Process Biochemistry 45 (2010) 706–713 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Involvement of oxidative stress and growth at high cell density in the viable but nonculturable state of Photorhabdus temperata ssp. temperata strain K122 Wafa Jallouli a, Nabil Zouari a,*, Samir Jaoua a,b a b Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box ‘1177’, 3038 Sfax, Tunisia Biological & Environmental Sciences Department College of Arts and Sciences Qatar University, P.O. Box. 2713, Doha, Qatar A R T I C L E I N F O A B S T R A C T Article history: Received 4 September 2009 Received in revised form 5 January 2010 Accepted 7 January 2010 Photorhabdus temperata ssp. temperata strain K122 represents a promising source of bioinsecticide. When cultured in an optimized medium, P. temperata exhibited restricted survival in terms of colonyforming ability on solid medium, which remained lower than the total cell counts. Membrane integrity assessment by flow cytometry showed that almost 100% of P. temperata cells were viable indicating that this bacterium enters in the viable but nonculturable state (VBNC). According to the double staining results, hydrogen peroxide was demonstrated to be responsible of P. temperata VBNC state. Addition of catalase or sodium pyruvate upon the inoculation of P. temperata on agar plates promoted the recovery of nonculturable cells up to 24 h incubation. Further, growth at high cell density enhanced the VBNC state of this bacterium. This should evidenced extracellular signals accumulation involved in quorum sensing mechanism. Elucidation of this state is interesting for both toxicity study and production of P. temperata useful as bioinsecticide. ß 2010 Elsevier Ltd. All rights reserved. Keywords: Photorhabdus temperata Nonculturable cells High cell density Catalase Sodium pyruvate H2O2 Flow cytometry 1. Introduction Microbial ecologists have long recognized that large proportions of the microbial populations inhabiting natural habitats appear to be nonculturable. Indeed, plate counts of cells typically indicate that far less than 1% of the total bacteria observed by direct microscopic examination can be grown on culture media [1]. The simplest explanation of these results was based on those reporting that nonculturable cells are dead, and indeed, this is the basic presumption of standard plate count methods for enumerating readily culturable cells [2]. An alternative explanation has been advanced considering the fact that nonculturable cells entered the ‘‘viable but nonculturable’’ (VBNC) state. A bacterial cell in the VBNC state may be defined as one which fails to grow on the routine bacteriological media on which it would normally grow and develop into a colony, but which is in fact alive [3]. The transition to the VBNC state represents probably a survival strategy that bacteria can adopt under adverse conditions (starvation, osmotic stress, oxidative stress, etc.. . .). Bacteria change their metabolic activity and cellular components, and in some cases, enter a non-growing stage. The existence of the VBNC state has been demonstrated in most Gram-negative bacteria like Echerichia coli [4], Vibrio cholerae [4], Salmonella enteridis [5] and Campylo- * Corresponding author. E-mail address: Nabil.Zouari@cbs.rnrt.tn (N. Zouari). 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.01.007 bacter jejuni [6,7]. P. luminescens and X. nematophilus were also shown to enter in the VBNC state [8]. By entering this state, colonyforming units (CFU) counts of P. luminescens declined to undetectable levels into 6 days following release in river water. In sterile river water, this decline was enhanced, since cells were detectable for only 2 days. In such samples where no colonyforming units were detected, the presence of viable cells was monitored using respiration assay. Indeed, ATP levels per cell were maintained at a constant value until 22 days indicating the presence of metabolically active cells [8]. Moreover, survival of P. luminescens in soil was also restricted since detection of cells was only possible during 7 days. It was reported that CFU counts of P. luminescens (ATCC 29999) fell drastically over a week when introduced into sterilized soil [9]. In our previous study, it was found that when growing in an optimized medium, P. temperata ssp. temperata strain K122, exhibited restricted survival in terms of colony-forming ability on the solid medium [10]. The large difference between total cell count in the medium and cells having the ability to develop into colonies in the solid medium indicated that a large population of cells was unable to form colonies. There is no information on the VBNC state of this