Culturability and viability techniques such as plate count on solid agar (PC), Most Probable Number (MPN) and Direct Viable Count-Fluorescence in Situ Hybridation (DVC FISH) were used to study the inactivation of Salmonella typhimurium by photo-Fenton process at pH 5.5. In the presence of only simulated solar irradiation (500 W·m-2), S. typhimurim showed that both culturability measured by MPN and viability (measured by DVC FISH) underwent just a slight decreasing of 2 and 1 log respectively after 240 min of light exposition while culturability measured by PC did not show any change. Results after 48 h of dark conditions did not reveal re-growth. However, when experiment was carried out in the presence of 2 mg L-1 of Fe3+ and 20 mg L-1 of H2O2 and pH 5.5, culturability was strongly affected after 240 min of simulated solar irradiation; nevertheless, viability was only slightly altered (~1 log). During dark period of 48 h changes on culturability and viability were not observed. On the other hand, it was also found that sugar metabolism was affected rather the amino-acids in S. typhimurium cells irradiated at different times upon photo-Fenton conditions. These findings might suggest for the first time that photo-Fenton process at pH 5.5 could induce viable but nonculturable state (VBNC) on waterborne S. typhimurium and that probably sugar metabolism damage could activate the VBNC state.
Photo-Fenton processes which are based in the light induced reaction of ferrous and/or ferric ions with hydrogen peroxide to generate reactive oxygen species such as protonated superoxide (HO2•) and •OH radical, have recently risen as a promising method to inactivate waterborne bacteria [1-5].
Recent studies reported that solar photo-Fenton process at near neutral pH (5.5) achieved a complete inactivation of wild Salmonella sp., a pathogen bacterial strain resistant to solar disinfection [
There are methodologies that can determinate the cell viability. Among them the coupling of Direct Viable count (DVC) with Fluorescence in situ Hybridation (FISH) seems to be effective to determinate viability of waterborne bacterial cells [
Herein, for the first time, culturability and viability were measured simultaneously by PC, MPN and DVCFISH of S. typhimurim being exposed to the photo-Fenton processes induced by simulated solar light irradiation at pH 5.5. It is suggested that this photochemical process could induce on the bacterial cells VBNC state avoiding their growing on solid agar and liquid media after 240 min of simulated solar irradiation. This fact is extremely important since it might allow evaluate precisely the ability and limitations of photo-Fenton process to inactive pathogen waterborne bacteria.
Pyrex glass bottles of 80 mL (3.5 cm in diameter and 10 cm high) were used as batch reactors. 2 mg L−1 of Fe3+ (FeSO4·7H2O (Merck-Germany)) and 20 mg L−1 of H2O2 (Merck-Germany 30% (v/v)) were added to 80 mL of an isotonic solution containing distilled water and NaCl (Sigma-Aldrich Germany) (8.0 g L−1), KCl (Merck Germany) (0.8 g L−1) in each reactor. The initial pH was adjusted by NaOH (Merck Germany) addition until reaching 5.5, not buffer was used. Solar irradiation was simulated by a Hanau Suntest solar simulator (Germany) having a wavelength spectral distribution with about 0.5% of emitted photons <300 nm (UV-C range) and about 7% between 300 and 400 nm (UV-B, A range). The emission spectrum between 400 and 800 nm follows the solar spectrum (
Dundee, UK). Pyrex bottles containing inoculated S. ty-phimurium were illuminated in the presence and absence of Fe3+ and H2O2 during 4 h and samples (1.0 mL) were taken at different time intervals and serial dilutions were performed in saline solution.
The irradiation experiments were performed at room temperature (25˚C) and the temperature of the solution increased up to approximately 30˚C during irradiation. All experiments were carried out in equilibrium agitating at 700 rpm in triplicate and the error bars were added on the graphs.
A strain of S. typhimurium ATCC 15490 (Washington DC, USA) was used during these experiments. Strain was preserved in a 30% glycerol solution to avoid membrane rupture during freezing storage. The bacterial reactivation was achieved by inoculating 10 mL of nutrient broth (Oxoid) with 100 µL of bacterial suspension, and then incubating for 18 h at 35˚C. A second inoculum was transferred from this culture to a new tube with 10 mL of nutrient broth and again incubated for 18 h at 35˚C. This second culture was washed by centrifuging at 3000 rpm for 10 min; the supernatant was carefully extracted so the pellet can be suspended and diluted in 10 mL of 0.1% peptonized bi-distillated water (Milli-Q water), agitated and centrifuged again. This procedure was repeated three times, leaving the last cell pellet suspended in 10 mL of 0.1% peptonized bi-distillated water. The final concentration of the washed cell suspension was adjusted according to the McFarland scale #0.5 at 1.5 × 108 CFU mL−1. Consecutive dilutions (10−1 - 10−4) of the adjusted cell suspension (1.5 × 108 CFU mL−1) were made in tubes with 9 mL of 0.1% peptonized water (Oxoid), and seeded onto nutrient agar to be incubated for 18 h at 35˚C to establish the initial concentration. This cell suspension was used to inoculate the batch reactors containing the isotonic saline distillated water with Fe3+ (FeSO4·7H2O (Merck-Germany)), and H2O2 (Merck-Germany), to initiate irradiation experiments.
