intervals for 10 days (15 degrees C) or 21 days (4 degrees C) of storage. Direct plating of samples with selective media was used to enumerate A. hydrophila. The organism was detected on most lots of vegetables as they were received from a commercial produce supplier. Without exception, the CAS system lengthened the time vegetables were subjectively considered acceptable for consumption. However, CAS did not significantly affect populations of A. hydrophila which survived or grew on inoculated vegetables.     

Growth kinetics of Aeromonas hydrophila in freshwaters supplemented with various organic and inorganic nutrients

Freshwaters of varying natural nutrient enrichment were used as growth media for the culture of an autochthonous, heterotrophic, freshwater bacterium, Aeromonas hydrophila. The growth rate of the bacterium in eutrophic waters was increased to the greatest extent by adding carbon, as glucose; generation times decreased by up to 65%. Additions of carbon and phosphorus increased the maximal cell densities by over 25-fold. In oligotrophic waters, bacterial growth was most strongly promoted by the simultaneous additions of carbon (as glucose) and phosphorus (as KH₂PO₄). In these waters, stationary phase densities were increased as much as 100-fold, with a corresponding 70% increase in growth rate. These data provide at least a partial explanation for the previously observed correlation between A. hydrophila densities and the trophic states of freshwaters.The authors are with the Department of Microbiology, Morrill Hall, University of Rhode Island, Kingston, Rhode Island 02881, USA     

Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus--a carnivore model

Biofilm of Aeromonas hydrophila was evaluated for oral vaccination of walking catfish (Clarias batrachus L.). Fish were fed with fish paste incorporating biofilm (BF) or free cells (FC) of A. hydrophila for 20 days and monitored for serum antibody production up to 60 days post-vaccination. Serum agglutinating antibody titre and relative percent survival (RPS) following challenge were found to be significantly higher in catfish fed with BF vaccine compared to that with FC.     

Growth of Aeromonas hydrophila on fresh vegetables stored under a controlled atmosphere.

The effects of controlled-atmosphere storage (CAS) on the survival and growth of Aeromonas hydrophila on fresh asparagus, broccoli, and cauliflower were examined. Two lots of each vegetable were inoculated with A. hydrophila 1653 or K144. A third lot served as an uninoculated control. Following inoculation, vegetables were stored at 4 or 15 degrees C under a CAS system previously shown to extend the shelf life of each commodity or under ambient air. Populations of A. hydrophila were enumerated on the initial day of inoculation and at various intervals for 10 days (15 degrees C) or 21 days (4 degrees C) of storage. Direct plating of samples with selective media was used to enumerate A. hydrophila. The organism was detected on most lots of vegetables as they were received from a commercial produce supplier. Without exception, the CAS system lengthened the time vegetables were subjectively considered acceptable for consumption. However, CAS did not significantly affect populations of A. hydrophila which survived or grew on inoculated vegetables.     

Survival of Salmonella enteritidis and Listeria monocytogenes on salad vegetables

The influence of initial head-spaces of air – 4.9% CO₂/2.1% O₂/93% N₂ and 5% CO₂/5.2% O₂/89.8% N₂ – on Salmonella enteritidis and Listeria monocytogenes, and on microbial association with shredded carrots and lettuce was studied at 4 °C. Both these pathogens survived but did not grow in any vegetable regardless of the packaging system used. Total viable count, lactic acid bacteria and pseudomonads were also monitored. Lactic acid bacteria were the predominant organisms in all samples. The pH dropped significantly during the storage of vegetables.     

Structural studies on the R-type lipopolysaccharide of Aeromonas hydrophila

A rough strain of Aeromonas hydrophila, AH-901, has an R-type lipopolysaccharide with the complete core. The following core structure was established by chemical degradations followed by sugar and methylation analyses along with ESIMS and NMR spectroscopy: [formula: see text] where D-alpha-D-Hep and l-alpha-D-Hep stand for D-glycero- and l-glycero-alpha-D-manno-heptose, respectively; Kdo stands for 3-deoxy-D-manno-oct-2-ulosonic acid; all monosaccharides are in the pyranose form; the degree of substitution with beta-D-Gal is approximately 50%. Lipid A of the lipopolysaccharide has a 1,4(')-bisphosphorylated beta-D-GlcN-(1-->6)-alpha-D-GlcN disaccharide backbone with both phosphate groups substituted with 4-amino-4-deoxyarabinose residues.     