bacterium. VBNC response in P. temperata strain K122 was never elucidated, physiologically. P. temperata represents a promising source of bioinsecticide [10]. When released by the nematodes into the hemolymph of the insect host, P. temperata propagate and kill it [11] by insecticidal toxins such as the toxin complexes (Tc) [12] and the ‘‘makes W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 caterpillars floppy’’ (Mcf) toxins [13]. Moreover, Photorhabdus toxins affect the fecundity of Frankliniella occidantalis (western flower thrips) [14]. P. temperata cells were also shown to exhibit oral toxicity independently from nematode larvae against the olive tree pathogenic insect Prays oleae [15], and the sugarcane stalk borer Diatraea saccharalis larvae [16]. Recently, we showed that P. temperata could be a suitable bioinsecticide to control by ingestion the pyralidae Mediterranean flour moth E. kuehniella larvae [10]. The ability of P. luminescens to survive and grow in culture medium has received limited attention, with several studies indicating that cell counts of P. luminescens strains Hm and NC1 reached a maximum at 24 h in 2% PP3 broth, and decreased steadily with about 1% of the cells which were viable at 192 h [17]. The elucidation of VBNC state of this bacterium is interesting for both production of bioinsecticide and toxicity study of VBNC cells. CFU count does not reflect real number of total viable cells and consequently could not determine exactly the number of cells produced in media or those used for evaluation of toxicity. The objective of this study is to identify the major parameters responsible for the inability of P. temperata to form colonies in the plate medium and evidenced also correlation between culturability loss and growth at high cell density in the optimized medium 2. Materials and methods 2.1. Organisms and medium This work was carried out with P. temperata ssp. temperata strain K122, courtesy offered by Dr. Mark Blight (CNRS, GIF sur Yvette, France) and Escherichia coli strain Top 10 (Amersham, France). Two media were used: Lauria–Bertani (LB) medium [18] and the optimized medium (OM medium) ensuring K122 nutritional requirements for biomass and pigments production [10] composed of (g/l): Na2HPO4, 2H2O 1.2, NH4Cl 1.07, KCl 0.35, C6H5O7Na3 2H2O 0.5, Na2SO4 0.28, MgCl2 0.12, CaCl2 0.05, NaCl 0.037, FeCl3
6H2O 0.0017, yeast extract 10, glucose monohydrate 5. Glucose stock solution (20%) was autoclaved separately and later added to the medium. The pH was adjusted to 7 before sterilization. 2.2. Inocula preparation A 48 h old P. temperata ssp. temperata strain K122 was isolated and dispersed into 3 ml of LB medium and incubated overnight at 30 8C. For E. coli Top10, a 24-h-old colony was isolated and dispersed into 3 ml of LB medium and incubated overnight at 37 8C. The culture was used to inoculate the culture medium to start with an initial optical density of 0.025 at 725 nm. Cultures were developed in 500 ml Erlenmeyer flasks containing 85 ml of the (OM medium) in a rotary shaker set at 200 rpm, incubated at 30 and 37 8C for P. temperata strain K122, and E. coli respectively. 2.3. Total direct count and biomass determination Total bacteria were counted by DAPI (40 ,6 diamidino-2-phenylindole, dihydrochloride) (Sigma) staining. Bacterial cells in culture medium were vacuumfiltered onto a polycarbonate black filter (Whatman; pore size 0.2 mm) in a funnel, then incubated with DAPI stock solution (50 mg/ml in sterilized water; final concentration; 5 mg/ml) for 5 min at room temperature in the dark. The solution in the funnel was vacuum-filtered, then the filter was air dried and mounted on glass microscope slides with non-fluorescence immersion oil (Olympus). The total number of bacteria was determined using an Olympus BH2 epifluorescence microscope (100 magnification). Total direct count was determined also by preparing dilutions of each sample and cells were counted microscopically using Thoma counting chamber by a binocular microscope (ZUZI) at 100-fold magnification. Approximately 10–15 cells per square and a minimum of 3 cells for each diluted sample were counted. Biomass was determined by measuring the optical density (OD) at 725 nm with a spectral photometer (Biorad). The cell dry weight (CDW) was determined gravimetrically after centrifugation of 5 ml culture broth at 7000 rpm for 15 min. The cell pellet was washed twice with distilled water and dried at 103 8C for 24 h (NF05-109). The values presented with results are the average of three replicates of three separate experiments for each cultural condition. 2.4. Culturable cell counts Culturable cell counts were determined by counting colony-forming units after different incubation times. 