100 μL from each dilution were seeded onto Petri dishes containing Nutrient Agar (Merck-Germany) and incubated at 37˚C ± 2˚C for 24 h to obtain the CFU measuring.
1 mL from each dilution were inoculated onto tubes with 9 mL of lactose broth (Difco) and incubated at 35˚C ± 2˚C for 24 h in order to initiate the presumptive phase. The positives for bacterial growth were then recovered in Rappaport broth (Merck-Germany) before being seeded on Hektoen (Merck-Germany), Xylose, Lysine Desoxycholate agar (XLD agar) (Difco-France) and Nutrient agar (Merck-Germany) to finalize with the confirmatory phase.
The FISH-DAPI coloration procedure suggested by Amman et al. [
The remaining 8.8 mL sample in each dilution tube were treated with Nalidixic acid (40 μg mL−1) and 1 mL of lactose broth (Difco-France) and yeast extract (DifcoFrance), then incubated in the dark for 18 h at 35 ± 2˚C. Cells were harvested by centrifugation at 5000 g. Depending on the expected concentration, 100 or 300 μL were re-suspended in 10 x phosphate buffered saline (PBS) solution to reach 1 mL and fixated with a paraformaldehyde fixation buffer 30% (v/v), stored overnight at 4˚C, and harvested by centrifugation at 1000 g for 10 min. The pellets were washed with PBS solution. This washing process was repeated two times to ensure the complete removal of p-formaldehyde, and the final pellets were re-suspended in 500 μL of 1 × PBS and 500 μL of ethanol 100%. 10 μL of the resulting cell suspension was transferred to each well of a 8-well slide, previously covered with gelatine (0.1% w/v) and KCr(SO4)2 (MerckGermany) (0.01% w/v). The loaded slides were dehydrated with ethanol and incubated for 2 h in the dark with 10 μL of hybridization solution pre-warmed at 45˚C and containing 24 ng∙μL−1of S. typhimurium probe [16,20], marked with 5’ Cy3 (prepared by Microsynth GmbH Switzerland). Finally, cells were washed with a washing solution at 45˚C and then rinsed with distilled water.
After hybridization, all samples were washed in distilled water and stained with 9 μL of DAPI (Organic Research-Ireland) (4’,6’-diamidino-2-phenylindole) 0.0001% (w/v) for 10 minutes, washed again in distilled water and dried (to aid cell localization, slides were cover with antifade citi-fluor solution). Microscopic cell count (DAPI) and oligonucleotide probe-positive count (Cy3) were performed using a Nikon Eclipse E800 microscope (Japan) equipped with a specific DAPI filter and G-2A filter sets. A minimum of 20 view squares were enumerated for each well; two wells were examined in total.
Four tubes containing 20 mL of mineral media containing distilled water and NaCl (Sigma-Aldrich Germany) (8.0 g∙L−1), KCl (Merck Germany) (0.8 g∙L−1) and 20% of Glucose (Merck Germany) or Peptone (DIFCO UK), respectively, were inoculated with 1 mL of irradiated bacteria. The turbidity at 650 nm was measured after 18, 24, and 96 h of incubation and the increase in 0.1 or more turbidity units was reported as positive growth.
The initial S. typhimurium concentration before irradiation events, evaluated with DVC FISH and MPN methods, achieved concentrations two logs higher than the PC method (
rate of cell recovery. In solid media (PC method) the bacteria growing is limited by the availability of water and nutrients close to the colony’s growing area while the liquid media (MPN and Nalidixic media) ensures constant and direct contact of cell with nutrients. This could explain why DVC-FISH and MPN gave initial concentrations higher than PC.
On the other hand, results obtained by MPN and PC (
In summary, the three methods used in this study (DVC FISH, MPN and PC) revealed that direct solar simulated irradiation had slight negative effect on the S. typhimurium population. It is well known that UV-C component (220 - 280 nm) of UV light is the most lethal to the microorganisms since it might produce chemical changes on the DNA components [