Báo cáo khoa học: Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead? ppt

Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead? Andrea Villarino 1,2 , Marie-Noe¨ lle Rager 3 , Patrick A. D. Grimont 1,2 and Odile M. M. Bouvet 2 1 Aquabiolab and 2 Unite ´ de Biodiversite ´ des Bacte ´ ries Pathoge ` nes E ´ mergentes, INSERM U389, Institut Pasteur, Paris, France; 3 Service de Re ´ sonance Magne ´ tique Nucle ´ aire UMR 7576, Ecole Nationale Supe ´ rieure de Chimie de Paris, France Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a conse- quence, the interpretation of the state of ÔnonculturableÕ cells is often ambiguous. Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost ÔculturabilityÕ but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden–Meyerhof and pentose-phosphate pathways. However, nonculturable cells obtained by irradi- ation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities. Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively. Keywords: NMR; radiation injury; viability; metabolism; Escherichia coli. Ultraviolet irradiation has been used in the disinfection of drinking water, wastewater and in air disinfection [1–3]. After disinfection, microorganisms are not detectable in standard culture media in which they have been previ- ously found to proliferate [4]. Thus, a bacterium is currently reported as dead when it does not yield visible growth in bacteriological media for a given time [5]. However, it has been suggested that the bacterial populations in water, when exposed to UV disinfection, might show a decrease in ÔculturabilityÕ, but in fact they could still be alive and able to cause disease [6]. Moreover, in aquatic systems among the various stresses to which bacteria are submitted, solar radiation (UV-B, 290–320 nm) seems to be the most important in causing the loss of culturability [7]. Wilber and Oliver [6] showed that, although both UV-treated Salmonella serotype Typhimurium and Escherichia coli lost culturability in standard culture media upon irradiation, they retained the capacity to respond to nutrients by cell elongation in the direct viable count (DVC) method. On the other hand, Caro et al. [8] observed that UV-treated Salmonella cells lost the capacity of cell elongation in the DVC method and lost culturability concomitantly with pathogenicity in mice. However, these cells were also considered to be alive because they retained respiratory activity, membrane integrity and DNA integrity. In a previous study, we considered that UV-treated E. coli cells that retained the same activities described by Caro et al. were dead because neither growth nor cell elongation or protein synthesis were detected [9]. Cells could be defined operationally as alive or dead depending on the method used to determine cell viability. Moreover, each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of the state of cells is often ambiguous [10,11]. Problems in the interpretation of the state of cells that have lost culturability are due, not only to the absence of consensus on the definition of bacterial death but also, to the lack of global studies showing their metabolic potential. The aim of the present study was to analyze the metabolic capacities of UV-induced nonculturable E. coli cells and to determine whether the level of UV-irradiation affects their metabolic potential and responsiveness. The capacity of cells to respond to the addition of nutrients determined by cell elongation, protein synthesis and glucose meta- bolism was analysed, as well as the effect of different aeration conditions on the regulation of metabolic fluxes through the Embden–Meyerhof and pentose phosphate pathways. Experimental procedures Bacterial strain and growth conditions Escherichia coli K-12S sensitive to bacteriophage lambda (strain CIP 54118) from the Collection de l’Institut Pasteur (Paris, France) was used [12]. Overnight cultures of E. coli K-12S were maintained long-term at )80 °CinTrypto Casein Soy broth (Sanofi Diagnostics Pasteur, Marnes-la Correspondence to O. M. M. Bouvet, Unite ´ des Pathoge ` nes et Fonctions des Cellules Epithe ´ liales Polarise ´ es, INSERM U510, Faculte ´ de Pharmacie, Universite ´ Paris XI, F-92296 Chaˆ tenay- Malabry, France. Fax: + 33 146835844, Tel.: + 33 146835843, E-mail: odile.bouvet@cep.u-psud.fr Abbreviations: UV1, low ultraviolet dose; UV2, high ultraviolet dose; DVC method, direct viable count method; qDVC method, quantita- tive direct viable count method; LB, Luria Bertani medium; EM, Embden–Meyerhof pathway; PP, pentose phosphate pathway. (Received 4 March 2003, revised 25 April 2003, accepted 6 May 2003) Eur. J. Biochem. 