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Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but... Purpose Aquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable ( VBNC) state, as a consequence of starvation in seawater at low tempera- tures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resus- citate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress. Methods Cell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating. Results The cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide. Conclusions The timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus. Keywords Dormancy, Resuscitation, Culturability, Viability, Hydrogen peroxide, Catalase, Flow cytometry, Oxidative stress *Correspondence: Elena Crotti elena.crotti@unimi.it Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 2 of 13 As mentioned above, several stress conditions, such as Introduction starvation and incubation at nonoptimal temperatures, Under unfavourable environmental conditions, micro- can induce the VBNC state. Within Vibrio genus, the bial cells may enter in a physiological reversible state appearance of VBNC cells has been intensively studied in of low metabolic activity known as quiescence or dor- the species V. vulnificus, shortly after incubation in cold mancy (Lennon and Jones 2011). This represents an seawater; cell resuscitation from this state has been dis- adaptive strategy for long-term survival of bacteria fac- covered upon exposure to higher temperatures prior to ing unfavourable environmental conditions (Ayrapetyan plating (Oliver 2005). The loss of culturability induced et  al. 2015). Examples of cellular processes leading to by starvation and low temperatures has been associated dormancy are sporulation in gram-positive bacteria with significant decrease in the expression of the peri - and the formation of persister cells or viable but non- plasmic catalase KatG and of catalase activity, which culturable (VBNC) cells in gram negatives (Lennon and affect the cell resistance to oxidative stress (Kong et  al. Jones 2011). Specifically, VBNC state is a dormant physi - 2004). Therefore, the growth of VBNC cells in rich media ological condition of resistance against stress factors, in that contain a certain amount of hydrogen peroxide, which bacterial cells are alive but no longer able to grow e.g. brain heart infusion (BHI), is hampered (Kong et  al. on laboratory media; they however retain the capabil- 2004). On the other hand, the quorum-sensing molecule ity to restart growth upon “resuscitation” under certain AI-2, as well as cell-free supernatants from V. vulnificus, favourable environmental conditions (Lennon and Jones Vibrio parahaemolyticus or Escherichia coli, can trig- 2011; Oliver 2005). Stress conditions, such as starvation ger the resuscitation process in both seawater samples and incubation at nonoptimal temperatures, can induce and experimental VBNC microcosms (Ayrapetyan et  al. the VBNC state in a wide range of bacteria, including 2014). It has been proposed that increased amounts of human pathogens (Oliver 2005, 2010). VBNC cells resist AI-2 stimulate the expression of the stress-related alter- to multiple antimicrobial treatments (e.g. antibiotics) and nate sigma factor rpoS (through the action of LuxR), easily bypass microbiological safety controls due to their restoring the expression of catalase (katG) and the cul- lack of culturability, representing thus a potential threat turable state (Ayrapetyan et al. 2014). However, studies in for human health (Nowakowska and Oliver 2013; Li et al. other Vibrio species suggest that the mechanisms behind 2014). For instance, wastewater disinfection treatments the loss of culturability could be far more complicated can induce the VBNC state (Cai et  al. 2021). The reuse (Li et  al. 2014). In V. cholerae, the induction of VBNC of such treated wastewater in agriculture and the lack of state and the following resuscitation cannot always be detection of VBNC cells, which is largely based on culti- obtained as for V. vulnificus, but depending on the time vation approach, represent a risk not only for the spread spent under the VBNC state, other factors should be pro- of pathogens but also of the antibiotic resistance genes vided, such as catalase or human colon cells extracts, or (ARGs) the dormant cells may harbour (Cai et  al. 2021; the cells should be co-cultured with colon cells, to res- Riva et  al. 2020). Furthermore, we have to consider that cue from the VBNC state (Imamura et  al. 2015). A cur- a number of bacteria for open-field environmental appli - rent view postulates that in V. cholerae, the VBNC state cations are in most cases poorly characterized in rela- is a condition that continuously changes over time, rather tion to their potential VBNC state that may be induced than a single-gene property (Imamura et  al. 2015). This under harsh conditions they may experience during view is supported by the occurrence of transcriptional their application in the field (Prakash et  al. 2013). Thus, changes in the first 12 h of incubation of V. harveyi in improving the knowledge on the mechanisms leading to seawater without nutrients (Montánchez et  al. 2014), or removing the VBNC state is necessary for both human with cells (which are still culturable) becoming smaller and animal health and to improve the biotechnological and adapting their gene expression (Kaberdin et al. 2015). exploitation of microorganisms. The reduction in size and the decrease of DNA quantity VBNC state has been extensively studied in the gram- have been extensively described also in VBNC cells of negative, aquatic bacteria of the genus Vibrio (Oliver V. parahaemolyticus (Falcioni et  al. 2008; Wagley et  al. 2005), which include waterborne or foodborne pathogens 2021). (e.g. Vibrio vulnificus, V. cholerae, Vibrio haemolyticus). The oxidative stress response has been repeatedly asso - Such bacteria inhabit marine, freshwater and estua- ciated with the VBNC state (Li et  al. 2014), though the rine environments and can be associated with shellfish role of hydrogen peroxide is still under debate. On one and other aquatic animals (O’Neil et  al. 1992; Lutz et  al. hand, VBNC cells of clinical strains of V. vulnificus have 2013). The current warming of the sea surfaces has been been reported to be highly sensitive to hydrogen per- connected with the spread of this genus and the emer- oxide, proposed as the main reason for the inability to gence of related human diseases (Vezzulli et al. 2016). grow on BHI (Kong et al. 2004; Nowakowska and Oliver P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 3 of 13 2013). On the other hand, VBNC cells of an environmen- preliminary washing, the gills were smashed in ster- tal strain of V. vulnificus have been shown to be more ile saline (9 g/L NaCl), and serial dilutions were plated resistant to this stress than the log-phase cells (Nowa- on Marine Agar (Difco) plates added with 100 μg/ml kowska and Oliver 2013). During the initial stages of cycloheximide to avoid eukaryotic growth. Plates were starvation, V. harveyi upregulates the genes responsible incubated aerobically at 30° C for 24–48 h. Once the for the oxidative stress resistance, detoxifying the endog- growth occurred, random colonies were selected and enous hydrogen peroxide generated as a side product of purified. The 16S rRNA gene of the isolates was ampli - the lipid turnover and repairing the damaged molecules fied through PCR using primers 27F (5′-TCG ACA TCG (Kaberdin et al. 2015).TTT ACG GCG TG-3′) and 1492R (5′-CTA CGG CTA CCT Considering cell response in relation to the oxidative TGT TAC GA-3′) (Mapelli et  al. 2013) and sequenced at stress caused by hydrogen peroxide, limited informa- Macrogen (South Korea). The obtained sequences were tion is available about changes occurring in VBNC and aligned against the curated database of EZBioCloud resuscitated cells. The aim of this study is thus to assess (https:// www. ezbio cloud. net/; Yoon et  al. 2017) to con- the relationship between cell phenotypic changes and firm their affiliation to Vibrio spp. Sequences were then response to oxidative stress of different Vibrio strains deposited in the ENA database under the accession num- during the transition to and from the VBNC state. To this ber ERZ12298639. Glycerol stocks were stored at −80 °C. aim, a first part of the work was dedicated to character - ize the phenotypic changes of 4 Vibrio strains (namely, Preparation of Vibrio microcosms and induction the laboratory strain Vibrio campbellii BAA-1117 and of the VBNC state the 3 environmental strains of Vibrio hepatarius UU21, Strains were grown overnight at 30 °C in tryptic soy broth Vibrio japonicus UU24 and Vibrio japonicus M5) during added with 25 g/L NaCl (TSB-NaCl). Bacterial cultures the incubation in cold seawater microcosms under starv- were used to inoculate (1% v/v) fresh TSB-NaCl broth. ing conditions. In the second part of the work, 2 out of As soon as the cultures reached the exponential growth 4 strains (i.e. Vibrio campbellii BAA-1117 and Vibrio phase (optical density, OD, 0.3–0.5), cells were washed japonicus UU24) were further monitored until the cells twice in sterile artificial sea water (ASW). To prepare completely entered in VBNC state, and their resuscita- ASW, 40 g of sea salts (Sigma-Aldrich, Milan, Italy) was tion was thus induced by incubation at 30 °C for differ - dissolved in 1 L of deionized water, and the solution was ent times before plating. These two strains were selected filtered with 0.22 μm pore size filters (Millipore, Milan, because the former was the quickest to enter into the Italy). Cells were counted using a Thoma chamber and VBNC state, while the latter was the one with a distin- diluted in sterile ASW to reach the concentration of guishable VBNC kinetic among the other isolates. In approximately 1 × 10 cells/ml in glass vials. Microcosms these strains, the relationship between resuscitation and prepared in ASW and thus, in starvation condition, were the oxidative stress caused by hydrogen peroxide was also stored at 4 °C in the dark; starvation and low tempera- investigated. ture were applied to induce the VBNC state. To check the entrance in VBNC state by cultivation, culturability trials Materials and methods were carried out by drop-plate technique. Briefly, cultur - Isolation and molecular identification of Vibrio spp. strains able cells collected from microcosms, initially shaken to One laboratory strain and three environmental isolates of allow the aggregate breakage and sample homogeniza- Vibrio spp. were used in the study. The laboratory strain tion, were counted in 10 μl of decimally diluted tripli- was Vibrio campbellii BAA-1117 (luxN::tn5Kan), with a cated aliquots plated on TSB-NaCl plates (added with 15 mutation in the LuxN receptor, which is involved in the g/L agar), incubated overnight at 30 °C. Viability staining quorum sensing mediated by homoserine lactones. Strain was assessed by flow cytometry, as reported below. ™ ™ BAA-1117 is a derived mutant of strain BAA-1116 that was reclassified as Vibrio campbellii according to Assessment of VBNC state and resuscitation microarray comparative genome hybridization and mul- In order to verify that the observed increase in cultur- tilocus sequence analysis (Lin et al. 2010). Three environ - able cells was due to resuscitation and not to the growth mental strains, UU21, UU24 and M5, were isolated from of an originally undetectable non-VBNC cell population, the gills of the fiddler crab Tubuca urvillei, sampled in we applied the procedure adapted by Whitesides and Oli- 2011 in Gazi Bay mangrove forest (−4.416, 39.51; Kenya). ver (1997). An aliquot of the microcosm was shaken and For bacterial isolation, the crab gills were dissected in serially diluted, and a sample of each of the dilutions, as proximity of a Bunsen burner flame, placed in a 2-ml well as of the undiluted sample, were plated (100 μl) on tube where the gills were washed in sterile physiological TSB-NaCl agar plates to verify the absence of growth. We solution (9 g/L NaCl) to remove external particles. After incubated the remaining dilutions overnight at 30 °C to Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 4 of 13 induce resuscitation, since temperature upshift has been solution was poured on each TSB-NaCl agar plate. The described to resuscitate VBNC cells, e.g. in V. vulnifucus plates were dried in a biosafety cabinet and immediately (Oliver 2005). The following day, we plated (100 μl) each inoculated with 100 μl of VBNC cells. The number of col - dilution on TSB-NaCl agar plates, and, after a further onies was evaluated after an overnight incubation at 30 overnight at 30 °C, the number of colonies was assessed. °C. Assuming that, if the