100 ml from each appropriate dilution of the culture sample were plated on LB medium or/and LB medium containing catalase or sodium 707 pyruvate and incubated at 30 8C during 48 h. The values presented with results are the average of three replicates of three separate experiments for each cultural condition. 2.5. Effect of anti ROS agents on culturability of P. temperata P. temperata ssp. temperata strain K122 cells were grown in the OM medium. After different incubation periods, aliquots were sampled and appropriate dilutions were plated onto LB agar and LB agar supplemented with catalase [19] or sodium pyruvate [20]. Catalase-LB agar plates were prepared by surface spreading 100 ml filter sterilized catalase solution (20,000 Units/ml) on 20 ml solidified LB agar medium. LB plates containing 40 mg of sodium pyruvate were prepared by surface spreading 100 ml filter sterilized solution (3.63 M) on 20 ml solidified LB agar medium. Study on the effect of other anti ROS agents was carried out using 100 ml filter sterilized solutions of 100 mM benzoic acid, 600 Units superoxide dismutase (SOD), 25 mM glutathione, 100 mM histidine, or 25 mM thiourea. Colony-forming units were counted after incubation at 30 8C during 48 h. The values presented with results are the average of three replicates of three separate experiments for each cultural condition 2.6. Susceptibility of P. temperata to H2O2 present in the liquid medium Photorhabdus cells were grown in the OM medium to an optical density of 0.2 measured at 725 nm. To 98 ml of culture broth, 2 ml of 100 mM H2O2 was added ensuring a final concentration of 2 mM H2O2 and incubated at room temperature. Aliquots were sampled each 15 min and appropriate dilutions were plated on LB agar and catalase-LB agar. The values presented with results are the average of three replicates of three separate experiments for each cultural condition. 2.7. Susceptibility of P. temperata to H2O2 present in the solid medium In order to assess K122 cells resistance to H2O2 in the solid medium, culture samples were diluted and plated on LB agar medium containing different amounts of a 3% H2O2 (2.5, 5, 10, 20, or 40 ml). The strain E. coli Top 10 was used as control. The values presented with results are the average of three replicates of three separate experiments for each cultural condition. 2.8. Catalase activity of P. temperata strain K122 Cells of P. temperata ssp. temperata strain K122 were incubated in the OM medium and aliquots of 10 ml of culture broth were sampled and cells were harvested by centrifugation at 12,000 rpm during 10 min, and frozen at 80 8C until analyzed for catalase activity. Cells were resuspended in 1 ml Kpi-EDTA buffer (50 mM potassium phosphate, 0.1 mM EDTA, pH 7.8) and lysed by sonication at 4 8C during 3 min. Catalase activity was determined in the supernatant after centrifugation at 15,000 rpm for 30 min, according to the method described by Beers and Sizer [21]. Protein concentrations were determined according to the method of Bradford with bovine serum albumin as a standard [22]. The values presented with results are the average of three replicates of three separate experiments for each cultural condition. 2.9. Flow cytometry Fluorescent measurements were made using Beackman Coulter EPICS XL flow cytometer with 488 nm excitation from an argon-ion laser at 15 mW. Samples were analyzed in the linear photomultiplier gains mode, a total of 10,000 cells being analyzed for each sample at a rate of 800–2000 cells/s. Samples taken from the culture were immediately diluted with PBS buffer pH 7.2 and stained with a mixture of two fluorescent dyes, propidium iodide (PI) (Roche) for the detection of dead bacteria, and dihydrorhodamine 123 (DHR 123) (Invitrogen) for ROS (reactive oxygen species) detection and particularly hydrogen peroxide [23]. The fluorescent dyes were used at a final concentration of 10 mg/ml and 15 mM, respectively. Sample stained by DHR 123 was incubated 30 min at 30 8C in the dark. At 5 min before reading, PI was added to the sample. Fluorescent filters and detectors were all standard with green fluorescence (Rhodamine 123) collected in the FL1 channel (525 nm), and red fluorescence (PI) collected in the FL3 channel (620 nm). All the experiments were repeated at least three times and representative data from single experiments are presented. To determine cell viability by flow cytometry, PI single stained was performed according to Hewitt et al. [24]. Heat stressed cells treated at 60 8C for 5 min and exponentially growing ones were used as positive and negative controls respectively [24]. 