270, 2689–2695 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03652.x Coquette, France) supplemented with glycerol (40%, v/v). Initially, cells were grown at 37 °C overnight in Luria– Bertani (LB) broth [13] transferred to a fresh medium at a dilution of 1 : 200 and grown to a late exponential phase (D 600 ¼ 0.6) in aerobic conditions. They were harvested by centrifugation at 3800 g for 15 min at 9 °Candwashed twice in phosphate buffer pH 7.4 (11 m M K 2 HPO 4 ,5m M KH 2 PO 4 ,120m M NaCl, 0.1 m M CaCl 2 ,0.5m M MgSO 4 ). Finally, they were diluted in the same buffer to a cell density of about 2 · 10 7 CFUÆmL )1 and used immediately for UV irradiation. UV irradiation To obtain nonculturable cells, a cell suspension containing (2 · 10 7 CFUÆmL )1 ) was irradiated as described previously [9]. Briefly, 6.5 mL of the cell suspension was placed in a sterile glass Petri dish (11 cm diameter) and irradiated with a 12-W (254 nm) germicidal lamp (Bioblock Scientific, Illkirch, France) at 25 °C with mild agitation. The lamp, at 13 cm from the Petri dish, was switched on 1 h before utilization and the intensity of radiation at the bottom of the Petri dish was controlled with an ultraviolet intensity meter (Bioblock Scientific). UV dose was calculated as the product of exposure time and the intensity at the bottom of the Petri dish (10 mJÆmin )1 Æcm )2 ). Cells were irradiated by two UV doses, UV1 dose (4 mJÆcm )2 ) corresponding to the first dose sufficient to obtain at least a six-log. reduction (i.e. 20 CFUÆmL )1 ) in the initial colony count and UV2 dose (80 mJÆcm )2 ) inducing about a seven-log. reduction (i.e. 2CFUÆmL )1 ). UV-treated bacteria were handled in dark- ness. After UV irradiation, treated cells were concentrated by centrifugation (3800 g) to a cell density of about 2 · 10 8 cellsÆmL )1 and used immediately in further experi- ments. For each experiment, a nonirradiated cell suspension at the same cell density was used as an untreated control. Culturability Samples (2 mL) of untreated or UV-treated cell suspension were incubated with or without 440 U of catalase (220 UÆmL )1 ) (Sigma) at room temperature. Aliquots (100 lL) were taken at different times and surface plated in triplicate on LB agar supplemented with or without catalase. Some experiments used both catalase and super- oxide dismutase (Sigma). The enzyme solutions used were filter-sterilized through 0.22 lm pore size membrane filter, and 0.2 mL were aseptically spread on the surface of agar media at a concentration of 2000 U per plate. Plates were then incubated in aerobic and anaerobic conditions at 37 °C for 48 h. Substrate responsiveness Substrate responsiveness of cells was determined by the direct viable count method (DVC) [14] in the conditions described previously with some modifications [9]. Cell samples were diluted (1/100, v/v) in LB medium containing nalidixic acid (40 lgÆmL )1 ) (Sigma). Cells that exceeded at least twice the mean length of cells before DVC were scored as elongated. The proportion of DVC positive cells was corrected by the proportion of elongated cells detected before the DVC method. At the same time, cells incubated in the same conditions but without nalidixic acid addition were also analysed. A quantitative DVC (qDVC) method was also used [15]. Elongated or nonelongated substrate-respon- sive cells were selectively lysed by spheroplast formation caused by incubation with nutrients, nalidixic acid and glycine (2% final concentration). This glycine effect leads to swollen cells with a very loose cell wall. The substrate- responsive cells were then lysed easily by a single freeze-thaw treatment. The number of cells responding to nutrients was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. Results were expressed as percentage of substrate-responsive cells with respect to the original colony count of untreated bacteria. Cell samples of DVC and qDVC were incubated in the dark at 37 °Cfor5hwith shaking (200 r.p.m). The