Development of a Listeria monocytogenes based vaccine against prostate cancer

Prostate specific antigen (PSA) is a likely immunotherapeutic target antigen for prostate cancer, the second leading cause of cancer-related death in American men. Previously, we demonstrated that attenuated strains of Listeria monocytogenes (Lm) can be used as effective vaccine vectors for delivery of tumor antigens causing regression of established tumors accompanied by strong immune responses toward these antigens in murine models of cancer. In the present study, we have developed and characterized a recombinant live attenuated L. monocytogenes/PSA (Lm–LLO–PSA) vaccine with potential use for the treatment of pCa. Human PSA gene was cloned into and expressed by an attenuated Lm strain. This recombinant bacterial vaccine, Lm–LLO–PSA was tested for stability, virulence, immunogenicity and anti-tumor effects in a murine model for pCa. Immunization with Lm–LLO–PSA was shown to lower the number of tumor infiltrating T regulatory cells and cause complete regression of over 80% of tumors formed by an implanted genetically modified mouse prostate adenocarcinoma cell line, which expressed human PSA. Lm–LLO–PSA was immunogenic in C57BL/6 mice and splenocytes from mice immunized with Lm–LLO–PSA showed significantly higher number of IFN-γ secreting cells over that of the naïve animals in response to a PSA H2Db-specific peptide, as measured by both, ELISpot and intracellular cytokine staining. In addition, using a CTL assay we show that the T cells specific for PSA were able to recognize and lyse PSA-peptide pulsed target cells in vitro. In a comparison study with two other PSA-based vaccines (a pDNA and a vaccinia vaccine), Lm–LLO–PSA was shown to be more efficacious in regressing established tumors when used in a homologues prime/boost regimen. Together, these results indicate that Lm–LLO–PSA is a potential candidate for pCa immunotherapy and should be further developed.     

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish

Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621 bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.     

Cultural conditions forAeromonas hydrophila affect the production of haemolysins with differing host specificities

Five strains ofAeromonas hydrophila were studied for production of haemolysin specific for erythrocytes of various animal species using three cultural methods. All the strains produced haemolysin for all the erythrocyte species when the organisms were cultured on blood agar.Using cellophane overlay method, all the strains produced haemolysin for fish erythrocytes and variable activity to mammalian erythrocytes. Only one strain produced haemolytic activity for various though not all of the erythrocyte species when grown in brain heart infusion broth.Data suggest thatA. hydrophila produces multiple haemolysins with specificities for erythrocytes of different animals. This was confirmed for trout and horse erythrocyte targeted haemolysins, by using iso-electric focussing separation and by measuring the effect of addition of ammonium sulphate to the growth medium.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Attachment Strength of Listeria monocytogenes and its Internalin-Negative Mutants

A single cell of Listeria monocytogenes attached on food contact surfaces can be a potential source of cross-contamination in a food-processing plant. To see whether internalin A (InlA) and B (InlB), major surface proteins on L. monocytogenes, play a significant role in the attachment process, wild-type L. monocytogenes EGD (LM_EGD) and its isogenic internalin-negative mutants (LM_EGDΔinlA, LM_EGDΔinlB, and LM_EGDΔinlAB) were used to determine attachment strength on inert glass surface. Western blot analysis using InlA and InlB antibodies confirmed the absence of InlA in LM_EGDΔinlA, InlB in LM_EGDΔinlB, and both InlA and InlB in LM_EGDΔinlAB. Regardless of initial attachment numbers, LM_EGD which expressed both InlA and InlB proteins exhibited the strongest attachment strength while the double mutant (LM_EGDΔinlAB) exhibited the weakest. The two single mutants (LM_EGDΔinlA and LM_EGDΔinlB) that expressed only one type of the internalins were shown to have intermediate attachment strength. These results suggest that both InlA and InlB expression play a significant role in the attachment strength of L. monocytogenes on glass surface.     

A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.     

Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.     

Analysis of survival rates and cellular fatty acid profiles of Listeria monocytogenes treated with supercritical carbon dioxide under the influence of cosolvents

In the present study, we identified several process variables that significantly affect the efficiency of supercritical carbon dioxide inactivation of the food-borne pathogen Listeria monocytogenes. Treatment with SC-CO(2) completely disabled the colony-forming activity of the cells (8-log reduction) within specific treatment time (10-50 min), pressure (80-150 bar), and temperature ranges (35-45 degrees C). Microorganism inactivation rates increased proportionally with pressure and temperature, but the inactivation rate decreased significantly when cells were suspended in phosphate-buffered saline rather than in physiological saline. Additionally, when the microbial cell suspension was 80-100% (w/w) of water, the SC-CO(2)-mediated reduction in CFU ml(-1) was 4-8 log higher at the same treatment conditions than in typical cell suspensions (a water content of 800-4000% [w/w]) or dry preparations that had only 2-10% (w/w) of water. The addition of a fatty acid, oleic acid, decreased the effectiveness of the microbial inactivation by SC-CO(2), but the addition of a surfactant, sucrose monolaurate, increased the effectiveness. Therefore, cosolvents for SC-CO(2), including water, a fatty acid, and a surfactant in this study, were found to greatly influence on the inactivation effectiveness. The extraction of cellular substances, such as nucleic acid- and protein-like materials and fatty acids, was monitored by spectrophotometry and GC/MS and increased with SC-CO(2) treatment time. Additionally, using scanning and transmission electron microscopies, we investigated morphological changes in the SC-CO(2)-treated cells. The effects of the variables we have described herein represent a significant contribution to our current knowledge of this method of inactivating food-borne pathogens.