2.10. Statistical analysis of results All the results related to determination of total cell counts, OD, CDW, CFU counts, effect of anti ROS agents, resistance to H2O2 and catalase activity were the average of three replicates of three separate experiments. They were statistically analyzed by SPSS software (version 100) using Duncan test performed after analysis of variance (ANOVA). 708 W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 Fig. 1. Entry of P. temperata ssp. temperata strain K122 grown in the OM medium in the viable but nonculturable state. (a) Changes observed in plate counting (*) 108 CFU/ml; direct microscopic counting using DAPI staining or Thoma counting chamber (~) 108 total cell count/ml, and total biomass (^) OD (725 nm); (&) dry cell weight (DCW)  10 (g/l). The values presented are the average of three replicates of three separate experiments. (b) Viability of P. temperata ssp. temperata strain K122 cells growing in the OM medium at 24 h incubation determined by PI single stained dot plot. Sample was analyzed in triplicate and representative data from single experiment are presented. 3. Results 3.1. Evidence of the viable but nonculturable state of P. temperata ssp. temperata strain K122 The strain K122 was cultured in liquid OM medium for 100 h (Fig. 1a). The determination of culturable cell counts by plating adequate diluted samples on the LB solid medium showed that at different incubation times, culturability was always less than the total cell counts determined microscopically. After 24 h incubation, CFU count was only 11% of the total cells, and then declined rapidly starting from 48 h. After such time in the OM medium, P. temperata cells were undetectable by plate counting. However, the total count determined directly either by DAPI staining, or using Thoma counting chamber, was stable (18  108 cells/ml) above 24 h incubation in the OM medium (Fig. 1a). Moreover, the assessment of biomass production by the determination of OD at 725 nm, and CDW showed similar result. PI single stained dot plot, performed at 24 h incubation, showed that almost 100% of cells were PI negatively stained indicating that they are viable cells (Fig. 1b). 3.2. Effect of cell density growth on culturability of P. temperata ssp. temperata strain K122 To study the effect of cell density growth on P. temperata strain K122 culturability, sample (20 ml) was collected and cells were removed by centrifugation (7000 rpm at 4 8C) and the supernatant was put back to the culture. This was performed during the growth of K122 in the OM medium at 14, 37, and 60 h incubation. Results of Fig. 2a showed that by decreasing cell density at 14 and 37 h incubation, total cell counts, determined microscopically, increased after a short lag phase, but remained lower than those obtained in the control culture. No significant increase in total cell counts was observed after removing cells at 60 h incubation. Following decrease in cell biomass at the three incubation times, CFU counts of the remaining cells fell drastically, but increased rapidly in relation to the growth phase reaching a maximum of 80  106, 40  106 and 10  106 cells/ml, corresponding respectively to 4.44%, 2.22%, and 0.55% of the total cells existing in the culture broth (Fig. 2b). On the other hand, results of Fig. 3 showed that, when P. temperata cells were grown in 5-, 10- or 20-folds diluted OM medium, total cell counts decreased down to 2  108, 9  107, and 4.8  107 cells/ml respectively (Fig. 3a). But, it is Fig. 2. Effect of biomass elimination at 14, 36, and 60 h of incubation on growth and culturability of P. temperata ssp. temperata: (^) control culture; (~) culture with low cell density. (a) Total cell count determined microscopically. (b) Culturable cells assessed by counting colony-forming units (CFU) from appropriate dilution of culture broth plated on LB agar. The values presented are the average of three replicates of three separate experiments. W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 709 Fig. 3. Effect of the OM medium dilution on growth and culturability of P. temperata ssp. temperata: (^) cells grown in the OM medium; (&) cells grown in 5-folds diluted OM medium; (~) cells grown in 10-folds diluted OM medium; (*) cells grown in 20-folds diluted OM medium. (a) Total cell count determined microscopically. (b) Culturable cells assessed by counting colony-forming units (CFU) from appropriate dilution of culture broth plated on LB agar. The values presented are the average of three replicates of three separate experiments. obviously clear that CFU counts remained stable for a long period reaching 78 h in 5-folds diluted OM medium, and up to 96 h incubation in 10- or 20-folds diluted OM medium (Fig. 3b). This observation occurred with low culturability of P. temperata strain K122 when plated on LB agar medium, since culturable cell counts did not exceed 25% of the total cells. 