cells were then fixed with 3% formalin (final concentration) to be enumerated by epi- fluorescence microscopy and analysed by flow cytometry. Epifluorescence microscopy and flow cytometry For cell enumeration, samples were filtered through poly- carbonate membrane filters (pore size, 0.2 lm, 25-mm diameter) (Milipore) and washed with phosphate buffer. These cells were detected by staining with propidium iodide (Sigma,StLouis,MO)at0.5lgÆmL )1 (final concentration) or by fluorescent in situ hybridization [16] with probe EUB 338 labelled with fluorescein isothiocyanate [17]. Filters were washed and mounted with Vectashield mount- ing medium (Vector, Burlingame, CA, USA) on glass microscope slides and stored in the dark at 4 °C until counted. Cells were counted with an Olympus BX-60 epifluorescence microscope (100-W mercury lamp) with a · 100 oil immersion fluorescent objective. Cells in 24 microscopic fields per filter were enumerated and averaged (about 400 cells for nonirradiated cells). For each sample, three filters were examined and maximal deviations from the mean were calculated. Modifications of E. coli size and granularity of untreated and UV-treated cells, before and after the DVC method described above, were analysed by flow cytometry [18]. Duplicate samples were analysed with a Becton Dickinson model FACScan cytometer equipped with a 15-mW, air- cooled argon ion laser (488 nm) by using CELL QUEST 3.3 software. The forward angle light scatter and side angle light scatter amplifier gains were set to linear and logarithmic mode, respectively. For each cell sample run, data for 10 000 events were collected. Protein synthesis Protein synthesis was analysed by incorporation of [ 35 S]methionine (Amersham Pharmacia Biotech) into pro- teins as described earlier [9]. Proteins were precipitated after 5 h of incubation at 37 °C in aerobic conditions in LB broth. The final concentration used for [ 35 S]methionine was 0.1 m M at 100 lCi. The precipitate was collected onto GF/C filters (0.45 lm), washed and radioactivity was counted in a scintillation counter. Protein synthesis was detected in duplicate samples and results were expressed as nmol of [ 35 S]methionine incorporated per lgofprotein.The 2690 A. Villarino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 detection limit of this method was 0.01 nmol [ 35 S]methio- nine per lg protein, corresponding to protein synthesis of about 10 6 CFUÆmL )1 . The maximal deviation from the mean of two independent experiments was calculated. Glucose uptake Duplicated samples of 2 mL of UV-treated and untreated cell suspension were incubated with or without 440 U of catalase (220 UÆmL )1 ). After 15 min of incubation at room temperature, 5 m M of glucose (final concentration) spiked with [ 14 C]glucose (10 lCi in the 2 mL of mix) (Amersham International) were added. The reaction mixtures were incubated in aerobic conditions at 37 °C with shaking (180 r.p.m). Aliquots were taken at different times, deposi- ted on GF/C filters (pore size, 0.45 lm; 2.5 cm diameter; Whatman, Maidstone, England) and then washed with phosphate buffer to remove nonincorporated [ 14 C]glucose. Each filter was dried and radioactivity was measured in a scintillation counter. Glucose uptake with catalase previ- ously inactivated in water at 100 °C for 30 min was used as a negative control. In order to avoid precipitation of heat- inactivated catalase in negative control experiments, phos- phate buffer without NaCl was used. The results obtained were expressed as nmol [ 14 C]glucose transported per lgof protein. The detection limit of this method was 0.5 nmol [ 14 C]glucose per lg protein corresponding to glucose uptake of about 10 7 CFUÆmL )1 .Themaximaldeviationfromthe mean of two independent experiments was calculated. Metabolic flux by 13 C NMR spectroscopy As in the case of glucose uptake, cell suspensions were incubated with or without catalase. Here, 13 Cglucose (Leman, St Quentin en Yvelines, France) labelled at C1 or C6 was used and the reaction mixtures were incubated 4 h in aerobic or anaerobic conditions. When glucose, labelled isotopically either in position C1 or C6, is added to bacterial suspension, the amount of label introduced in acetate C2 depends on the activity of the pentose phosphate (PP) pathway. The equations