increase in CFU counts was due to The production of catalase by the four Vibrio strains the growth of originally undetectable non-VBNC cells was visually assessed using the straightforward method rather than to the resuscitation, at least one non-VBNC described by Iwase et  al. (2013). Briefly, a calibration cell should have been initially present in each dilution curve was constructed with defined units of catalase in from which we cultivated cells, we then calculated the a solution made of 1% (v/v) Triton X-100 and undiluted theoretical initial concentration of these non-VBNC cells hydrogen peroxide (30% v/v); the height of oxygen-form- in the undiluted sample and verified that this value was ing foam was measured using a ruler. Samples of bacte- higher than our detection result (Whitesides and Oliver rial cells (10 mg), prepared using overnight cultures, were 1997). resuspended in physiological saline and subjected to the For other experiments of resuscitation from the VBNC same procedure (Iwase et al. 2013). state, we incubated the bacterial aliquots at 30 °C for different time spans prior to plating on TSB-NaCl agar Growth in the presence of hydrogen peroxide of VBNC plates and let them grow overnight. or resuscitated cells To determine the sensitivity of the strains to hydrogen Viability staining and flow cytometry peroxide, growth curves of standard-growing cells of Two aliquots of 100 μl were collected from each micro- the strains BAA-1117 and UU24 were obtained using ™ ® cosm and stained separately with SYTO 9 or propidium the Infinite 200 PRO microplate reader (TECAN Italia iodide (PI) from the LIVE-DEAD BacLight kit (L7012, S.r.l, Milan, Italy). With the term “standard-growing cells/ Life Technologies, Milan, Italy). Both live, and dead bac- cultures”, we identified cells of bacterial strains routinely terial cell membranes are permeable to SYTO 9, which grown in the lab (i.e. not induced in VBNC state or resus- binds DNA and RNA. PI only enters dead or damaged citated from VBNC state). Ten microlitres of the cell sus- cells. The staining solutions were prepared by adding pension (10 cell/ ml) was inoculated in 90 μl TSB-NaCl 3 μl of each dye to 1 ml Milli-Q water, according to the medium in each well of a 384-well microplate using an manufacturer protocol; then, 100 μl of the staining solu- ep-Motion liquid handler (Eppendorf, Milan, Italy). Dif- tions was mixed with each microcosm aliquot. Samples ferent concentrations of hydrogen peroxide (from 0 to 0.6 were analysed in flow cytometry using a BD Accuri C6 mM) were prepared by adding to each well 2 μl of hydro- device (BD Biosciences, Milan, Italy). For each sample, gen peroxide solution at the appropriate concentrations 30,000 events were acquired, and a non-stained sample diluting 30% (v/v) hydrogen peroxide (Sigma-Aldrich, from each microcosm was always included in the analy- Milan, Italy) in deionized water. The microtiter plates sis. The excitation laser was set at 488 nm. Green fluores - were incubated at 30 °C for 24 h, and the optical density cence signal of SYTO 9 was detected with a 530/30 nm was measured every 15 mins. We calculated the growth filter, while red fluorescence from PI was measured with curves by averaging the OD measurements of 8 replicates a 585/40 nm filter. In our experimental setup, the use of and subtracting the average OD value of negative con- PI to trace dead cells based on red fluorescence was not trols (non-inoculated wells). We performed an end-point useful; we could conversely detect a green fluorescent analysis counting the number of wells showing the bacte- stain using PI (see “Results” section). The obtained data rial growth at the end of the incubation time. were analysed using BD Accuri C6 Plus software ver- The capability of cells from microcosms to grow in the sion 1.0 (BD Biosciences, Milan, Italy). Flow cytometry presence of hydrogen peroxide was monitored in micro- analyses were used to evaluate phenotypic characteristics titer plates using the protocol reported above. Specifi - of Vibrio strains in VBNC state and after resuscitation. cally, we considered (i) cells collected from microcosms at day 3, before the induction of the VBNC state (inocu- Plates treated with catalase and determination of strains’ lum: 10 μl; hydrogen peroxide concentrations from 0 to catalase production 0.600 mM), (ii) VBNC cells (inoculum: 10 μl; hydrogen Catalase from bovine liver (C9322) was purchased from peroxide concentrations from 0 to 0.250 mM) and (iii) Sigma-Aldrich (Milan, Italy), dissolved in phosphate resuscitated VBNC cells (inoculum: 10 μl; hydrogen per- buffer pH 7 at the concentration of 1 mg/ml and filter oxide concentrations from 0 to 0.250 mM), following the sterilized. In order to obtain the same catalase activity per incubation at 30 °C for different resuscitation times. plate as described by Kong et al. (2004), 1 ml of catalase P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 5 of 13 Resuscitation after stress hydrogen peroxide isopropanol demonstrated that in our experimental Two-millilitre aliquots of the microcosms were prepared setup, PI was unable to confer a red fluorescence to the in sterile tubes. To each one, the appropriate volume of dead cells in the microcosms. It is noteworthy to remark hydrogen peroxide/water solution was added to reach that Vibrio cells, grown in routine laboratory conditions the final hydrogen peroxide concentration of 0.007 mM, and killed by heat or isopropanol, showed red fluores - 0.02 mM and 0.05 mM. After overnight incubation at 30 cence with PI staining, highlighting that the lack of red °C, an aliquot of cell suspension was plated, while a sec- fluorescence for the PI-treated killed cells was specific for ond one was treated with catalase (C9322 Sigma-Aldrich; cells collected from microcosms. Moreover, no green flu - adding 25 μl of catalase solution to 1 ml aliquot: stock 1 orescence signals were detected in PI-treated killed cells, mg/ml) and incubated at 30 °C until plating the follow- demonstrating that the PI-green fluorescence was related ing day. Cell phenotypic characteristics were analysed by to live cells. flow cytometry, as reported above. In V. campbellii BAA-1117 , all the cells showed increased value of green fluorescence upon staining with Results PI already at day 33, while for the other strains, two pop- Starvation in cold seawater induces phenotypic changes ulations appeared: one with lower size cells and higher Vibrio strains isolated from the mangrove crab gills were green fluorescence and the other with bigger cells but less identified as Vibrio hepatarius UU21, Vibrio japonicus fluorescent upon staining with PI (Fig. 1I–L; Fig. S2). UU24 and Vibrio japonicus M5 by partial 16S rRNA gene Microcosms of V. campbellii BAA-1117 and V. sequencing and alignment against the curated database japonicus UU24 were monitored until they completely EZBioCloud (Table  1; Fig. S1). To monitor phenotypic entered into VBNC state (Fig.  1). Strain BAA-1117 changes in the cells subjected to starvation and cold completely lost the capability to generate colonies on stresses, we analysed, by flow cytometry, the cells from TSB-NaCl plates at day 53 while strain UU24 at day 108 microcosms at different times during a time span of 50 (Figs.  1M and 2N). VBNC-induced cells of the 2 strains days, upon staining with the LIVE-DEAD BacLight kit; BAA-1117 and UU24 showed, by flow cytometry, cell at each analysed time point, we plated the cells to also populations with smaller size, lower DNA content and assess their culturability. higher green fluorescence upon staining with PI at days For all the strains, we observed the same trends, 50 and 124 of incubation, respectively (Fig. 1; Fig. S2). although with a different timing. In all the microcosms, a cell shrinkage as decreasing of the FSC-A parameter was Resuscitation completely reverted the cell phenotype observed by flow cytometry (Fig.  1A–D), while in case of for strain BAA‑1117 V. campbellii BAA-1117 , this process was already com- To resuscitate the cells, we applied the protocol described pleted at day 33, and for strains V. hepatarius UU21 and by Whitesides and Oliver (1997) incubating an aliquot of V. japonicus UU24 and M5, we observed a dwarf popu- the microcosm at 30 °C overnight; plating the cell sus- lation at day 47/50, even if large cells were still present pension after this treatment resulted in colony growth (Fig.  1A–D; Fig. S2). Similarly, the green fluorescence on TSB-NaCl agar medium. In order to demonstrate that signal detected upon staining with SYTO 9, which is the observed colonies appeared after resuscitation rather proportional to DNA abundance into the cells, decreased than being related to the growth of a previously undetect- rapidly in V. campbellii BAA-1117 and more slowly in able fraction of non-VBNC-induced cells (i.e. to verify the other strains (Fig. 1E–H). that all the cells included into microcosms were in VBNC Concomitantly to the reduction in size and DNA quan- state), we serially diluted the VBNC-induced microcosm tity, we recorded an upshift of the green fluorescence prior to incubation at 30 °C and still in starvation con- upon staining with propidium iodide (PI) in the dwarf ditions, and we verified that cells resuscitated in all the cells or the smallest fraction of cells (in size) (Fig.  1I–L; dilutions. Dilutions were indeed plated immediately to Fig. S2). Experiments with cells artificially killed by verify the absence and presence of CFUs, respectively, heat shock (10 min at 100 °C) or by adding 50% (v/v) before and following the resuscitation process at 30 °C. Resuscitation occurred in all dilutions incubated at 30 °C, and in the case of V. campbellii BAA-1117 , the final Table 1 Identification of the isolates obtained in the study colony counts reached levels which reflected the dilution applied. For instance, for this strain after 48 h of resusci- Isolate EZBioCloud type strain 4 3 2 tation, we counted ~9 × 10 , 9 × 10 and 9 × 10 cells/ UU24 Vibrio japonicus JCM 31412 ml in the dilutions 1:100, 1:1000 and 1:10,000, respec- UU21 Vibrio hepatarius LMG 20362 tively (Whitesides and Oliver 1997). Assuming that they M5 Vibrio japonicus JCM 31412 can be grown from a single non-VBNC parental cell, Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 6 of 13 Fig. 1 Change in size, fluorescence and culturability of Vibrio spp. strains entering in VBNC state. Changes detected by flow cytometry. A–D Cell size (FSC-A). E–H DNA quantity (green fluorescence (FL1-A) of cells stained with SYTO 9). I–L Green fluorescence (FL1-A) of cells stained with propidium iodide (PI). Over time, the cell size decreases, as well as the DNA quantity, which is proportional to the green fluorescence of SY TO 9; concomitantly, the green fluorescence induced by PI increases. M–N Decrease in culturability during incubation at 4 °C, according to the CFU numbers detected by plating cells on solid medium. CFU counts are reported as L og (CFU + 1). A, E, I and M V. campbellii BAA-1117 strain. B, F, J and N V. japonicus UU24 strain. C, G and K V. hepatarius UU21 strain. D, H and L V. japonicus M5 strain. Black line: microcosm at 3 days. Green line: microcosm at 50 days. Purple line: microcosm at 124 days there should have been in the original microcosm at least trend of resuscitation in the case of V. japonicus UU24: 9 × 10 culturable cells/ml, which were not detected in we were still able to count the resuscitated cells in the the plating before the treatment. We observed a similar more diluted samples, but we noticed a confluent growth P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 7 of 13 Fig. 2 Change in size, fluorescence and culturability of Vibrio spp. strains exiting from VBNC state. Resuscitation observed by flow cytometry. A–B Cell size (FSC-A). C–D DNA quantity (green fluorescence (FL1-A) of cells stained with SYTO 9). E–F Green fluorescence (FL1-A) of cells stained with propidium iodide (PI). Changes detected before for cell size, DNA quantity and propidium iodide fluorescence (Fig. 1) are reverted in a different extent in the two strains. Resuscitation detected by culturability. G–H Increase in culturability, according to the CFU numbers detected plating cells on solid medium, after the incubation at 30 °C. The process of resuscitation happens exponentially. Counts (CFU/ml) are reported as L og (CFU + 1). A, C, E and G V. campbellii BAA-1117 strain. Black line: no incubation at 30 °C. Green line: after 17 h of incubation at 30 °C. Purple: after 21 h of incubation at 30 °C. B, D, F and H V. japonicus UU24 strain. Blue line: no incubation at 30 °C. Black line: after 1 h of incubation at 30 °C. Green line: after 24 h of incubation at 30 °C. Purple line: after 64 h of incubation at 30 °C of cells by plating