3.3. Involvement of secreted metabolites on P. temperata ssp. temperata culturability loss In order to study the involvement of secreted metabolites on P. temperata culturability loss, 20 ml of culture broth were discarded from a culture at three incubation times (17, 40, and 86 h) and replaced immediately by 20 ml of cell-free supernatant harvested at 24 h incubation from a separate culture. For the control culture, 20 ml of culture broth was removed and replaced by 20 ml of a fresh medium. Growth and culturability of produced cells were followed (Fig. 4). It was clear that growth resumed after a lag period of 6 h if the replacement by cell-free supernatant has been performed at 17 h incubation. In contrast, in the control culture, cell counts increased immediately at such time, but a lag period of 1 h, and 3 h appeared if replacement was performed at 40 and 86 h, respectively. At such incubation times, cells were unable to grow in the case of replacements by cell-free supernatant (Fig. 4a). In that case, culturability loss was more enhanced and CFU counts declined even at 17 h incubation (Fig. 4b) although the log phase observed after this time (Fig. 4a). In the control culture, determination of culturable cell counts showed an increase in CFU counts at each log phase induced after substitution of fresh medium. This effect was less pronounced for substitution of fresh medium during stationary phase (40 and 86 h incubation). Similar experiments were performed with heat treated supernatant (80 8C, 5 min) recovered at 24 h incubation from a separate culture (Fig. 4). Results showed that growth of the remaining cells was similar to that obtained without heat treating (Fig. 4a), but their culturability was highly improved, since 30 h incubation, compared to the control culture performed with substitution of culture broth by a fresh medium (Fig. 4b). However, culturable cell counts always remained lower than the direct total cell counts determined microscopically. 3.4. Effect of anti ROS agents on P. temperata ssp. temperata culturability To investigate the inability of P. temperata cells to form colonies on the solid medium, anti ROS (reactive oxygen species) were introduced in the plating medium. 40 mg of sodium pyruvate per plate allowed 100% of cells to form colonies since CFU counts were Fig. 4. Effect of secreted proteins on P. temperata culturability loss growth: (^) culture broth replacement by 20 ml fresh medium; (~) culture broth replacement by 20 ml supernatant (harvested from a culture aged of 24 h) at 17, 40 and 86 h incubation; (&) culture broth replacement by 20 ml heated supernatant at 17, 40 and 86 h incubation. (a) Total cell count determined microscopically. (b) Culturable cells assessed by counting colony-forming units (CFU) from appropriate dilution of culture broth plated on LB agar. The values presented are the average of three replicates of three separate experiments. 710 W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 Table 1 Culturability of P. temperata exposed to 2 mM H2O2. (a) Cells were plated onto LB agar. (b) Cells were plated onto LB agar supplemented with catalase. The values presented are the average of three replicates of three separate experiments. 105 CFU/ml H2O2 (a) 0 min 15 min 30 min 45 min 60 min Fig. 5. Effect of anti ROS addition in LB plates on P. temperata strain K122 culturability. (&) Total cell count determined microscopically; Cells are plated onto LB agar (); LB agar supplemented with: (~) catalase; (^) sodium pyruvate; (&) thiourea; (~) benzoic acid; ( ) SOD; (+) histidine; (*) glutathione. The values presented are the average of three replicates of three separate experiments. almost equal to total cell counts determined microscopically (Fig. 5). This was true only with cells sampled before 24 h incubation. After that, there was a continuous decline to complete loss of culturability. The highest effect of sodium pyruvate was achieved at 40 mg per plate. Higher concentrations, up to 320 mg per plate, did not improve culturability of cells sampled after 24 h incubation (results not shown). The possible involvement of hydrogen peroxide in P. temperata ssp. temperata strain K122 culturability loss was studied. Catalase, an enzyme which breaks down hydrogen peroxide [19], was added to LB plates by surface spreading. Spreading 2000 Units/ml of catalase on LB plates yielded similar counts to those obtained on LB plates containing 40 mg sodium pyruvate (Fig. 5). At higher catalase concentrations, up to 8000 Units per plate, CFU counts were not improved (results not shown). Several ROS inhibitors (600 Units SOD, 25 mM glutathione, 100 mM histidine, 25 mM thiourea, 100 mM benzoic acid) were tested and shown with no effect on the culturability of P. temperata strain K122 on LB plates (Fig. 5). 