Distribution of Aeromonas hydrophila in natural and man-made thermal effluents.

Densities of Aeromonas hydrophila showed distinct thermal optima (25 to 35 degrees C) and thermal maxima (45 degrees C) when measured along thermal gradients created by geothermal and nuclear reactor effluents. Survival of A. hydrophila never exceeded 48 h at temperatures of greater than 45 degrees C. Thermophilic strains could not be isolated at any site.     

SDS-PAGE heat-shock protein profiles of environmental Aeromonas strains

Aeromonas microorganisms normally grow at temperatures between 5 degrees C and 45 degrees C and therefore should have high thermotolerance. Thus it was of interest to find out whether A. hydrophila, A. caviae and A. veronii biovar sobria serovars respond to abrupt temperature changes with a heat shock-like response. To this end the present study was undertaken to determine whether Aeromonas species exhibits a heat shock response to different temperatures and time factors. The response of Aeromonas serovars to 24 h and 48 h of thermal stress at 25 degrees C, 42 degrees C and 50 degrees C involved the synthesis of 12-18 heat shock proteins (HSPs) bands with molecular weights ranging between 83.5-103.9 kDa in the high HSP molecular mass and 14.5-12.0 as low molecular mass HSP. Electrophoretic analysis of the HSPs showed that the serovars do not cluster very tightly and also that they are distinct from each other.     

Heat resistance and growth of Salmonella enteritidis, Listeria monocytogenes and Aeromonas hydrophila in whole liquid egg

Salmonella enteritidis ATCC,13067, Listeria monocytogenes ATCC,19116 and Aeromonas hydrophila ATCC,7965 strains were evaluated for growth and thermal resistance in liquid whole egg (LWE). Each strain grew well in LWE at temperatures between 4 and 30 degrees C, except S. enteritidis which grew weakly at 4 and 10 degrees C. Maximum populations for each strain increased with increasing growth temperature. The thermal destruction of each strain was determined in six liquid products. The egg products used were LWE, LWE with 5, 10 and 15% NaCl and LWE with 5 and 10% sucrose. L. monocytogenes tended to be more heat resistant than S. enteritidis and A. hydrophila. The highest kill rates were noted in LWE, while survival was best in those products supplemented with NaCl. Radiation D10 values of strains in LWE were 0.18, 0.39 and 0.49 kGy for A. hydrophila, S. enteritidis and L. monocytogenes, respectively.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Influence of modified-atmosphere storage on the growth of uninjured and heat-injured Aeromonas hydrophila.

The growth of uninjured and heat-injured Aeromonas hydrophila incubated at 5 degrees C (22 days) and 30 degrees C (31 h) under air, N2, and CO2 was investigated. At 30 degrees C, the growth patterns of cells on brain heart infusion agar incubated under air and N2 were similar, although slight differences in the lengths of the lag phases and the final populations were detected. The lag phases of cells incubated under air and N2 were substantially longer at 5 degrees C than at 30 degrees C. The population of uninjured A. hydrophila incubated at 5 degrees C under air and N2 remained constant, whereas the number of injured cells declined before the exponential growth phase. Growth at 5 degrees C was enhanced when uninjured and heat-injured A. hydrophila were incubated under N2. At 30 degrees C, cells incubated under CO2 exhibited noticeably longer lag phases and lower growth rates than did cells incubated under air and N2. The viable populations of uninjured and heat-injured cells incubated at 5 degrees C under CO2 declined steadily throughout incubation.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila.

The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48 degrees-65 degrees C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7 degrees C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30 degrees C) were twice as resistant as those in the early stage (5 h at 30 degrees C), and the maximum D-value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z-values significantly. The heat resistance of cells incubated for 48 h at 30 degrees C increased (twice) after holding at 7 degrees C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30 degrees C) did not influence their D-values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 to 4.0 reduced D-values by a factor of 5. Although the strain studied was heat-sensitive (D55 degrees C = 0.17 min; z = 5.11 degrees C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