those less diluted ones for which it was showed a decrease of fluorescence (Fig.  2 A, C, E; Fig. supposed to find single colonies; no cell colonies were S3). Concomitantly, the green fluorescence upon staining however detected in the plating before the resuscitation with SYTO 9 increased (Fig. 2C). The same reversal was treatment. Nevertheless, it remains to be elucidated why observed in V. japonicus UU24, but was not completed in strain UU24 showed a confluent growth when those dilu - the monitored time (Fig.  2B, D, F; Fig. S3); the PI-green tions were resuscitated. fluorescence gradually decreased (Fig.  2F), but cells were Resuscitation of V. campbellii BAA-1117 and V. still distributed in two populations of different fluores - japonicus UU24 was monitored over time. Microcosms’ cence after 64 h (Fig. S3). DNA quantity and cell size did aliquots were incubated at 30 °C along different times, not increase significantly (Fig. 2B and D). after which the cells were observed by flow cytometry and plated (Fig. 2G–H). In both strains, the CFU increase Catalase induced the resuscitation of VBNC cells was correlated to the time of incubation at 30 °C (resus- According to Kong and co-workers (2004), we veri- citation time) with an exponential relationship, until fied if the treatment of TSB-NaCl plates with catalase CFU number reached a plateau. However, flow cytom - could restore the culturability of VBNC cells. This was etry revealed a different behaviour of the two strains. carried out with aliquots of cells from the microcosms In V. campbellii BAA-1117 , the decrease in size and of V. japonicus UU24 at 179 days and V. campbellii DNA quantity and the increase in PI-green fluorescence BAA-1117 at 31 days. Although the latter was not yet resulted completely to revert after resuscitation with the completely in VBNC state, the use of catalase greatly process completed between 17 and 21 h (Fig. S3). After improved the culturability of the cells in both cases. 17 h, two populations were still detectable: one smaller However, the number of colonies obtained with this pro- in size and with an increased fluorescence upon stain - cedure was 100 or 1000 fold lower than the number of ing with PI and one larger in size (resuscitated) which colonies obtained by resuscitation at 30 °C (Fig. 3). Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 8 of 13 Fig. 3 Culturability of V. campbellii BAA-1117 (light grey) and V. japonicus UU24 (dark grey). Different treatments were considered: (i) after resuscitation on TSB-NaCl agar plates, (ii) without resuscitation on TSB-NaCl agar plates treated with catalase and (iii) without resuscitation on TSB-NaCl agar plates. Cells from V. campbellii BAA-1117 microcosm were not yet completely in VBNC state (31 days). Cells from V. japonicus UU24 microcosm were in VBNC state (179 days). Use of catalase greatly improved the culturability of the cells for both strains. Significant differences were observed among treatments for each strain (t-test, p < 0.05). Counts (CFU/ml) are reported as L og (CFU + 1) VBNC cells are sensitive to hydrogen peroxide in the presence of hydrogen peroxide concentrations To assess the capability of VBNC cells to grow in the from 0.05 to 0.25 mM (Fig.  4). According to our data, presence of oxidative stress, we first challenged stand - the capability to cope with oxidative stress was gradu- ard-growing cells with different concentrations of hydro - ally acquired with the resuscitation time. After 7–16 gen peroxide (Fig. S4). We noticed that the response to and 5–20 h of resuscitation, respectively, V. campbellii hydrogen peroxide was discrete, which means that only BAA-1117 and V. japonicus UU24 displayed a behav- in some wells we could observe bacterial growth. The iour similar to non-VBNC cells. After 20 h of resusci- growth rate could be also influenced by the presence of tation, V. campbellii BAA-1117 resulted even more hydrogen peroxide (i.e. we observed a delay of growth in resistant to oxidative stress, growing in all the wells the presence of certain concentrations of hydrogen per- containing 0.1 mM hydrogen peroxide (Fig. 4). oxide, as shown in Fig. S5). Thus, to assess the strains’ response to hydrogen peroxide, we counted the number of wells in which strains’ growth was observed. Standard- Hydrogen peroxide prevents resuscitation by damaging growing cells of V. campbellii BAA-1117 and V. japoni- cells that cannot be rescued by catalase in the plate growth cus UU24 showed a different sensitivity to hydrogen medium peroxide. Strain BAA-1117 growth failed in 25% of the To further investigate the relationship between resus- wells added with 0.1 mM hydrogen peroxide, and it was citation and sensitivity to hydrogen peroxide, we added consistently inhibited with 0.2 mM hydrogen peroxide hydrogen peroxide (0.007, 0.02 and 0.05 mM) to micro- (Fig. S4). Conversely, strain UU24 showed a higher resist- cosms’ aliquots prior to the incubation of VBNC cells at ance to oxidative stress induced by hydrogen peroxide: 30 °C for resuscitation. According to Kong et al. (2004), at 0.1 mM hydrogen peroxide, it always grew as good as a hydrogen peroxide concentration of 0.007 mM, which in the absence of hydrogen peroxide, while 0.225–0.250 is present in the standard cultural media, is enough mM hydrogen peroxide could prevent the growth of the to prevent cell growth. We verified that the 0.007 mM strain in 25% of the wells (Fig. S4). This behaviour was concentration prevented the resuscitation (culturabil- observed for cells collected from the microcosms at day 3 ity) of VBNC cells of V. campbellii BAA-1117 , while (i.e. cells not yet entered into VBNC state). for V. japonicus, UU24 concentrations higher than 0.02 Conversely, when in VBNC state, cells of both strains mM were necessary. Cells did not show an increase in could not grow in the presence of any of the hydrogen size nor anyone of the changes described before for peroxide concentrations applied. Sensitivity to hydro- the resuscitation process (Fig. 5). To verify if they were gen peroxide was then checked by exposing the VBNC still capable to resuscitate after relieving the oxida- cells at different periods of resuscitation at 30 °C and tive stress, we added catalase and plated the bacterial P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 9 of 13 Fig. 4 Heat maps showing the growth of V. campbellii BAA-1117 (A) and V. japonicus UU24 (B). Growth was studied in the presence of increasing hydrogen peroxide concentrations after different resuscitation times (incubation at 30 °C) in a microtiter plate. X-axis: hours of incubation at 30 °C; Y-axis: H O concentrations (mM). Different colours represent number of wells in which growth has been detected based on the increase of OD. 2 2 Both the strains acquired the capability to overcome oxidative stress gradually during resuscitation, though with a different timing and against different concentrations of hydrogen peroxide Fig. 5 Size and fluorescence of the VBNC-induced cells of V. campbellii BAA-1117 and V. japonicus UU24. Cells were incubated at 30 °C to induce resuscitation in the presence of different hydrogen peroxide concentrations, i.e. oxidative stress. Black line: cells incubated in the presence of 0.007 mM hydrogen peroxide. Green line: cells incubated in the presence of 0.02 mM hydrogen peroxide. Purple line: cells “resuscitated” at 30 °C and inserted to indicate the hypothetical shift in case of resuscitation. If resuscitation had occurred, black and green lines would have been merged with purples ones. A, B and C V. campbellii BAA-1117 . While the resuscitated cells increased in cell size and DNA quantity and decreased in green fluorescence when stained with PI (please check Fig. 1), stressed cells preserved the phenotypic features of VBNC cells. A Size (FSC-A). The size of stressed cells did not increase as that of resuscitated cells. B Green fluorescence (FL1-A) of cells stained with SYTO 9. The quantity of DNA in stressed cells did not increase. C Green fluorescence of cells stained with propidium iodide. The fluorescence of stressed cells did not decrease. D V. japonicus UU24; green fluorescence of cells stained with propidium iodide. While in resuscitated cells a big population has lost the fluorescence, in stressed cells, this population is much smaller. While cells stressed with 0.007 mM hydrogen peroxide were still able to resuscitate and grow, cells stressed with higher concentrations of hydrogen peroxide are not. Size and DNA quantity did not show in this strain any appreciable difference between resuscitated, VBNC and stressed cells Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 10 of 13 suspensions; cells could not resuscitate after a further damaged cells. However, observing the fluorescence of overnight incubation in presence of catalase, probably PI-stained cells in the green channel (FL1), we found that because they were permanently damaged by the pro- the dwarf, low-DNA population that appeared as a con- longed exposure to the oxidant. sequence of prolonged incubation in cold ASW recorded an increase in green fluorescence: this could be possibly Catalase production linked to some changes in the cells exposed to starvation, Only V. campbellii BAA-1117 showed high catalase which affect the fluorophore labelling (Parada et al. 2016; production (207 units for 10 mg cells) with the selected Kaberdin et al. 2015; Montánchez et al. 2014). The green method (Iwase et al. 2013), while for the other three envi- fluorescence appeared only in the aliquots stained with ronmental strains (UU21, UU24 and M5), catalase pro- PI, but not in the non-stained ones, and such observation duction appeared negligible if compared to the one of V. was consistently repeated for all the strains. Moreover, campbellii BAA-1117 . during the resuscitation process, the green fluorescence was related to cells exiting from VBNC state, while in V. Discussion campbellii BAA-1117 , the decrease in fluorescence has The time to enter a VBNC state is highly variable in the been combined with other changes; in V. japonicus UU24, genus Vibrio, 3 (Kong et  al. 2004) to 4 days (Whitesides this was the only phenotypic feature useful to trace the and Oliver 1997) for V. vulnificus and 60 (Asakura et  al. resuscitation process. Indeed V. japonicus-resuscitated 2007) to 77 days (Imamura et al. 2015; Senoh et al. 2015) cells did not increase their size or DNA quantity but lost for V. cholerae. V. harveyi cells were followed for 21 days, the PI-induced green fluorescence. This phenomenon is but the induction of VBNC state was not reported as rather difficult to explain but yet consistent in our data. complete (Parada et al. 2016; Kaberdin et al. 2015; Mon- Further investigations are required to clarify this obser- tánchez et  al. 2014). Variable times are reported also for vation; indeed, the discovery of a marker of VBNC or V. parahaemolyticus from 9 (González-Escalona et  al. resuscitated Vibrio cells would be of great interest both 2006) to 69 days (Falcioni et  al. 2008). Such differences from public health and marine ecology perspectives. may be attributed to the specificity of the strains or of the The well-known “great plate count anomaly” states that experimental conditions. Our experiments, performed only a minor fraction of the bacterial species in the envi- on three Vibrio strains isolated from environmental ronment is culturable (Connon and Giovannoni 2002). samples and one collection strain (V. campbellii BAA- Our findings pointed out that, also in case of culturable 1117 ), confirmed that the time to enter a VBNC state strains and dormant cells, plate count could underes- is variable for the different species tested and can be a timate the bacterial load of an environmental sample. very slow process, involving for some strains only a frac- While all the cells of V. campbellii BAA-1117 acquired tion of the cells. All V. campbellii BAA-1117 cells rap- the features of VBNC state in a short time span (53 days), idly and almost simultaneously lost their culturability and among the considered strains, for our environmental acquired specific features when observed by flow cytom - strains, the VBNC state entrance appeared to be a slower etry (Fig.  1). Consistent with the observations reported process (e.g. 108 days for strain UU24) than for the labo- in Falcioni et al. (2008), the size of VBNC cells markedly ratory strain and eventually involved only a fraction of decreased, as well as DNA content (Trevors et  al. 2010). the total cell population. However, as reported above, the At the same time, when stained with PI, VBNC cells time to enter a VBNC state is highly variable in Vibrio acquired an unexpected green fluorescence, which in our genus and reported to be, for instance, 3–4 days for