3.5. Susceptibility of P. temperata ssp. temperata to hydrogen peroxide in the plate medium The sensitivity of P. temperata strain K122 to hydrogen peroxide in the LB solid medium was compared to that of E. coli strain Top 10, used as control. P. temperata cells cultured in the OM medium formed colonies on solid LB medium on which a solution of 3% H2O2 was spread up to 2.5 ml/plate, but did not form colonies on plates supplemented with 5 ml of 3% H2O2. Cells of E. coli grown at the same conditions formed colonies on plates supplemented with up to 20 ml of 3% H2O2, but did not form colonies on plates supplemented with more H2O2. To determine whether H2O2 accumulation, is a result of LB medium preparation [25] or the result of cell metabolism, culturability of P. temperata ssp. temperata cells plated onto LB agar and LB agarose was assessed. Similar CFU counts were obtained in both cases (results not shown). 72 73 70 75 72 105 CFU/ml +H2O2 (a) 70 59 10 0.4 0 105 CFU/ml H2O2 (b) 320 319 317 318 317 105 CFU/ml +H2O2 (b) 321 315 258 214 209 medium containing 2 mM H2O2, even when 2000 Units/ml catalase were added in the plate medium (Table 1). As expected, using catalase at 2000 Units/ml in the solid medium allowed 100% of P. temperata cells to form colonies, with no added H2O2 in the OM medium. Growth was followed in the liquid OM medium supplemented with 2000 Units/ml catalase after 23 h incubation. CFU counts, determined in LB solid medium supplemented with 2000 Units/ml catalase, and total cell counts determined microscopically, were similar to those obtained in the OM medium with no added catalase. When catalase was substituted by sodium pyruvate in the liquid OM medium and in the LB solid medium, similar results were obtained (results not shown). 3.7. Catalase activity of P. temperata ssp. temperata strain K122 Determination of catalase activity of P. temperata cells grown in the OM medium showed that cells from the stationary phase (above 24 h incubation) exhibited similar catalase activity for 96 h incubation in the OM medium (Fig. 6). 3.8. Cell physiology study by flow cytometry Cells analysis by flow cytometry demonstrated that during the growth of P. temperata strain K122 in the OM medium there was a progressive change in P. temperata physiological state with respect to PI/DHR double staining. In the dot plots presented in Fig. 7, DHR+/PI+ stained cells were dead cells with ROS accumulation, while DHR+/PI stained cells were healthy viable cells with ROS accumulation. Finally DHR /PI stained cells were viable cells with no accumulation of ROS. In the culture broth sampled at 24 h incubation, two populations of cells could be identified that differ in intracellular ROS accumulation. All the cells were viable (PI negatively stained). After 48 h incubation, cells analysis by flow cytometry revealed a change in cells physiological state. At this 3.6. Susceptibility of P. temperata ssp. temperata strain K122 to hydrogen peroxide in the liquid medium Culturability assessment of P. temperata ssp. temperata cells showed a decrease in CFU counts after 15 min incubation in liquid Fig. 6. Catalase activity of P. temperata strain K122 grown in the OM medium. The values presented are the average of three replicates of three separate experiments. W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 711 Fig. 7. Double staining PI/DHR dot plots of P. temperata strain K122 cells growing in the OM medium. Samples were taken at: (a) 24 h, (b) 48 h, (c) 72 h. The experiment was repeated at least three times and representative data from single experiment is presented. time, the totality of cells were viable and accumulated ROS (PI / DHR+). After 72 h incubation, the PI/DHR double staining showed that 97.1% of the cells were DHR+/PI , while only 2.63% were DHR+/PI+ (dead cells accumulating ROS). 