Inhibitory activity of spices and essential oils on psychrotrophic bacteria

This study was designed to evaluate "in vitro" the inhibitory effects of spices and essential oils on the growth of psycrotrophic food-borne bacteria: Aeromonas hydrophila, Listeria monocytogenes and Yersinia enterocolitica. The sensitivity to nine spices and their oils (chilli, cinnamon, cloves, ginger, nutmeg, oregano, rosemary, sage, thyme) was studied. Antibacterial activity was evaluated on liquid and solid medium. Spices: 1% concentration of each spice was added separately to Triptic Soy Broth and then inoculated to contain 10(8)/ml organism and held to 4 degrees C for 7 days. Populations of test organism were determined on Triptic Soy Agar. Oils: Inhibition of growth was tested by using the paper disc agar diffusion method (at 35, 20 and 4 degrees C) and measuring their inhibition zone. MIC was determined by the broth microdilution method. Some culinary spices produce antibacterial activity: inhibition of growth ranged from complete (cinnamon and cloves against A. hydrophila) to no inhibition. Antibacterial inhibition zone ranged from 8 mm to 45 mm: thyme essential oil showed the greatest inhibition against A. hydrophila.     

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 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.     

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Membrane filter procedure for enumeration of Aeromonas hydrophila in fresh waters.

A membrane filter method (mA) for the enumeration of Aeromonas hydrophila in natural water samples was developed. The complex, primary medium employs trehalose as a fermentable carbohydrate and ampicillin and ethanol as selective inhibitors. After 20 h of incubation at 37 degrees C, an in situ mannitol fermentation test followed by an in situ oxidase test is used to further differentiate A. hydrophila from other aquatic and terrestrial microorganisms present in freshwaters. The primary medium decreases background microbial growth by about two orders of magnitude. The recoveries on mA medium from suspensions of A. hydrophila prepared from pure cultures and held for 24 h at 15 degrees C exceeded 95% of the recoveries on brain-heart infusion agar spread plates. The confirmation rate for colonies designated A. hydrophila was 98%, whereas 11% of the presumptively negative colonies were, in fact, A. hydrophila. Recoveries of A. hydrophila from fresh, surface water samples exceeded recoveries by the other methods examined.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis

In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966(T) of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4 degrees C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37 degrees C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD(50)). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD(50) dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.     

Thermoregulation in the Angolan free-tailed bat Mops condylurus: A small mammal that uses hot roosts.

The Angolan free-tailed bat (Mops condylurus) uses roosts that often exceed 40 degrees C, an ambient temperature (Ta) that is lethal to many microchiropterans. We measured the physiological responses of this species at Ta's from 15 degrees to 45 degrees C. Torpor was commonly employed during the day at the lower Ta, but the bats generally remained euthermic at night, with a mean body temperature (Tb) of 35.2 degrees C. Metabolic rate reflected the pattern of Tb, increasing with falling Ta at night but decreasing during the day. Metabolic rate and evaporative losses were lower in torpid than in euthermic bats. Body temperature increased at each Ta >35 degrees C and was 43 degrees C at Ta of 45 degrees C. At Ta of 40 degrees C bats increased dry thermal conductance and evaporative heat loss compared to lower Ta. At 45 degrees C dry thermal conductance was lower than at 40 degrees C and evaporative heat loss was 132% of metabolic heat production. At high Ta there was only a slight increase in metabolic rate despite the employment of evaporative cooling mechanisms and an increase in Tb. Collectively our results suggest that M. condylurus is well suited to tolerate high Ta, and this may enable it to exploit thermally challenging roost sites and to colonise habitats and exploit food sources where less stressful roosts are limiting.     

Production of exotoxins by Aeromonas spp. at 5 degrees C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5 degrees C for 7-10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37 degrees C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5 degrees C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria, are of potential public health significance in meats stored at refrigeration temperature.     

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.     

Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

Formation of viable but non-culturable state (VBNC) of Aeromonas hydrophila and its virulence in goldfish, Carassius auratus

In this study we investigated the viable but non-culturable (VBNC) state of Aeromonas hydrophila and its virulence in goldfish. Aeromonas hydrophila cultured in a 0.35% NaCl solution at pH 7.5 and at 25 degrees C for 50 days showed the VBNC state. In the VBNC state we were unable to detect viable bacteria by the plate count method but we did find 10(4) cells/ml by the direct viable count microscopical method after staining with fluorescein diacetate and ethidium bromide. The virulence comparison in goldfish showed that bacteria cultured at 25 degrees C for 1 day in a 0.35% NaCl solution were more virulent than bacteria cultured for 28 days. VBNC bacteria showed lower virulence in goldfish compared to 28-day-cultured bacteria by intraperitoneal injection. The results from the study suggest that A. hydrophila can remain in the aquatic environment for prolonged periods in the VBNC state but those cells are not pathogenic to goldfish.