V. experiments appeared to be consistent with VBNC state vulnificus and 60–77 days for V. cholerae (Kong et  al. induction (as discussed later). In the other three strains 2004; Whitesides and Oliver 1997; Asakura et  al. 2007; (UU24, M5 and UU21), we observed the same behaviour, Imamura et al. 2015; Senoh et al. 2015). Thus, even if we even though with a remarkably slower rate (Fig. 1). count a number of colonies of a certain species from an PI is a well-known red-fluorescent cationic dye used environmental sample, we cannot exclude that other pop- to detect cells with damaged membranes. Although it ulations are present in the sample, and other approaches is not clear if cells could actually recover after the dam- should be paralleled to cultivation for confirmation. age (Davey and Hexley, 2011), it is commonly used to When VBNC cells of V. campbellii BAA-1117 were detect dead cells, often as part of live/dead kits (Falcioni resuscitated, the exact reversion of the phenotypic fea- et al. 2008; González-Escalona et al. 2006; Imamura et al. tures detected in the process of VBNC state induction 2015). However, under the experimental conditions was observed (Baffone et  al. 2006; Li et  al. 2014). For adopted in this study, we did not detect any red fluores - this strain, which was the quickest to enter into VBNC cence upon staining with PI over time in ASW micro- state in our experimental conditions, the resuscitation cosms. u Th s, we were not able to assess the number of was also a rapid process involving almost all the cells at P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 11 of 13 the same time. The resuscitation of V. japonicus UU24 oxidative stress. VBNC cells incubated at 30 °C with the was instead slower and gradual (Fig.  2). Although they addition of hydrogen peroxide did not show any phe- became again culturable, cells only partially reverted the notypic change as reported in case of resuscitated cells VBNC state phenotype, particularly considering cell size (Fig.  5). u Th s, to assess if they retained the capability to and DNA quantity. As observed for the entrance into resuscitate or they were dead, we added catalase, and VBNC state, resuscitation of our environmental strains we incubated them at 30 °C for an additional overnight. involved only one subpopulation. This confirms previous After plating, we did not observe any colony, suggesting evidence about the fact that the mechanisms of the resus- that the exposure to hydrogen peroxide killed the VBNC citation process vary across strains (Li et  al. 2014). This cells before they could resuscitate. implies that every attempt to improve the culturability of environmental bacteria based on a resuscitation protocol Conclusions should be aware of the variability among strains. Timing Data highlighted that hydrogen peroxide is an important and protocols applied to restore the culturability could factor preventing the growth of VBNC cells in plates, affect each strain differently; attempts of resuscitating although it was not sufficient to explain the differen - simultaneously a mixed community, for example from an tial response of the tested strains. Its detrimental effects environmental sample prior to a cultivation-based sur- varied in magnitude for the two considered strains of V. vey, could hence result in different proportions of resus - campbellii BAA-1117 and V. japonicus UU24, which citated cells from each of the strains in the sample. both resulted more sensitive as VBNC cells rather than VBNC cells of V. campbellii and V. japonicus can be as growing cells. In this regard, future studies could be also regrown by supplementing the plates with catalase to focused on the metabolic pathways involved in the resus- remove the hydrogen peroxide that is naturally present in citation process of bacteria, when exposed to the oxida- the medium (Arana et al. 1992), even if with a lower effi - tive stress imposed by hydrogen peroxide, considering ciency than by incubation at 30 °C. Therefore, the oxida - different times of resuscitation and monitoring the bacte - tive stress induced by the presence of hydrogen peroxide rial gene expression and proteome profiles. Moreover, it is not the only factor implied in the loss of culturability will be important to understand the mechanism explain- of these cells, in particular in the case of V. japonicus (Li ing why for some species only a subset of clonal popula- et  al. 2014). To clarify the strains’ behaviour in the pres- tions enter the VBNC state. ence of hydrogen peroxide, we measured the sensitivity In this study, we also observed that Vibrio cells incu- to this compound of both strains during growth in stand- bated in microcosms acquired, over time, a green fluo - ard conditions and their ability to produce catalase. Inter- rescence when stained with PI, which then reverted with estingly, although more sensitive to hydrogen peroxide, resuscitation. Interestingly, we found that for V. japonicus V. campbellii BAA-1117 showed a catalase production UU24, the decrease in PI-green fluorescence was the only far higher than the environmental strains analysed in this phenotypic feature useful to trace the resuscitation pro- study, suggesting that they could have different mecha - cess. Considering that this behaviour is rather difficult to nisms of protection from oxidative stress. However, after explain, but consistent in our data, we should remark that resuscitation, the oxidative stress resistance is gradu- the discovery of a marker of VBNC Vibrio cells would ally acquired along with the time of incubation at 30 °C be of great interest, and that further investigations are (Fig. 4). needed to clarify this observation. Kong and colleagues (2004) measured that hydrogen peroxide concentration in plates of Heart Infusion Agar Supplementary Information was 0.007 mM. We verified that the addition of this The online version contains supplementary material available at https:// doi. hydrogen peroxide concentration to the microcosms’ ali- org/ 10. 1186/ s13213- 022- 01703-6. quots, prior to incubation at 30 °C, prevented the resus- citation and growth of V. campbellii, which was more Additional file 1: Figure S1. Multi-alignment analysis of the partial 16S rRNA gene sequences of the 4 Vibrio strains used in this study. The multi- sensitive to hydrogen peroxide, but not of V. japonicus, alignment was performed with Clustal Omega (EMBL-EBI; https:// www. which required concentrations approximately three times ebi. ac. uk/ Tools/ msa/ clust alo/; version 1.2.4). Figure S2. Appearance of higher. Nevertheless, in both cases, the concentration dwarf populations for the 4 Vibrio spp. strains. Along with a prolonged incubation in cold ASW, a dwarf population appeared, characterized by that could prevent the resuscitation was far lower than an increased green fluorescence upon staining with PI. A, B: V. campbel- the one that could inhibit growth of standard cultures, TM lii BAA-1117 . C, D: V. hepatarius UU21. E, F: V. japonicus M5. G, H, I: V. confirming the hypothesis that VBNC cells are more sen - japonicus UU24. Plots show the size (x axis) and the PI-green fluorescence (y axis) of the microcosm cells after 3 days of incubation (A, C, E, G) or after sitive to oxidative stress (Nowakowska and Oliver 2013). TM 47 days (B, D, F, H). While all V. campbellii BAA-1117 cells are dwarf and This effect was sharper in V. campbellii , which possi- fluorescent with PI after 47 days, for the other strains two populations bly relied more on catalase for the protection against are detectable. For V. japonicus UU24, the dwarfing process is completed Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 12 of 13 References in 124 days (I). Figure S3. Size (x axis) and green fluorescence (y axis) Arana I, Muela A, Iriberri J, Egea L, Barcina I (1992) Role of hydrogen peroxide TM upon staining with propidium iodide of V. campbellii BAA-1117 and V. in loss of culturability mediated by visible light in Escherichia coli in a japonicus UU24 during the resuscitation process. A, B, C: VBNC-induced freshwater ecosystem. Appl Environ Microbiol 58:3903–3907. https:// doi. TM cells of strain BAA-1117 after 0, 17 and 21 hours of incubation at 30°C, org/ 10. 1128/ aem. 58. 12. 3903- 3907. 1992 respectively. D, E, F: VBNC-induced cells of strain UU24 after 1, 24 and 64 Asakura H, Ishiwa A, Arakawa E, Makino S, Okada Y, Yamamoto S, Igimi S (2007) hours of incubation at 30°C, respectively. During incubation at 30°C in Gene expression profile of Vibrio cholerae in the cold stress-induced “resuscitation” process, a less fluorescent population appeared, which in viable but non-culturable state. Environ Microbiol 9:869–879. https:// doi. TM strain BAA-1117 resulted bigger in size. Figure S4. Sensitivity of strains V. org/ 10. 1111/j. 1462- 2920. 2006. 01206.x TM campbellii BAA-1117 (A) and V. japonicus UU24 (B) to hydrogen peroxide Ayrapetyan M, Williams TC, Oliver JD (2014) Interspecific quorum sensing (0 mM – 0.6 mM), visualized by counting the microtiter wells in which the mediates the resuscitation of viable but nonculturable vibrios. Appl Envi- bacterial growth was observed. Overnight cultures of cells (not induced ron Microbiol 80:2478–2483. https:// doi. org/ 10. 1128/ AEM. 00080- 14 to enter into VBNC state) were used. Figure S5. Growth curves of strains Ayrapetyan M, Williams TC, Oliver JD (2015) Bridging the gap between viable TM V. campbellii BAA-1117 (A) and V. japonicus UU24 (B) in presence of but non-culturable and antibiotic persistent bacteria. Trends Microbiol increasing quantities of hydrogen peroxide (0 mM – 0.6 mM). The graphs 23(1):1–13. https:// doi. org/ 10. 1016/j. tim. 2014. 09. 004 indicate the growth curves generated by the mean O.D. value detected Baffone W, Casaroli A, Citterio B, Pierfelici L, Campana R, Vittoria E et al (2006) in 8 independent microtiter wells (replicates) for each hydrogen peroxide Campylobacter jejuni loss of culturability in aqueous microcosms and concentration considered. ability to resuscitate in a mouse model. Int J Food Microbiol 107:83–91. https:// doi. org/ 10. 1016/j. ijfoo dmicro. 2005. 08. 015 Cai Y, Liu J, Li G, Wong PK, An T (2021) Formation mechanisms of viable but Acknowledgements nonculturable bacteria through induction by light-based disinfection and Authors kindly thank Prof. Raffaella Di Cagno for providing V. campbellii BAA- their antibiotic resistance gene transfer risk: a review. Crit Rev Environ Sci 1117 strain. Technol. https:// doi. org/ 10. 1080/ 10643 389. 2021. 19323 97 Connon SA, Giovannoni SJ (2002) High-throughput methods for culturing Authors’ contributions microorganisms in very-low-nutrient media yield diverse new marine EMP and EC conceived the study. EMP, SA, FM, ZZ, MF and EC performed the isolates. Appl Environ Microbiol 68(8):3878–3885. https:// doi. org/ 10. 1128/ experiments and analysed the data. MF sampled the crabs. EMP wrote the AEM. 68.8. 3878- 3885. 2002 first version of the manuscript. SB and DD supported the research. All authors Davey HM, Hexley P (2011) Red but not dead? Membranes of stressed Saccha- critically revised the manuscript and have approved its content. romyces cerevisiae are permeable to propidium iodide. Environ Microbiol 13:163–171. https:// doi. org/ 10. 1111/j. 1462- 2920. 2010. 02317.x Funding Falcioni T, Papa S, Campana R, Manti A, Battistelli M, Baffone W (2008) State The research leading to these results has received funding from the European transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow Union’s Seventh Framework Programme (FP7/2007–2013) under grant agree- cytometry. Cytom Part B - Clin Cytom 74:272–281. https:// doi. org/ 10. ment no. 311975 (MACUMBA). The publication reflects the views only of the 1002/ cyto.b. 20427 authors, and the European Union cannot be held responsible for any use González-Escalona N, Fey A, Höfle MG, Espejo RT, Guzmán C (2006) Quantita- which may be made of the information contained therein. DD has been sup- tive reverse transcription polymerase chain reaction analysis of Vibrio ported by the King Abdullah University of Science and Technology Competi- cholerae cells entering the viable but non-culturable state and starvation tive Research Grant Program 2018-OSR-CRG 2018-3739. in response to cold shock. Environ Microbiol 8:658–666. https:// doi. org/ 10. 1111/j. 1462- 2920. 2005. 00943.x Availability of data and materials Imamura D, Mizuno T, Miyoshi S, Shinoda S (2015) Stepwise changes in viable Data related to DNA sequencing are made available through public databases but nonculturable Vibrio cholerae cells. Microbiol Immunol 59:305–310. as specified in the “Materials and methods ” section. https:// doi. org/ 10. 1111/ 1348- 0421. 12246 Iwase T, Tajima A, Sugimoto S, Okuda K, Hironaka I, Kamata Y, Takada K, Mizunoe Y (2013) A simple assay for measuring catalase activity: a visual Declarations approach. Sci Rep 3:3081. https:// doi. org/ 10. 1038/ srep0 3081 Kaberdin VR, Montánchez I, Parada C, Orruño M, Arana I, Barcina I (2015) Ethics approval and consent to participate Unveiling the metabolic pathways associated with the adaptive Decapod crustaceans (such as lobsters and crabs) are exempt from current reduction of cell size during Vibrio harveyi persistence in seawater European legislation that protects animals used for scientific purposes microcosms. Microb Ecol 70:689–700. https:// doi. org/ 10. 1007/ because they are non-sentient and thus incapable of suffering even though s00248- 015- 0614-7 the care and use of the experimental animals complied with local animal Kong I-S, Bates TC, Hülsmann A, Hassan H, Smith BE, Oliver JD (2004) Role of welfare laws, guidelines and policies. catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiol Ecol 50:133–142. https:// doi. org/ 10. 1016/j. femsec. 2004. 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Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