4. Discussion The VBNC state in Photorhabdus species was first reported by Morgan et al. [8], with evidences that nonculturable Photorhabdus cells living in water are viable cells, retaining a constant ATP level until 22 days of incubation. In the present study, CFU counts of P. temperata strain K122, cultured in the OM medium, decreased dramatically while almost 100% of cells were viable. At 24 h incubation, CFU count was only 11% of the total cell counts, whereas the totality of cells were healthy viable according to the PI single staining. This finding confirms that this bacterium enter the viable but nonculturable state (VBNC). This state was never described for P. temperata ssp. temperata strain K122, but has been demonstrated in numerous bacteria cultured from environmental sources or from starved or cold stored laboratory microcosms such as those described for E. coli [26], Micrococcus luteus [27], V. vulnificus [28]. In batch culture, few bacteria were characterized to enter the VBNC state. Viable counts of E. coli cells started to decline after 48 h incubation in LB medium [29]. Mycobacterium smegmatis cells grown under suboptimal conditions in batch culture, adopted a stable nonculturable state after 3–4 days incubation in stationary phase [30]. Renye et al. [31] demonstrated that Streptococcus mutans grown in a chemically defined medium (FMC) survived for about 3 days. Here, we demonstrated that growth at high cell density is also involved in the enhancement of culturability loss of P. temperata ssp. temperata, since CFU counts of such cells, grown at low cell density in diluted OM medium, remained stable up to 96 h incubation, but were lower than total cell counts, determined microscopically. Moreover, following biomass elimination by removing a fraction of cells growing at high cell density in the OM medium at 14, 37, and 60 h incubation, CFU counts of the remaining cells fell drastically even though they were viable. Vulic and Kolter [29] reported that in LB medium, high cell density underlined viability loss of E. coli strain MG 1655. However, when grown in diluted LB medium, viable counts of E. coli determined by the assessment of CFU counts stay constant for at least 5 days. This was explained by the fact that medium metabolites secreted at high cell density, affect viability. This phenomenon could be related to quorum sensing mechanism exhibited at high cell density [32]. Indeed, many Gram-negative bacteria use quorum sensing mechanism for monitoring their population density and coordinating gene expression in response to changes in cell density [33]. It was reported that genes involved in cell growth and division are down-regulated by quorum sensing in E. coli O157:H7 [34]. In P. luminescens TT01, quorum sensing mechanism regulates more than 300 targets including cpm gene expression responsible for the production of carbapenem antibiotic [35], biofilm formation, virulence, expression of several transcriptional regulators, oxidative stress resistance [32]. But quorum sensing was never reported to be correlated to the decrease of culturability in P. luminescens. The investigation of the involvement of secreted metabolites in the VBNC state of P. temperata, clearly showed that the substitution of culture broth at different incubation times by cell-free supernatant harvested from a separate culture, accelerated the VBNC response and decreased the growth capacity although the log phase observed after 17 h of incubation. These findings could confirm the involvement of extracellular signals accumulated in the supernatant after growth at high cell density in decreasing cell capacity to grow and to form colonies in the solid medium. In this case, the growth phase could not be the only factor affecting the culturability, but factors related to nutrient concentrations and accumulated extracellular metabolites might contribute to such loss of culturability. Heat treatment of the supernatant led to culturability count improvement since 30 h incubation. Thus, generation of P. temperata ssp. temperata cells, no longer able to form colonies in the solid medium after 24 h incubation, could be due to growth at high cell density, leading to the production of extracellular signals affecting culturability. Similarly, Vulic and Kolter [29] have reported the implication of extracellular signals produced at high cell density growth in E. coli viability loss. Finally, we demonstrated that extracellular signals accumulation cannot be interrupted by the addition of ethanol or butanol in the culture broth of P. temperata after 24 h incubation (results not shown). Such alcohols are known to delay viability loss of E. coli [29]. On the other hand, the involvement of reactive oxygen species in the VBNC state of P. temperata, was clearly showed since the supplementation of LB plates with either catalase (2000 Units/ml) or sodium pyruvate, degrading hydrogen peroxide, allowed 100% of P. temperata cells to form colonies in the plate medium. This clearly evidenced that hydrogen peroxide is involved in the inability of P. temperata cells to form colonies in the LB solid medium. The use of other ROS inhibitors like SOD, glutathione, histidine, thiourea, and benzoic acid in the plate medium, did