used for estimating the relative activities of the PP or Embden–Meyerhof (EM) pathway were: y ) x ¼ PP, x ¼ EM, where x was the C2 enrich- ment of the acetate measured from [1- 13 C]glucose and y the C2 enrichment of the acetate measured from [6- 13 C]glucose. Perchloric acid extraction was performed to prevent a possible alteration of the secretion of the metabolites due to the UV-treatment. The reaction was stopped by addition of 240 lL of perchloric acid at 4 °C. The samples were vortexed for 2 min, placed in ice for 15 min, vortexed again for 2 min and finally centrifuged at room temperature at 8000 g for 15 min. Acid extracts were neutralized to pH 7 withNaOHandstoredat)20 °C until NMR analysis. All NMR data were recorded at 303K on a Bruker Avance 400 spectrometer using a 10-mm broad-band probe. Neutralized extracts were introduced in a 8-mm NMR tube, itself inserted in a 10-mm NMR tube containing D 2 O. 13 CNMR spectra recorded at 100.13 MHz were acquired during 1 h (2400 scans) with a composite pulse decoupling. Exponen- tial filtering of 3 Hz was applied prior to Fourier transfor- mation. Chemical shifts were referred to the a-C1 resonance of D -glucose (93.1 p.p.m). The acetate concentration and other metabolites formed (glucose, lactate, ethanol) were determined by NMR analysis and enzymatic assays (Boeh- ringer, Mannheim, Germany) as described previously [19]. Results Loss of culturability after UV-treatment The physiological state of nonculturable E. coli cells obtained by irradiation with a low (UV1) and a high (UV2) UV dose was examined. Immediately following the UV treatment, no decrease in the total number of cells was observed. The total cell count was 2.8 · 10 8 cellsÆmL )1 (± 4%) for untreated and UV1- or UV2-treated cells. After both UV treatments the great majority of the population ( 10 8 cellsÆmL )1 ) became nonculturable on LB agar plates while a minor percentage remained culturable (0.001– 0.0001%). However, no interference from these few cultur- able cells (UV survivors) was observed in further experi- ments because their number remained much lower than the detection limit of the method used. Loss of culturability on nutrient media could be explained by direct and indirect damage to nucleic acids produced by UV radiation [20]. Direct effects of UV radiation at 254 nm on nucleic acids include, for example, photodimerization between adjacent pyrimidine bases. Indirect effects result when reactive oxygen species such as hydrogen peroxide are generated. They also react with DNA, damaging bases, breaking strands and cross-linking DNA and protein [2]. In our experiments, catalase and superoxide dismutase were added to the medium to enhance culturability by protecting against the effects of free radicals. However, no increase in colony count on LB agar plates of UV1- and UV2-treated cells previously incubated in phosphate buffer containing 220 UÆmL )1 of catalase for 2 h, 4 h, or 24 h was observed. Furthermore, neither the addition of 2000 U catalase or both catalase and superoxide dismutase on LB agar plates nor incubation in anaerobic conditions reversed this result. After prolonged incubation (5 days) no further colony development could be observed. During the first 24 h of incubation time without nutrients after UV1-irradiation, the proportion of total and UV- survivor cells remained constant. However, in the case of cells treated with the UV2 dose, a decrease of about 30% in the original total cell number indicated the existence of cell lysis. This decrease in the total cell number was followed by a small increase in the number of culturable cells, which, after 24 h of incubation, reached almost 0.1% of the initial value. The regrowth is most probably explained by growth of the minor percentage of UV-survivors cells at the expense of nutrients liberated by UV2 lysed cells. Cell lysis could be explained by loss of the ability of UV2-treated cells to modify their autolysins. Inhibition of murein synthesis and loss of the electrical or pH gradient of cellular membranes have been described as ways to trigger lysis due to the uncontrolled autolytic action of murein hydrolases [21,22]. Response to nutrients Epifluorescence microscopy was used to determine whether, immediately after the UV-treatment, cells that lost cultura- bility had the ability to produce cell elongation with the Ó FEBS 2003 Are nonculturable E. coli cells alive or dead? (Eur. J. Biochem. 