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Abstract

Purpose Aquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable ( VBNC) state, as a consequence of starvation in seawater at low tempera- tures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resus- citate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress. Methods Cell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating. Results The cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide. Conclusions The timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus. Keywords Dormancy, Resuscitation, Culturability, Viability, Hydrogen peroxide, Catalase, Flow cytometry, Oxidative stress *Correspondence: Elena Crotti elena.crotti@unimi.it Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 2 of 13 As mentioned above, several stress conditions, such as Introduction starvation and incubation at nonoptimal temperatures, Under unfavourable environmental conditions, micro- can induce the VBNC state. Within Vibrio genus, the bial cells may enter in a physiological reversible state appearance of VBNC cells has been intensively studied in of low metabolic activity known as quiescence or dor- the species V. vulnificus, shortly after incubation in cold mancy (Lennon and Jones 2011). This represents an seawater; cell resuscitation from this state has been dis- adaptive strategy for long-term survival of bacteria fac- covered upon exposure to higher temperatures prior to ing unfavourable environmental conditions (Ayrapetyan plating (Oliver 2005). The loss of culturability induced et  al. 2015). Examples of cellular processes leading to by starvation and low temperatures has been associated dormancy are sporulation in gram-positive bacteria with significant decrease in the expression of the peri - and the formation of persister cells or viable but non- plasmic catalase KatG and of catalase activity, which culturable (VBNC) cells in gram negatives (Lennon and affect the cell resistance to oxidative stress (Kong et  al. Jones 2011). Specifically, VBNC state is a dormant physi - 2004). Therefore, the growth of VBNC cells in rich media ological condition of resistance against stress factors, in that contain a certain amount of hydrogen peroxide, which bacterial cells are alive but no longer able to grow e.g. brain heart infusion (BHI), is hampered (Kong et  al. on laboratory media; they however retain the capabil- 2004). On the other hand, the quorum-sensing molecule ity to restart growth upon “resuscitation” under certain AI-2, as well as cell-free supernatants from V. vulnificus, favourable environmental conditions (Lennon and Jones Vibrio parahaemolyticus or Escherichia coli, can trig- 2011; Oliver 2005). Stress conditions, such as starvation ger the resuscitation process in both seawater samples and incubation at nonoptimal temperatures, can induce and experimental VBNC microcosms (Ayrapetyan et  al. the VBNC state in a wide range of bacteria, including 2014). It has been proposed that increased amounts of human pathogens (Oliver 2005, 2010). VBNC cells resist AI-2 stimulate the expression of the stress-related alter- to multiple antimicrobial treatments (e.g. antibiotics) and nate sigma factor rpoS (through the action of LuxR), easily bypass microbiological safety controls due to their restoring the expression of catalase (katG) and the cul- lack of culturability, representing thus a potential threat turable state (Ayrapetyan et al. 2014). However, studies in for human health (Nowakowska and Oliver 2013; Li et al. other Vibrio species suggest that the mechanisms behind 2014). For instance, wastewater disinfection treatments the loss of culturability could be far more complicated can induce the VBNC state (Cai et  al. 2021). The reuse (Li et  al. 2014). In V. cholerae, the induction of VBNC of such treated wastewater in agriculture and the lack of state and the following resuscitation cannot always be detection of VBNC cells, which is largely based on culti- obtained as for V. vulnificus, but depending on the time vation approach, represent a risk not only for the spread spent under the VBNC state, other factors should be pro- of pathogens but also of the antibiotic resistance genes vided, such as catalase or human colon cells extracts, or (ARGs) the dormant cells may harbour (Cai et  al. 2021; the cells should be co-cultured with colon cells, to res- Riva et  al. 2020). Furthermore, we have to consider that cue from the VBNC state (Imamura et  al. 2015). A cur- a number of bacteria for open-field environmental appli - rent view postulates that in V. cholerae, the VBNC state cations are in most cases poorly characterized in rela- is a condition that continuously changes over time, rather tion to their potential VBNC state that may be induced than a single-gene property (Imamura et  al. 2015). This under harsh conditions they may experience during view is supported by the occurrence of transcriptional their application in the field (Prakash et  al. 2013). Thus, changes in the first 12 h of incubation of V. harveyi in improving the knowledge on the mechanisms leading to seawater without nutrients (Montánchez et  al. 2014), or removing the VBNC state is necessary for both human with cells (which are still culturable) becoming smaller and animal health and to improve the biotechnological and adapting their gene expression (Kaberdin et al. 2015). exploitation of microorganisms. The reduction in size and the decrease of DNA quantity VBNC state has been extensively studied in the gram- have been extensively described also in VBNC cells of negative, aquatic bacteria of the genus Vibrio (Oliver V. parahaemolyticus (Falcioni et  al. 2008; Wagley et  al. 2005), which include waterborne or foodborne pathogens 2021). (e.g. Vibrio vulnificus, V. cholerae, Vibrio haemolyticus). The oxidative stress response has been repeatedly asso - Such bacteria inhabit marine, freshwater and estua- ciated with the VBNC state (Li et  al. 2014), though the rine environments and can be associated with shellfish role of hydrogen peroxide is still under debate. On one and other aquatic animals (O’Neil et  al. 1992; Lutz et  al. hand, VBNC cells of clinical strains of V. vulnificus have 2013). The current warming of the sea surfaces has been been reported to be highly sensitive to hydrogen per- connected with the spread of this genus and the emer- oxide, proposed as the main reason for the inability to gence of related human diseases (Vezzulli et al. 2016). grow on BHI (Kong et al. 2004; Nowakowska and Oliver P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 3 of 13 2013). On the other hand, VBNC cells of an environmen- preliminary washing, the gills were smashed in ster- tal strain of V. vulnificus have been shown to be more ile saline (9 g/L NaCl), and serial dilutions were plated resistant to this stress than the log-phase cells (Nowa- on Marine Agar (Difco) plates added with 100 μg/ml kowska and Oliver 2013). During the initial stages of cycloheximide to avoid eukaryotic growth. Plates were starvation, V. harveyi upregulates the genes responsible incubated aerobically at 30° C for 24–48 h. Once the for the oxidative stress resistance, detoxifying the endog- growth occurred, random colonies were selected and enous hydrogen peroxide generated as a side product of purified. The 16S rRNA gene of the isolates was ampli - the lipid turnover and repairing the damaged molecules fied through PCR using primers 27F (5′-TCG ACA TCG (Kaberdin et al. 2015).TTT ACG GCG TG-3′) and 1492R (5′-CTA CGG CTA CCT Considering cell response in relation to the oxidative TGT TAC GA-3′) (Mapelli et  al. 2013) and sequenced at stress caused by hydrogen peroxide, limited informa- Macrogen (South Korea). The obtained sequences were tion is available about changes occurring in VBNC and aligned against the curated database of EZBioCloud resuscitated cells. The aim of this study is thus to assess (https:// www. ezbio cloud. net/; Yoon et  al. 2017) to con- the relationship between cell phenotypic changes and firm their affiliation to Vibrio spp. Sequences were then response to oxidative stress of different Vibrio strains deposited in the ENA database under the accession num- during the transition to and from the VBNC state. To this ber ERZ12298639. Glycerol stocks were stored at −80 °C. aim, a first part of the work was dedicated to character - ize the phenotypic changes of 4 Vibrio strains (namely, Preparation of Vibrio microcosms and induction the laboratory strain Vibrio campbellii BAA-1117 and of the VBNC state the 3 environmental strains of Vibrio hepatarius UU21, Strains were grown overnight at 30 °C in tryptic soy broth Vibrio japonicus UU24 and Vibrio japonicus M5) during added with 25 g/L NaCl (TSB-NaCl). Bacterial cultures the incubation in cold seawater microcosms under starv- were used to inoculate (1% v/v) fresh TSB-NaCl broth. ing conditions. In the second part of the work, 2 out of As soon as the cultures reached the exponential growth 4 strains (i.e. Vibrio campbellii BAA-1117 and Vibrio phase (optical density, OD, 0.3–0.5), cells were washed japonicus UU24) were further monitored until the cells twice in sterile artificial sea water (ASW). To prepare completely entered in VBNC state, and their resuscita- ASW, 40 g of sea salts (Sigma-Aldrich, Milan, Italy) was tion was thus induced by incubation at 30 °C for differ - dissolved in 1 L of deionized water, and the solution was ent times before plating. These two strains were selected filtered with 0.22 μm pore size filters (Millipore, Milan, because the former was the quickest to enter into the Italy). Cells were counted using a Thoma chamber and VBNC state, while the latter was the one with a distin- diluted in sterile ASW to reach the concentration of guishable VBNC kinetic among the other isolates. In approximately 1 × 10 cells/ml in glass vials. Microcosms these strains, the relationship between resuscitation and prepared in ASW and thus, in starvation condition, were the oxidative stress caused by hydrogen peroxide was also stored at 4 °C in the dark; starvation and low tempera- investigated. ture were applied to induce the VBNC state. To check the entrance in VBNC state by cultivation, culturability trials Materials and methods were carried out by drop-plate technique. Briefly, cultur - Isolation and molecular identification of Vibrio spp. strains able cells collected from microcosms, initially shaken to One laboratory strain and three environmental isolates of allow the aggregate breakage and sample homogeniza- Vibrio spp. were used in the study. The laboratory strain tion, were counted in 10 μl of decimally diluted tripli- was Vibrio campbellii BAA-1117 (luxN::tn5Kan), with a cated aliquots plated on TSB-NaCl plates (added with 15 mutation in the LuxN receptor, which is involved in the g/L agar), incubated overnight at 30 °C. Viability staining quorum sensing mediated by homoserine lactones. Strain was assessed by flow cytometry, as reported below. ™ ™ BAA-1117 is a derived mutant of strain BAA-1116 that was reclassified as Vibrio campbellii according to Assessment of VBNC state and resuscitation microarray comparative genome hybridization and mul- In order to verify that the observed increase in cultur- tilocus sequence analysis (Lin et al. 2010). Three environ - able cells was due to resuscitation and not to the growth mental strains, UU21, UU24 and M5, were isolated from of an originally undetectable non-VBNC cell population, the gills of the fiddler crab Tubuca urvillei, sampled in we applied the procedure adapted by Whitesides and Oli- 2011 in Gazi Bay mangrove forest (−4.416, 39.51; Kenya). ver (1997). An aliquot of the microcosm