not enhance culturability, indicating again that it is hydrogen peroxide which is involved in culturability loss of P. temperata strain K122 (Fig. 5). Several studies have provided evidences for the involvement of reactive oxygen species in the VBNC response of some Gram-negative bacteria [36,37]. Development of a hydrogen peroxide sensitive population of culturable cells has been observed 712 W. Jallouli et al. / Process Biochemistry 45 (2010) 706–713 in numerous bacteria like E. coli, and V. vulnificus [37]. Susceptibility of P. temperata strain K122 to hydrogen peroxide in the plate medium revealed that such cells were hypersensitive to H2O2 contrarily to E. coli Top 10 used as control. Similarly, in liquid medium containing 2 mM H2O2 P. temperata ssp. temperata strain K122, underwent a decrease in culturability after 15 min, even when catalase was added in the plate medium. Such decrease in CFU count was confirmed by monitoring membrane integrity (result not shown). In that case the PI single stained dot plot demonstrated that 35% of the cells were dead by adding H2O2. In contrast, Kong et al. [38] reported that by using 2 mM H2O2, no effect on wild type cells of V. vulnificus at room temperature was observed, while the oxy R mutant (containing very low catalase activity) underwent a marked decrease in culturability. Similar results were reported with catalase deficient mutant of Helicobacter pylori which is hypersensitive to H2O2 [39]. On the other side, the continuous decline of CFU counts after 24 h incubation could be explained by accumulation of extracellular signals, involved in quorum sensing, exhibited at high cell density. Indeed, when P. temperata cells were grown in diluted OM medium and plated on LB agar supplemented with catalase or sodium pyruvate, 100% culturability was recovered up to 96 h incubation, which is not the case of growth in the not diluted OM medium. Hydrogen peroxide degrading agents added to the liquid culture broth of P. temperata at 23 h incubation (growth at high cell density) did not improve culturability of plated cells. Indeed, this agrees with our findings that catalase activity (40 Units/mg proteins) was stable during the incubation period over 24 h incubation. This evidenced sensitivity of P. temperata cells to ROS agents and to growth at high cell density. This conclusion is supported by the findings of Krin et al. [32], who reported that in P. luminescens TT01, quorum sensing mechanism controls ROS protection by increasing oxidative stress resistance. Stability of catalase activity during the growth of P. temperata in the OM medium indicates that cellular catalase failed to eliminate ROS which result in much ROS accumulation. Similar results were reported during methanol metabolism by Pichia pastoris [40]. Assessment of P. temperata ssp. temperata culturability on LB agar and LB agarose which should contain almost 244 and 76.5 ng/ ml H2O2, respectively, according to Vulic and Kolter [29], showed similar culturable cell counts in both cases, suggesting that inability of P. temperata to form colonies onto LB agar was not due to H2O2 which should appear during sterilization in medium but probably to accumulation of H2O2 as a ROS agent by cells during metabolism. In contrast, Kong et al. [38] have reported that colony formation of V. vulnificus was highly inhibited on HI agar, but was not inhibited on HI agarose. Such a consequence was less dramatic in the liquid medium, as hydrogen peroxide should diffuse away from the cells, in contrast to cells which are plated, were the toxic ROS would remain close to cells and make them enable to form colonies [38]. Furthermore, on the basis of flow cytometric analysis, different physiological state of P. temperata cells could be distinguished during the growth in the OM medium. At 24 h incubation, only 13.5% of the cells were viable and do not accumulate H2O2 during metabolism corresponding to cells which are able to form colonies after plating in the LB solid medium. At 48 h incubation, almost all cells were viable and accumulated ROS leading to the inability to form colonies. However, at 72 h incubation, 2.63% of cells are dead by accumulation of ROS indicating that high oxidative stress induces cell damage and can even lead to cell death. In this study, we demonstrated that the bacterium P. temperata enters the VBNC state. Enhancement of this state was showed to be due to the accumulation of extracellular signals produced at high cell density. In this work, we also demonstrated that inability of P. temperata cells to form colonies is caused by hydrogen peroxide accumulation during metabolism. 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