270) 2691 DVC method (incubation with nutrients and nalidixic acid). After DVC of UV1-treated cells, DVC positive cells that exceeded at least twice the mean length of E. coli K-12 were observed (Fig. 1, B1 and B2). Nevertheless, a few slightly elongated cells were observed even without nalidixic addi- tion. However, this change in cell size was not detectable or quantifiable even after analysis of a great number of cells by flow cytometry (data not shown). To determine whether slightly elongated or nonelongated UV1-treated cells responded to nutrient addition, an improved DVC method (qDVC) was used. With this method, substrate-responsive cells were selectively lysed by spheroplast formation caused by incubation with nutrients, nalidixic acid and glycine. It is known that glycine interferes with several steps in pepti- doglycan synthesis for bacterial cell wall formation [23], and this effect leads to swollen cells with a very loose cell wall. The substrate-responsive cells were lysed easily by a freezing treatment in liquid nitrogen and then thawed at room temperature. With this method, the proportions of substrate responsive cells obtained were 90% for untreated and 60% for UV1-treated cells. Cell lysis was not observed in the negative controls without glycine addition. When DVC (Fig. 1, C1 and C2) and qDVC were performed using UV2- treated cells, substrate responsive cells were not detected. With these cells, no elongated cells were detected after DVC, and after qDVC cell lysis was detected in both samples, with or without glycine addition. To obtain more evidence of the response to nutrients, the incorporation of [ 14 C]glucose into cells and the effect of exogenous catalase on glucose uptake were studied (Fig. 2). For untreated cells, glucose incorporation in the absence of catalase reached a steady state level of about 3.6 lmol [ 14 C]glucose per lg protein after 4 h of incubation. UV1- treated cells incorporated 2.5 lmol [ 14 C]glucose per lg protein corresponding to 69% of the glucose incorporated by untreated cells. However, for UV2-treated cells, a large decrease in the maximal glucose incorporation (0.6 lmol [ 14 C]glucose per lg protein) was observed, corresponding to only 17% of the glucose incorporated by untreated cells (Fig. 2A). When the same experiments were carried out in the presence of catalase, an increase in glucose uptake of about 60% was observed (Fig. 2B) in untreated and UV1 and UV2-treated cells. For UV2-treated cells this increase was observed only during the first 4 h of glucose incorpor- ation. After this time, glucose uptake with or without catalase addition decreased (data not shown), undoubtedly explained by the beginning of cell lysis described above. In all cases, no increase in glucose uptake was observed when experiments were carried out with catalase previously inactivated at 100 °C. In nongrowth conditions, the Fig. 1. Visualization of cells. Visualization by fluorescent in situ hybridization of untreated cells (A), UV1-treated cells (B) and UV2-treated cells (C) before (A1, B1, C1) and after DVC method (A2, B2, C2). Fig. 2. Glucose uptake. Glucose uptake in aerobic conditions (A), non irradiated cells ( • ,NI),UV1-treatedcells(j, UV1) and UV2-treated cells (m, UV2). Glucose uptake in aerobic conditions after 4 h of incubation with or without exogenous catalase (B). The detection limit of this method was 0.5 nmol [ 14 C] glucose per g protein corresponding to glucose uptake of about 10 7 CFUÆml )1 (10% of the initial number of cells). 2692 A. Villarino et al. (Eur. J. Biochem. 270) Ó FEBS 2003 imbalance during glucose uptake between cell metabolism and the arrest of cell division could be favorable to peroxide generation and accumulation. In E. coli,peroxidearises primarily from the auto-oxidation of components of its respiratory chain [24], and the presence of peroxide induces membrane damage [25]. Thus, prevention by exogenus catalase of peroxide damage could explain the observed increase in glucose uptake. However, this effect was observed indifferently in untreated and both UV1- and UV2- treated cells, showing no relation with the degree of UV-damage. To obtain more information on the physiological state of UV-treated cells, protein synthesis was analysed (Fig. 3). After 1.5 h of incubation with [ 35 S]methionine, UV1-treated cells synthesized less protein than untreated cells. Then, both untreated and UV1-treated cells reached a maximal incorporation of about 1.5 nmol of [ 35 S]methionine per lg of protein. As expected, no [ 35 S]methionine incorporation in proteins for UV2-treated cells was detected. Glucose metabolism under different aeration conditions The capacity of E. coli cells to metabolize glucose was investigated in whole cells using 13 C-NMR spectroscopy. 