was shaken and For bacterial isolation, the crab gills were dissected in serially diluted, and a sample of each of the dilutions, as proximity of a Bunsen burner flame, placed in a 2-ml well as of the undiluted sample, were plated (100 μl) on tube where the gills were washed in sterile physiological TSB-NaCl agar plates to verify the absence of growth. We solution (9 g/L NaCl) to remove external particles. After incubated the remaining dilutions overnight at 30 °C to Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 4 of 13 induce resuscitation, since temperature upshift has been solution was poured on each TSB-NaCl agar plate. The described to resuscitate VBNC cells, e.g. in V. vulnifucus plates were dried in a biosafety cabinet and immediately (Oliver 2005). The following day, we plated (100 μl) each inoculated with 100 μl of VBNC cells. The number of col - dilution on TSB-NaCl agar plates, and, after a further onies was evaluated after an overnight incubation at 30 overnight at 30 °C, the number of colonies was assessed. °C. Assuming that, if the increase in CFU counts was due to The production of catalase by the four Vibrio strains the growth of originally undetectable non-VBNC cells was visually assessed using the straightforward method rather than to the resuscitation, at least one non-VBNC described by Iwase et  al. (2013). Briefly, a calibration cell should have been initially present in each dilution curve was constructed with defined units of catalase in from which we cultivated cells, we then calculated the a solution made of 1% (v/v) Triton X-100 and undiluted theoretical initial concentration of these non-VBNC cells hydrogen peroxide (30% v/v); the height of oxygen-form- in the undiluted sample and verified that this value was ing foam was measured using a ruler. Samples of bacte- higher than our detection result (Whitesides and Oliver rial cells (10 mg), prepared using overnight cultures, were 1997). resuspended in physiological saline and subjected to the For other experiments of resuscitation from the VBNC same procedure (Iwase et al. 2013). state, we incubated the bacterial aliquots at 30 °C for different time spans prior to plating on TSB-NaCl agar Growth in the presence of hydrogen peroxide of VBNC plates and let them grow overnight. or resuscitated cells To determine the sensitivity of the strains to hydrogen Viability staining and flow cytometry peroxide, growth curves of standard-growing cells of Two aliquots of 100 μl were collected from each micro- the strains BAA-1117 and UU24 were obtained using ™ ® cosm and stained separately with SYTO 9 or propidium the Infinite 200 PRO microplate reader (TECAN Italia iodide (PI) from the LIVE-DEAD BacLight kit (L7012, S.r.l, Milan, Italy). With the term “standard-growing cells/ Life Technologies, Milan, Italy). Both live, and dead bac- cultures”, we identified cells of bacterial strains routinely terial cell membranes are permeable to SYTO 9, which grown in the lab (i.e. not induced in VBNC state or resus- binds DNA and RNA. PI only enters dead or damaged citated from VBNC state). Ten microlitres of the cell sus- cells. The staining solutions were prepared by adding pension (10 cell/ ml) was inoculated in 90 μl TSB-NaCl 3 μl of each dye to 1 ml Milli-Q water, according to the medium in each well of a 384-well microplate using an manufacturer protocol; then, 100 μl of the staining solu- ep-Motion liquid handler (Eppendorf, Milan, Italy). Dif- tions was mixed with each microcosm aliquot. Samples ferent concentrations of hydrogen peroxide (from 0 to 0.6 were analysed in flow cytometry using a BD Accuri C6 mM) were prepared by adding to each well 2 μl of hydro- device (BD Biosciences, Milan, Italy). For each sample, gen peroxide solution at the appropriate concentrations 30,000 events were acquired, and a non-stained sample diluting 30% (v/v) hydrogen peroxide (Sigma-Aldrich, from each microcosm was always included in the analy- Milan, Italy) in deionized water. The microtiter plates sis. The excitation laser was set at 488 nm. Green fluores - were incubated at 30 °C for 24 h, and the optical density cence signal of SYTO 9 was detected with a 530/30 nm was measured every 15 mins. We calculated the growth filter, while red fluorescence from PI was measured with curves by averaging the OD measurements of 8 replicates a 585/40 nm filter. In our experimental setup, the use of and subtracting the average OD value of negative con- PI to trace dead cells based on red fluorescence was not trols (non-inoculated wells). We performed an end-point useful; we could conversely detect a green fluorescent analysis counting the number of wells showing the bacte- stain using PI (see “Results” section). The obtained data rial growth at the end of the incubation time. were analysed using BD Accuri C6 Plus software ver- The capability of cells from microcosms to grow in the sion 1.0 (BD Biosciences, Milan, Italy). Flow cytometry presence of hydrogen peroxide was monitored in micro- analyses were used to evaluate phenotypic characteristics titer plates using the protocol reported above. Specifi - of Vibrio strains in VBNC state and after resuscitation. cally, we considered (i) cells collected from microcosms at day 3, before the induction of the VBNC state (inocu- Plates treated with catalase and determination of strains’ lum: 10 μl; hydrogen peroxide concentrations from 0 to catalase production 0.600 mM), (ii) VBNC cells (inoculum: 10 μl; hydrogen Catalase from bovine liver (C9322) was purchased from peroxide concentrations from 0 to 0.250 mM) and (iii) Sigma-Aldrich (Milan, Italy), dissolved in phosphate resuscitated VBNC cells (inoculum: 10 μl; hydrogen per- buffer pH 7 at the concentration of 1 mg/ml and filter oxide concentrations from 0 to 0.250 mM), following the sterilized. In order to obtain the same catalase activity per incubation at 30 °C for different resuscitation times. plate as described by Kong et al. (2004), 1 ml of catalase P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 5 of 13 Resuscitation after stress hydrogen peroxide isopropanol demonstrated that in our experimental Two-millilitre aliquots of the microcosms were prepared setup, PI was unable to confer a red fluorescence to the in sterile tubes. To each one, the appropriate volume of dead cells in the microcosms. It is noteworthy to remark hydrogen peroxide/water solution was added to reach that Vibrio cells, grown in routine laboratory conditions the final hydrogen peroxide concentration of 0.007 mM, and killed by heat or isopropanol, showed red fluores - 0.02 mM and 0.05 mM. After overnight incubation at 30 cence with PI staining, highlighting that the lack of red °C, an aliquot of cell suspension was plated, while a sec- fluorescence for the PI-treated killed cells was specific for ond one was treated with catalase (C9322 Sigma-Aldrich; cells collected from microcosms. Moreover, no green flu - adding 25 μl of catalase solution to 1 ml aliquot: stock 1 orescence signals were detected in PI-treated killed cells, mg/ml) and incubated at 30 °C until plating the follow- demonstrating that the PI-green fluorescence was related ing day. Cell phenotypic characteristics were analysed by to live cells. flow cytometry, as reported above. In V. campbellii BAA-1117 , all the cells showed increased value of green fluorescence upon staining with Results PI already at day 33, while for the other strains, two pop- Starvation in cold seawater induces phenotypic changes ulations appeared: one with lower size cells and higher Vibrio strains isolated from the mangrove crab gills were green fluorescence and the other with bigger cells but less identified as Vibrio hepatarius UU21, Vibrio japonicus fluorescent upon staining with PI (Fig. 1I–L; Fig. S2). UU24 and Vibrio japonicus M5 by partial 16S rRNA gene Microcosms of V. campbellii BAA-1117 and V. sequencing and alignment against the curated database japonicus UU24 were monitored until they completely EZBioCloud (Table  1; Fig. S1). To monitor phenotypic entered into VBNC state (Fig.  1). Strain BAA-1117 changes in the cells subjected to starvation and cold completely lost the capability to generate colonies on stresses, we analysed, by flow cytometry, the cells from TSB-NaCl plates at day 53 while strain UU24 at day 108 microcosms at different times during a time span of 50 (Figs.  1M and 2N). VBNC-induced cells of the 2 strains days, upon staining with the LIVE-DEAD BacLight kit; BAA-1117 and UU24 showed, by flow cytometry, cell at each analysed time point, we plated the cells to also populations with smaller size, lower DNA content and assess their culturability. higher green fluorescence upon staining with PI at days For all the strains, we observed the same trends, 50 and 124 of incubation, respectively (Fig. 1; Fig. S2). although with a different timing. In all the microcosms, a cell shrinkage as decreasing of the FSC-A parameter was Resuscitation completely reverted the cell phenotype observed by flow cytometry (Fig.  1A–D), while in case of for strain BAA‑1117 V. campbellii BAA-1117 , this process was already com- To resuscitate the cells, we applied the protocol described pleted at day 33, and for strains V. hepatarius UU21 and by Whitesides and Oliver (1997) incubating an aliquot of V. japonicus UU24 and M5, we observed a dwarf popu- the microcosm at 30 °C overnight; plating the cell sus- lation at day 47/50, even if large cells were still present pension after this treatment resulted in colony growth (Fig.  1A–D; Fig. S2). Similarly, the green fluorescence on TSB-NaCl agar medium. In order to demonstrate that signal detected upon staining with SYTO 9, which is the observed colonies appeared after resuscitation rather proportional to DNA abundance into the cells, decreased than being related to the growth of a previously undetect- rapidly in V. campbellii BAA-1117 and more slowly in able fraction of non-VBNC-induced cells (i.e. to verify the other strains (Fig. 1E–H). that all the cells included into microcosms were in VBNC Concomitantly to the reduction in size and DNA quan- state), we serially diluted the VBNC-induced microcosm tity, we recorded an upshift of the green fluorescence prior to incubation at 30 °C and still in starvation con- upon staining with propidium iodide (PI) in the dwarf ditions, and we verified that cells resuscitated in all the cells or the smallest fraction of cells (in size) (Fig.  1I–L; dilutions. Dilutions were indeed plated immediately to Fig. S2). Experiments with cells artificially killed by verify the absence and presence of CFUs, respectively, heat shock (10 min at 100 °C) or by adding 50% (v/v) before and following the resuscitation process at 30 °C. Resuscitation occurred in all dilutions incubated at 30 °C, and in the case of V. campbellii BAA-1117 , the final Table 1 Identification of the isolates obtained in the study colony counts reached levels which reflected the dilution applied. For instance, for this strain after 48 h of resusci- Isolate EZBioCloud type strain 4 3 2 tation, we counted ~9 × 10 , 9 × 10 and 9 × 10 cells/ UU24 Vibrio japonicus JCM 31412 ml in the dilutions 1:100, 1:1000 and 1:10,000, respec- UU21 Vibrio hepatarius LMG 20362 tively (Whitesides and Oliver 1997). Assuming that they M5 Vibrio japonicus JCM 31412 can be grown from a single non-VBNC parental cell, Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 6 of 13 Fig. 1 Change in size, fluorescence and culturability of Vibrio spp. strains entering in VBNC state. Changes detected by flow cytometry. A–D Cell size (FSC-A). E–H DNA quantity (green fluorescence (FL1-A) of cells stained with SYTO 9). I–L Green fluorescence (FL1-A) of cells stained with propidium iodide (PI). Over time, the cell size decreases, as well as the DNA quantity, which is proportional to the green fluorescence of SY TO 9; concomitantly, the green fluorescence induced by PI increases. M–N Decrease in culturability during incubation at 4 °C, according to the CFU numbers detected by plating cells on solid medium. CFU counts are reported as L og (CFU + 1). A, E, I and M V. campbellii BAA-1117 strain. B, F, J and N V. japonicus UU24 strain. C, G and K V. hepatarius UU21 strain. D, H and L V. japonicus M5 strain. Black line: microcosm at 3 days. Green line: microcosm at 50 days. Purple line: microcosm at 124 days there should have been in the original microcosm at least trend of resuscitation in the case of V. japonicus UU24: 9 × 10 culturable cells/ml, which were not detected in we were still able to count the resuscitated cells in the the plating before the treatment. We observed a similar more diluted samples, but we noticed a confluent growth P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 7 of 13 Fig. 2 Change in size, fluorescence and culturability of Vibrio spp. strains