The ability of motile Aeromonas spp. to survive in drinking water (mineral and tap water) and in sea water was experimentally tested. Clinically isolated cytotoxic strains of A. hydrophila, A. caviae and A. sobria were selected for this study. After contamination of water samples, the survival of Aeromonas strains was studied for at least three months using viable counts. The results obtained show that the survival of the Aeromonas spp. varies considerably depending on species and water type. For all three species, the survival time was longest in mineral water, where viable bacteria of each strain were still detected after 100 d. Moreover, A. hydrophila and A. caviae also re-grew on the first day. In tap water all strains showed marked survival, although to a lesser extent than in mineral water. Aeromonas cells showed a rapid decline in sea water (90% reduction in viable cells after about two d) and thus seem to be more sensitive to saline/marine stress than chlorination.

T h e survival of mesophilic Aeronionas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. T w o cytotoxic (Aer. /zydrophila and Aer. cazirre) and one invasive (Aer. sohrzu) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas, the number of living cells was determined over at least 5 months. F o r all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. T h e times necessary to achieve a 9So% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer.Igdrophilu and Aer. sobrin, while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas-associated human infections.Grrc,spponili.nre to : l h (;io,xrii Briinili. Istitirto ill Igrme. Unizwsrta Dqli Stud1 dr llrliino.

Escherichia coli cells challenged with low or high concentrations of hydrogen peroxide are killed via two different mechanisms and respond with morphological changes which are also dependent on the extracellular concentration of the oxidant. Treatment with low concentrations (<2.5 mM) of H2O2 is followed by an extensive cell filamentation which is dependent on the level of H2O2 or the time of exposure. In particular, addition of 1.75 mM H2O2 results in a growth lag of approximately 90 min followed by partial increase in optical density, which was mainly due to the onset of the filamentous response. In fact, microscopic analysis of the samples obtained from cultures incubated with the oxidant for various time intervals has revealed that this change in morphology becomes apparent after 90 min of exposure to H2O2 and that the length of the filaments gradually increases following longer time intervals. Analysis of the ability of these cells to form colonies has indicated a loss in viability in the first 90 min of exposure followed by a gradual recovery in the number of cells capable of forming colonies. Measurement of lactate dehydrogenase in culture medium (as a marker for membrane damage) has revealed that a small amount of this enzyme was released from the cells at early times ( < 150 min) but not after longer incubation periods (300 min). Cells exposed to high concentrations of H2O2 (> 10 mM) do not filament and their loss of viability is associated with a marked reduction in cell volume. In fact, treatment with 17.5 mm H2O2 resulted in a time-dependent decrease of the optical density, clonogenicity, and cellular volume. In addition, these effects were paralleled by a significant release in the culture medium of lactate dehydrogenase thus suggesting that the reduced cell volume may be dependent on membrane damage followed by loss of intracellular material. This hypothesis is supported by preliminary results obtained in electron microscopy studies. In conclusion, this study further demonstrates that the response of E. coli to hydrogen peroxide is highly dependent on the concentration of H2O2 and further stresses the point that low or high concentrations of the oxidant result in the production of different species leading to cell death via two different mechanisms and/or capable of specifically affecting the cell shape.

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Escherichia coli lethality by hydrogen peroxide is characterized by two modes of killing. In this paper we have found that hydroxyl radicals (OH.) generated by H2O2 and intracellular divalent iron are not involved in the induction of mode one lethality (i.e. cell killing produced by concentrations of H2O2 lower than 2.5 mM). In fact, the OH radical scavengers, thiourea, ethanol and dimethyl sulfoxide, and the iron chelator, desferrioxamine, did not affect the survival of cells exposed to 2.5 mM H2O2. In addition cell vulnerability to the same H2O2 concentration was independent on the intracellular iron content. In contrast, mode two lethality (i.e. cell killing generated by concentrations of H2O2 higher than 10 mM) was markedly reduced by OH radical scavengers and desferrioxamine and was augmented by increasing the intracellular iron content. It is concluded that OH. are required for mode two killing of E. coli by hydrogen peroxide.