13 C-NMR studies were performed in untreated and UV- treated cells incubated for 4 h in aerobic and anaerobic conditions and the concentrations of fermentative products were measured by enzymatic assays. Results with enriched [1- 13 C]glucose are shown in Fig. 4. In anaerobic conditions for untreated and UV1-treated cells, a similar NMR spectrum was obtained. Acetate (A), lactate (L) and ethanol (E) were the main products formed, at levels of about 20, 90 and 10 mol per 100 mol of metabolized glucose, respectively. However, for UV2-treated cells, less glucose was consumed, a lower concentration of lactate and acetate was observed and no ethanol was detected (Fig. 4). The fact that ethanol is not detected in UV2-treated cells could be explained by the reduced glucose consumption or most probably by the incapacity of these cells to synthesize proteins. In anaerobic conditions, only cells that can synthesize the pyruvate formate lyase de novo can form ethanol [26]. In aerobic conditions, acetate was the only product detected in cellular extracts, the concentration for untreated and UV1-treated cells being about 90 mol per 100 mol of metabolized glucose. For UV2-treated cells, 70 mol of acetate per 100 mol of metabolized glucose were detected. In E. coli, glucose is metabolized via the EM and PP pathways [27,28]. In order to determine whether UV-treated cells incubated in different aeration conditions were able to modify their metabolic flux of glucose, the activities of the EM and PP pathways were studied. Glucose metabolism through these two pathways was quantified separately by using glucose substrates with a 13 C label at different carbon atoms. An easy and versatile method to determinate the acetate concentration by NMR after incubation with [6- 13 C]glucose or [1- 13 C]glucose was used to quantify separately the EM and PP competing pathway contribution. If glucose labelled isotopically either in C1 or C6 positions is added to bacterial suspensions, the amount of label introduced in acetate C2 will depend on the activity of the PP pathway. In fact, when [1- 13 C]glucose is used as the carbon source, part of the 13 C label is lost as CO 2 in the phosphogluconate dehydrogenase step of the PP pathway, whereas the other part of the 13 C label is incorporated into acetate C2 via the EM pathway. On the other hand, when [6- 13 C]glucose is used as the carbon source, all the 13 Clabel is incorporated into acetate C2 via the EM and PP pathways [29]. Even though the described procedure is a simplified flux estimation because other possible CO 2 -liberating reac- tions are neglected [30] it allowed a first estimation of the flux differences between untreated and UV-treated cells. For untreated and both UV1- and UV2-treated cells, the relative activities of the EM and PP pathways in anaerobic conditions were about 91% and 9%, respectively (Table 1). In untreated cells, the initial rate of glucose consumption was 7 nmol per lg protein per min and a similar rate was observed in UV1- and UV2-treated cells. Considering aerobic conditions, only untreated and UV1-treated cells had the capacity to modify metabolic flux through both pathways, the relative activity of the EM and PP pathways Fig. 3. Protein synthesis. Protein synthesis of untreated cells ( • ), UV1-treated cells (j), UV2-treated cells (m), detected by incorpor- ation of [ 35 S]methionine. The detection limit of this method was 0.01 nmol of [ 35 S]methionine per g protein corresponding to protein synthesis of about 10 6 CFUÆml )1 (1% of the initial number of cells). Fig. 4. 13 C-NMR spectra. 