exiting from VBNC state. Resuscitation observed by flow cytometry. A–B Cell size (FSC-A). C–D DNA quantity (green fluorescence (FL1-A) of cells stained with SYTO 9). E–F Green fluorescence (FL1-A) of cells stained with propidium iodide (PI). Changes detected before for cell size, DNA quantity and propidium iodide fluorescence (Fig. 1) are reverted in a different extent in the two strains. Resuscitation detected by culturability. G–H Increase in culturability, according to the CFU numbers detected plating cells on solid medium, after the incubation at 30 °C. The process of resuscitation happens exponentially. Counts (CFU/ml) are reported as L og (CFU + 1). A, C, E and G V. campbellii BAA-1117 strain. Black line: no incubation at 30 °C. Green line: after 17 h of incubation at 30 °C. Purple: after 21 h of incubation at 30 °C. B, D, F and H V. japonicus UU24 strain. Blue line: no incubation at 30 °C. Black line: after 1 h of incubation at 30 °C. Green line: after 24 h of incubation at 30 °C. Purple line: after 64 h of incubation at 30 °C of cells by plating those less diluted ones for which it was showed a decrease of fluorescence (Fig.  2 A, C, E; Fig. supposed to find single colonies; no cell colonies were S3). Concomitantly, the green fluorescence upon staining however detected in the plating before the resuscitation with SYTO 9 increased (Fig. 2C). The same reversal was treatment. Nevertheless, it remains to be elucidated why observed in V. japonicus UU24, but was not completed in strain UU24 showed a confluent growth when those dilu - the monitored time (Fig.  2B, D, F; Fig. S3); the PI-green tions were resuscitated. fluorescence gradually decreased (Fig.  2F), but cells were Resuscitation of V. campbellii BAA-1117 and V. still distributed in two populations of different fluores - japonicus UU24 was monitored over time. Microcosms’ cence after 64 h (Fig. S3). DNA quantity and cell size did aliquots were incubated at 30 °C along different times, not increase significantly (Fig. 2B and D). after which the cells were observed by flow cytometry and plated (Fig. 2G–H). In both strains, the CFU increase Catalase induced the resuscitation of VBNC cells was correlated to the time of incubation at 30 °C (resus- According to Kong and co-workers (2004), we veri- citation time) with an exponential relationship, until fied if the treatment of TSB-NaCl plates with catalase CFU number reached a plateau. However, flow cytom - could restore the culturability of VBNC cells. This was etry revealed a different behaviour of the two strains. carried out with aliquots of cells from the microcosms In V. campbellii BAA-1117 , the decrease in size and of V. japonicus UU24 at 179 days and V. campbellii DNA quantity and the increase in PI-green fluorescence BAA-1117 at 31 days. Although the latter was not yet resulted completely to revert after resuscitation with the completely in VBNC state, the use of catalase greatly process completed between 17 and 21 h (Fig. S3). After improved the culturability of the cells in both cases. 17 h, two populations were still detectable: one smaller However, the number of colonies obtained with this pro- in size and with an increased fluorescence upon stain - cedure was 100 or 1000 fold lower than the number of ing with PI and one larger in size (resuscitated) which colonies obtained by resuscitation at 30 °C (Fig. 3). Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 8 of 13 Fig. 3 Culturability of V. campbellii BAA-1117 (light grey) and V. japonicus UU24 (dark grey). Different treatments were considered: (i) after resuscitation on TSB-NaCl agar plates, (ii) without resuscitation on TSB-NaCl agar plates treated with catalase and (iii) without resuscitation on TSB-NaCl agar plates. Cells from V. campbellii BAA-1117 microcosm were not yet completely in VBNC state (31 days). Cells from V. japonicus UU24 microcosm were in VBNC state (179 days). Use of catalase greatly improved the culturability of the cells for both strains. Significant differences were observed among treatments for each strain (t-test, p < 0.05). Counts (CFU/ml) are reported as L og (CFU + 1) VBNC cells are sensitive to hydrogen peroxide in the presence of hydrogen peroxide concentrations To assess the capability of VBNC cells to grow in the from 0.05 to 0.25 mM (Fig.  4). According to our data, presence of oxidative stress, we first challenged stand - the capability to cope with oxidative stress was gradu- ard-growing cells with different concentrations of hydro - ally acquired with the resuscitation time. After 7–16 gen peroxide (Fig. S4). We noticed that the response to and 5–20 h of resuscitation, respectively, V. campbellii hydrogen peroxide was discrete, which means that only BAA-1117 and V. japonicus UU24 displayed a behav- in some wells we could observe bacterial growth. The iour similar to non-VBNC cells. After 20 h of resusci- growth rate could be also influenced by the presence of tation, V. campbellii BAA-1117 resulted even more hydrogen peroxide (i.e. we observed a delay of growth in resistant to oxidative stress, growing in all the wells the presence of certain concentrations of hydrogen per- containing 0.1 mM hydrogen peroxide (Fig. 4). oxide, as shown in Fig. S5). Thus, to assess the strains’ response to hydrogen peroxide, we counted the number of wells in which strains’ growth was observed. Standard- Hydrogen peroxide prevents resuscitation by damaging growing cells of V. campbellii BAA-1117 and V. japoni- cells that cannot be rescued by catalase in the plate growth cus UU24 showed a different sensitivity to hydrogen medium peroxide. Strain BAA-1117 growth failed in 25% of the To further investigate the relationship between resus- wells added with 0.1 mM hydrogen peroxide, and it was citation and sensitivity to hydrogen peroxide, we added consistently inhibited with 0.2 mM hydrogen peroxide hydrogen peroxide (0.007, 0.02 and 0.05 mM) to micro- (Fig. S4). Conversely, strain UU24 showed a higher resist- cosms’ aliquots prior to the incubation of VBNC cells at ance to oxidative stress induced by hydrogen peroxide: 30 °C for resuscitation. According to Kong et al. (2004), at 0.1 mM hydrogen peroxide, it always grew as good as a hydrogen peroxide concentration of 0.007 mM, which in the absence of hydrogen peroxide, while 0.225–0.250 is present in the standard cultural media, is enough mM hydrogen peroxide could prevent the growth of the to prevent cell growth. We verified that the 0.007 mM strain in 25% of the wells (Fig. S4). This behaviour was concentration prevented the resuscitation (culturabil- observed for cells collected from the microcosms at day 3 ity) of VBNC cells of V. campbellii BAA-1117 , while (i.e. cells not yet entered into VBNC state). for V. japonicus, UU24 concentrations higher than 0.02 Conversely, when in VBNC state, cells of both strains mM were necessary. Cells did not show an increase in could not grow in the presence of any of the hydrogen size nor anyone of the changes described before for peroxide concentrations applied. Sensitivity to hydro- the resuscitation process (Fig. 5). To verify if they were gen peroxide was then checked by exposing the VBNC still capable to resuscitate after relieving the oxida- cells at different periods of resuscitation at 30 °C and tive stress, we added catalase and plated the bacterial P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 9 of 13 Fig. 4 Heat maps showing the growth of V. campbellii BAA-1117 (A) and V. japonicus UU24 (B). Growth was studied in the presence of increasing hydrogen peroxide concentrations after different resuscitation times (incubation at 30 °C) in a microtiter plate. X-axis: hours of incubation at 30 °C; Y-axis: H O concentrations (mM). Different colours represent number of wells in which growth has been detected based on the increase of OD. 2 2 Both the strains acquired the capability to overcome oxidative stress gradually during resuscitation, though with a different timing and against different concentrations of hydrogen peroxide Fig. 5 Size and fluorescence of the VBNC-induced cells of V. campbellii BAA-1117 and V. japonicus UU24. Cells were incubated at 30 °C to induce resuscitation in the presence of different hydrogen peroxide concentrations, i.e. oxidative stress. Black line: cells incubated in the presence of 0.007 mM hydrogen peroxide. Green line: cells incubated in the presence of 0.02 mM hydrogen peroxide. Purple line: cells “resuscitated” at 30 °C and inserted to indicate the hypothetical shift in case of resuscitation. If resuscitation had occurred, black and green lines would have been merged with purples ones. A, B and C V. campbellii BAA-1117 . While the resuscitated cells increased in cell size and DNA quantity and decreased in green fluorescence when stained with PI (please check Fig. 1), stressed cells preserved the phenotypic features of VBNC cells. A Size (FSC-A). The size of stressed cells did not increase as that of resuscitated cells. B Green fluorescence (FL1-A) of cells stained with SYTO 9. The quantity of DNA in stressed cells did not increase. C Green fluorescence of cells stained with propidium iodide. The fluorescence of stressed cells did not decrease. D V. japonicus UU24; green fluorescence of cells stained with propidium iodide. While in resuscitated cells a big population has lost the fluorescence, in stressed cells, this population is much smaller. While cells stressed with 0.007 mM hydrogen peroxide were still able to resuscitate and grow, cells stressed with higher concentrations of hydrogen peroxide are not. Size and DNA quantity did not show in this strain any appreciable difference between resuscitated, VBNC and stressed cells Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 10 of 13 suspensions; cells could not resuscitate after a further damaged cells. However, observing the fluorescence of overnight incubation in presence of catalase, probably PI-stained cells in the green channel (FL1), we found that because they were permanently damaged by the pro- the dwarf, low-DNA population that appeared as a con- longed exposure to the oxidant. sequence of prolonged incubation in cold ASW recorded an increase in green fluorescence: this could be possibly Catalase production linked to some changes in the cells exposed to starvation, Only V. campbellii BAA-1117 showed high catalase which affect the fluorophore labelling (Parada et al. 2016; production (207 units for 10 mg cells) with the selected Kaberdin et al. 2015; Montánchez et al. 2014). The green method (Iwase et al. 2013), while for the other three envi- fluorescence appeared only in the aliquots stained with ronmental strains (UU21, UU24 and M5), catalase pro- PI, but not in the non-stained ones, and such observation duction appeared negligible if compared to the one of V. was consistently repeated for all the strains. Moreover, campbellii BAA-1117 . during the resuscitation process, the green fluorescence was related to cells exiting from VBNC state, while in V. Discussion campbellii BAA-1117 , the decrease in fluorescence has The time to enter a VBNC state is highly variable in the been combined with other changes; in V. japonicus UU24, genus Vibrio, 3 (Kong et  al. 2004) to 4 days (Whitesides this was the only phenotypic feature useful to trace the and Oliver 1997) for V. vulnificus and 60 (Asakura et  al. resuscitation process. Indeed V. japonicus-resuscitated 2007) to 77 days (Imamura et al. 2015; Senoh et al. 2015) cells did not increase their size or DNA quantity but lost for V. cholerae. V. harveyi cells were followed for 21 days, the PI-induced green fluorescence. This phenomenon is but the induction of VBNC state was not reported as rather difficult to explain but yet consistent in our data. complete (Parada et al. 2016; Kaberdin et al. 2015; Mon- Further investigations are required to clarify this obser- tánchez et  al. 2014). Variable times are reported also for vation; indeed, the discovery of a marker of VBNC or V. parahaemolyticus from 9 (González-Escalona et  al. resuscitated Vibrio cells would be of great interest both 2006) to 69 days (Falcioni et  al. 2008). Such differences from public health and marine ecology perspectives. may be attributed to the specificity of the strains or of the The well-known “great plate count anomaly” states that experimental conditions. Our experiments, performed only a minor fraction of the bacterial species in the envi- on three Vibrio strains isolated from environmental ronment is culturable (Connon and Giovannoni 2002). samples and one collection strain (V. campbellii BAA- Our findings pointed out that, also in case of culturable 1117 ), confirmed that the time to enter a VBNC state strains and dormant cells, plate count could underes- is variable for the different species tested and can be a timate the bacterial load of an environmental sample. very slow process, involving for some strains only a frac- While all the cells of V. campbellii BAA-1117 acquired tion of the cells. All V. campbellii BAA-1117 cells rap- the features of VBNC state in a short time span (53 days), idly and almost simultaneously lost their culturability and among the considered strains, for our environmental acquired specific features when observed by flow cytom - strains, the VBNC state entrance appeared to be a slower etry (Fig.  1). Consistent with the observations reported process (e.g. 108 days for strain UU24) than for the labo- in Falcioni et al. (2008), the size of VBNC cells markedly ratory strain and eventually involved only a fraction of decreased, as well as DNA content (Trevors et  al. 2010). the total cell population. However, as reported above, the At the same time, when stained with PI, VBNC cells time to enter a VBNC state is highly variable in Vibrio acquired an unexpected green fluorescence, which in our genus and reported to be, for instance, 3–4 days for V. experiments appeared to be consistent with VBNC state vulnificus and 60–77 days for V. cholerae (Kong et  al. induction (as discussed later). In the other three strains 2004; Whitesides and Oliver 1997; Asakura et  al. 2007; (UU24, M5 and UU21), we observed the same behaviour, Imamura et al. 2015; Senoh et al. 2015). Thus, even if we even though with a remarkably slower rate (Fig. 1). count a number of colonies of a certain species from an PI is a well-known red-fluorescent cationic dye used environmental sample, we cannot exclude that other pop- to detect cells with damaged membranes. Although it ulations are present in the sample, and other approaches is not clear if cells could actually recover after the dam- should be paralleled to cultivation for confirmation. age (Davey and Hexley, 2011), it is commonly used to When VBNC cells of V. campbellii BAA-1117 were detect dead cells, often as part of live/dead kits (Falcioni resuscitated, the exact reversion of the phenotypic fea- et al. 2008; González-Escalona et al. 2006; Imamura et al. tures detected in the process of VBNC state induction 2015). However, under the experimental conditions was observed (Baffone et  al. 2006; Li et  al. 2014). For adopted in this study, we did not detect any red fluores - this strain, which was the quickest to enter into VBNC cence upon staining with PI over time in ASW micro- state in our experimental conditions, the resuscitation cosms. u Th s, we were not able to assess the number of was also a rapid process involving almost all the cells at P rosdocimi et al. Annals of Microbiology (2023) 73:1 Page 11 of 13 the same time. The resuscitation of V. japonicus UU24 oxidative stress. VBNC cells incubated at 30 °C with the was instead slower and gradual (Fig.  2). Although they addition of hydrogen peroxide did not show any phe- became again culturable, cells only partially reverted the notypic change as reported in case of resuscitated cells VBNC state phenotype, particularly considering cell size (Fig.  5). u Th s, to assess if they retained the capability to and DNA quantity. As observed for the entrance into resuscitate or they were dead, we added catalase, and VBNC state, resuscitation of our environmental strains we incubated them at 30 °C for an additional overnight. involved only one subpopulation. This confirms previous After plating, we did not observe any colony, suggesting evidence about the fact that the mechanisms of the resus- that the exposure to hydrogen peroxide killed the VBNC citation process vary across strains (Li et  al. 2014). This cells before they could resuscitate. implies that every attempt to improve the culturability of environmental bacteria based on a resuscitation protocol Conclusions should be aware of the variability among strains. Timing Data highlighted that hydrogen peroxide is an important and protocols applied to restore the culturability could factor preventing the growth of VBNC cells in plates, affect each strain differently; attempts of resuscitating although it was not sufficient to explain the differen - simultaneously a mixed community, for example from an tial response of the tested strains. Its detrimental effects environmental sample prior to a cultivation-based sur- varied in magnitude for the two considered strains of V. vey, could hence result in different proportions of resus - campbellii BAA-1117 and V. japonicus UU24, which citated cells from each of the strains in the sample. both resulted more sensitive as VBNC cells rather than VBNC cells of V. campbellii and V. japonicus can be as growing cells. In this regard, future studies could be also regrown by supplementing the plates with catalase to focused on the metabolic pathways involved in the resus- remove the hydrogen peroxide that is naturally present in citation process of bacteria, when exposed to the oxida- the medium (Arana et al. 1992), even if with a lower effi - tive stress imposed by hydrogen peroxide, considering ciency than by incubation at 30 °C. Therefore, the oxida - different times of resuscitation and monitoring the bacte - tive stress induced by the presence of hydrogen peroxide rial gene expression and proteome profiles. Moreover, it is not the only factor implied in the loss of culturability will be important to understand the mechanism explain- of these cells, in particular in the case of V. japonicus (Li ing why for some species only a subset of clonal popula- et  al. 2014). To clarify the strains’ behaviour in the pres- tions enter the VBNC state. ence of hydrogen peroxide, we measured the sensitivity In this study, we also observed that Vibrio cells incu- to this compound of both strains during growth in stand- bated in microcosms acquired, over time, a green fluo - ard conditions and their ability to produce catalase. Inter- rescence when stained with PI, which then reverted with estingly, although more sensitive to hydrogen peroxide, resuscitation. Interestingly, we found that for V. japonicus V. campbellii BAA-1117 showed a catalase production UU24, the decrease in PI-green fluorescence was the only far higher than the environmental strains analysed in this phenotypic feature useful to trace the resuscitation pro- study, suggesting that they could have different mecha - cess. Considering that this behaviour is rather difficult to nisms of protection from oxidative stress. However, after explain, but consistent in our data, we should remark that resuscitation, the oxidative stress resistance is gradu- the discovery of a marker of VBNC Vibrio cells would ally acquired along with the time of incubation at 30 °C be of great interest, and that further investigations are (Fig. 4). needed to clarify this observation. Kong and colleagues (2004) measured that hydrogen peroxide concentration in plates of Heart Infusion Agar Supplementary Information was 0.007 mM. We verified that the addition of this The online version contains supplementary material available at https:// doi. hydrogen peroxide concentration to the microcosms’ ali- org/ 10. 1186/ s13213- 022- 01703-6. quots, prior to incubation at 30 °C, prevented the resus- citation and growth of V. campbellii, which was more Additional file 1: Figure S1. Multi-alignment analysis of the partial 16S rRNA gene sequences of the 4 Vibrio strains used in this study. The multi- sensitive to hydrogen peroxide, but not of V. japonicus, alignment was performed with Clustal Omega (EMBL-EBI; https:// www. which required concentrations approximately three times ebi. ac. uk/ Tools/ msa/ clust alo/; version 1.2.4). Figure S2. Appearance of higher. Nevertheless, in both cases, the concentration dwarf populations for the 4 Vibrio spp. strains. Along with a prolonged incubation in cold ASW, a dwarf population appeared, characterized by that could prevent the resuscitation was far lower than an increased green fluorescence upon staining with PI. A, B: V. campbel- the one that could inhibit growth of standard cultures, TM lii BAA-1117 . C, D: V. hepatarius UU21. E, F: V. japonicus M5. G, H, I: V. confirming the hypothesis that VBNC cells are more sen - japonicus UU24. Plots show the size (x axis) and the PI-green fluorescence (y axis) of the microcosm cells after 3 days of incubation (A, C, E, G) or after sitive to oxidative stress (Nowakowska and Oliver 2013). TM 47 days (B, D, F, H). While all V. campbellii BAA-1117 cells are dwarf and This effect was sharper in V. campbellii , which possi- fluorescent with PI after 47 days, for the other strains two populations bly relied more on catalase for the protection against are detectable. For V. japonicus UU24, the dwarfing process is completed Prosdocimi et al. Annals of Microbiology (2023) 73:1 Page 12 of 13 References in 124 days (I). Figure S3. Size (x axis) and green fluorescence (y axis) Arana I, Muela A, Iriberri J, Egea L, Barcina I (1992) Role of hydrogen peroxide TM upon staining with propidium iodide of V. campbellii BAA-1117 and V. in loss of culturability mediated by visible light in Escherichia coli in a japonicus UU24 during the resuscitation process. A, B, C: VBNC-induced freshwater ecosystem. Appl Environ Microbiol 58:3903–3907. https:// doi. TM cells of strain BAA-1117 after 0, 17 and 21 hours of incubation at 30°C, org/ 10. 1128/ aem. 58. 12. 3903- 3907. 1992 respectively. D, E, F: VBNC-induced cells of strain UU24 after 1, 24 and 64 Asakura H, Ishiwa A, Arakawa E, Makino S, Okada Y, Yamamoto S, Igimi S (2007) hours of incubation at 30°C, respectively. During incubation at 30°C in Gene expression profile of Vibrio cholerae in the cold stress-induced “resuscitation” process, a less fluorescent population appeared, which in viable but non-culturable state. Environ Microbiol 9:869–879. https:// doi. TM strain BAA-1117 resulted bigger in size. Figure S4. 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Technol. https:// doi. org/ 10. 1080/ 10643 389. 2021. 19323 97 Connon SA, Giovannoni SJ (2002) High-throughput methods for culturing Authors’ contributions microorganisms in very-low-nutrient media yield diverse new marine EMP and EC conceived the study. EMP, SA, FM, ZZ, MF and EC performed the isolates. Appl Environ Microbiol 68(8):3878–3885. https:// doi. org/ 10. 1128/ experiments and analysed the data. MF sampled the crabs. EMP wrote the AEM. 68.8. 3878- 3885. 2002 first version of the manuscript. SB and DD supported the research. All authors Davey HM, Hexley P (2011) Red but not dead? Membranes of stressed Saccha- critically revised the manuscript and have approved its content. romyces cerevisiae are permeable to propidium iodide. Environ Microbiol 13:163–171. https:// doi. org/ 10. 1111/j. 1462- 2920. 2010. 02317.x Funding Falcioni T, Papa S, Campana R, Manti A, Battistelli M, Baffone W (2008) State The research leading to these results has received funding from the European transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow Union’s Seventh Framework Programme (FP7/2007–2013) under grant agree- cytometry. Cytom Part B - Clin Cytom 74:272–281. https:// doi. org/ 10. ment no. 311975 (MACUMBA). The publication reflects the views only of the 1002/ cyto.b. 20427 authors, and the European Union cannot be held responsible for any use González-Escalona N, Fey A, Höfle MG, Espejo RT, Guzmán C (2006) Quantita- which may be made of the information contained therein. DD has been sup- tive reverse transcription polymerase chain reaction analysis of Vibrio ported by the King Abdullah University of Science and Technology Competi- cholerae cells entering the viable but non-culturable state and starvation tive Research Grant Program 2018-OSR-CRG 2018-3739. in response to cold shock. Environ Microbiol 8:658–666. https:// doi. org/ 10. 1111/j. 1462- 2920. 2005. 00943.x Availability of data and materials Imamura D, Mizuno T, Miyoshi S, Shinoda S (2015) Stepwise changes in viable Data related to DNA sequencing are made available through public databases but nonculturable Vibrio cholerae cells. Microbiol Immunol 59:305–310. as specified in the “Materials and methods ” section. https:// doi. org/ 10. 1111/ 1348- 0421. 12246 Iwase T, Tajima A, Sugimoto S, Okuda K, Hironaka I, Kamata Y, Takada K, Mizunoe Y (2013) A simple assay for measuring catalase activity: a visual Declarations approach. 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Int J Syst Evol Microbiol. 67:1613–1617. https:// doi. org/ 10. 1099/ ijsem.0. 001755 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : lished maps and institutional affiliations. fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Annals of MicrobiologySpringer Journals

Published: Jan 4, 2023

Keywords: Dormancy; Resuscitation; Culturability; Viability; Hydrogen peroxide; Catalase; Flow cytometry; Oxidative stress

References