13 C-NMR spectra of untreated, UV1- and UV2-treated cells after 4 h of incubation with [1– 13 C] glucose in anaerobic conditions. The glucose anomers, a and b are visible as well as three end products of glucose metabolism, acetate (A); lactate (L) and ethanol (E). Ó FEBS 2003 Are nonculturable E. coli cells alive or dead? (Eur. J. Biochem. 270) 2693 being  44% and 56% (Table 1), respectively. In these cells, an increase of at least fourfold in the rate of glucose consumption was observed (about 30 nmol per lg protein per min). In contrast, UV2-treated cells were unable to respond to variations in aeration conditions. These cells showed a similar metabolic flux through both pathways and rate of glucose consumption in aerobic and anaerobic conditions. In this study, the flux estimation was deter- mined by MNR in cells incubated in nongrowing condi- tions. Nevertheless, the same method applied to E. coli grown anaerobically gives similar flux values (22% by the PP pathway) [31]. Using more comprehensive methods such as GC-MS, it has been confirmed recently, that in growing cells, the oxidative PP pathway is still active under anaerobic conditions and decreases with decreasing oxygen availability [32]. As described above, during glucose incorporation in nongrowth conditions, peroxide was generated in cells. It was expected that both the EM and PP pathway activity would be affected by peroxide, which freely diffuses into cells, harming cell proteins. Furthermore, in vitro assays showed that peroxide inhibited the activity of several E. coli K-12 enzymes such as phosphogluconate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase and acetate kinase (data not shown). However, we obtained the same flux through the EM and PP pathways in experiments where peroxide was degraded or not through the addition of exogenous catalase. This result, along with the evidence of retention of protein synthesis described above, might be indicative of preservation in UV1-treated nonculturable cells of intracellular catalase activity, which prevents intra- cellular damage to cells. Indeed, a homeostatic regulation of intracellular hydrogen peroxide concentration by the pro- duction of intracellular catalase, but in culturable E. coli cells, has already been observed [33]. Discussion Cells were defined operationally as alive or dead depending on the method used to determine cell viability. For example, using the capacity of cell division or elongation as a criterion for bacterial life, cells treated with UV1 and UV2 doses could be diagnosed as dead cells. In contrast, if the capacity to transport or metabolize glucose is used as a criterion of bacterial life, cells treated with UV1 and UV2 doses could be diagnosed as living cells. This study suggests that global information on intracellular stability (protein synthesis, metabolic flux) is needed to define with less ambiguity the physiological state of nonculturable cells. However, in the absence of consensus on the definition of bacteria death (independent of culturability), UV1-treated cells could be diagnosed as simply in a metabolically active state and not as living cells. After addition of nutrients, nonculturable cells obtained by UV1 irradiation maintained the capacity to synthesize proteins and peptidoglycan. They also retained both a high level of glucose transport and the capacity to metabolize glucose at the same rates as those of nontreated cells. Moreover, UV1-treated cells retained the capacity to modify their metabolic flux through the EM and PP pathways after variation of aeration conditions. On the other hand, nonculturable cells obtained by irradiation with the UV2 dose were clearly in a metabolically inactive state. UV2-treated cells not only showed a gradual loss of cell integrity, they also lost the capacity to respond to nutrient addition by cell elongation or protein synthesis and the capacity to modify their metabolic flux in glucose metabo- lism after variation of aeration conditions. UV2-treated cells retained only residual metabolic activity and showed several levels of metabolic imbalance. To clarify the medical significance (when pathogenic) of bacteria that lose culturability, further studies should be performed to examine the persistence of these active cells and their capacity to repair their damage, to produce important metabolites (e.g. toxins) and restart cell division. 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