3.1. Response of Sa17 Steady State Cells to NaCl and Sorbic Acid
Regarding growth of Sa17 the presence of 0.15% sorbic acid and the combination of 2% NaCl and 0.15% sorbic acid somewhat reduced growth initially but overall no major differences could be observed between the different treatments (
Figure 2). The lower pH (5–5.5) in cultures with sorbic acid compared to cultures with NaCl (6–6.5) is most likely the reason for this minor initial reduction. By the fourth hour of incubation, cells grown in the presence of 2% NaCl and in BHI with no additives, appeared to reach the highest OD compared to the other treatments.
S. aureus is known to be a highly osmo-tolerant microorganism, and the obtained results confirm its ability to grow as efficiently under the used NaCl concentrations, as in BHI with no additives [
2].
Figure 2.
Growth of Sa17 steady state cells under exposure to 2% NaCl (◊, solid black line), 0.15% sorbic acid (●, solid light grey line), 2% NaCl + 0.15% sorbic acid (■, solid dark grey line) in batch cultivation conditions. Controls with MMC (×, dashed line) and BHI with no additives (▲, dotted line) were included. Results are obtained from two independent experiments. The Y axis represents OD measured at 620 nm and the X axis the time in hours. Time point 0 h represents the moment when the steady state cells (OD 5.5) were harvested, transferred into flasks and subjected to the various treatments.
Figure 2.
Growth of Sa17 steady state cells under exposure to 2% NaCl (◊, solid black line), 0.15% sorbic acid (●, solid light grey line), 2% NaCl + 0.15% sorbic acid (■, solid dark grey line) in batch cultivation conditions. Controls with MMC (×, dashed line) and BHI with no additives (▲, dotted line) were included. Results are obtained from two independent experiments. The Y axis represents OD measured at 620 nm and the X axis the time in hours. Time point 0 h represents the moment when the steady state cells (OD 5.5) were harvested, transferred into flasks and subjected to the various treatments.
Investigation of the RF and
sea gene copies to assess the impact of the treatments on prophage induction and
sea gene regulation is demonstrated in
Figure 3. Interestingly, it was observed that 2% NaCl increased the levels of RF and
sea gene copies, in a pattern similar to that of the positive control with MMC. Although a less pronounced impact of NaCl compared to that of MMC was obtained, the results indicated that the investigated concentration could trigger prophage induction in an analogous manner as previously observed for the phage-inducing agent [
13,
14]. This is further supported by the observation that in the control condition of BHI with no additives, the RF and
sea gene copy levels remained low throughout the four hours of incubation. The impact of NaCl on prophage induction has also been observed in a study by Harris
et al. [
24], where NaCl concentrations relevant to meat production were investigated for their effect on the levels of the prophage-encoded Shiga toxins of
E. coli O157:H7. It was demonstrated that 2% NaCl triggered prophage induction and increased the levels of
stx2A transcripts and
stx2A DNA abundance, leading to enhanced toxin production.
In contrast to NaCl, the use of 0.15% sorbic acid on Sa17 steady state cells had little impact on the levels of RF and
sea gene copies. The relative ratios for both RF and
sea gene remained below five and was at the most in total about seven folds lower than the levels observed from 2% NaCl (
Figure 3). Similar levels were detected for the combination of 2% NaCl and 0.15% sorbic acid. This finding was interesting as it appeared that use of sorbic acid suppressed the effect of NaCl on prophage induction observed earlier.
Figure 3.
RF (bars) and
sea gene (symbols) copies of Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
) and (Δ), 0.15% sorbic acid (
) and (♦), 2% NaCl + 0.15% sorbic acid (
) and (
×) and the controls of MMC (
) and (○) and BHI with no additives (□) and (■), obtained from two independent experiments. The left
Y axis represents the relative ratio levels, as calculated from Cq values, of RF and
sea gene for MMC control while the right
Y axis represents the relative ratio of RF and
sea gene for NaCl, sorbic acid, NaCl + sorbic acid and BHI with no additives. The
X axis represents the time in hour.
Figure 3.
RF (bars) and
sea gene (symbols) copies of Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
) and (Δ), 0.15% sorbic acid (
) and (♦), 2% NaCl + 0.15% sorbic acid (
) and (
×) and the controls of MMC (
) and (○) and BHI with no additives (□) and (■), obtained from two independent experiments. The left
Y axis represents the relative ratio levels, as calculated from Cq values, of RF and
sea gene for MMC control while the right
Y axis represents the relative ratio of RF and
sea gene for NaCl, sorbic acid, NaCl + sorbic acid and BHI with no additives. The
X axis represents the time in hour.
The absence of effect of sorbic acid on the phage was remarkable as it is known that weak acids can activate the SOS response mechanism and therefore prophage induction would be expected to occur [
16]. In a study by Wallin-Carlquist
et al. [
23] acetic acid was related to prophage induction as it was observed that it boosted
sea expression in the
S. aureus strains, Mu50 and Sa45, at pH 6 and 5.5 respectively. The low levels of RF and
sea gene copies observed in the present study could be attributed to the low pH, close to 5, that characterized the respective cultures. The antimicrobial activity of weak acids lays on their general ability to lower both the extracellular pH in addition to permeating the cell membrane and decreasing the intracellular pH with subsequent inhibition of essential metabolic and anabolic processes [
15]. Therefore at pH 5, the cell could stimulate most of its energy towards re-establishing its homeostasis and thus other processes including protein synthesis and activation of response systems would be stalled. Weinrick
et al. [
25], in an investigation regarding the effects of mild acid on gene expression in
S. aureus, observed that at pH 5.5 the majority of phage-encoded genes were down-regulated compared to pH 7.5.
The physiological state of cells at the time of exposure to a stress factor, and the stress conditions previously encountered by the cell, are known to affect the response of an organism towards further stress imposed. In this study, cells entered stationary phase after the first hour of incubation under the batch conditions, and the response to sorbic acid could therefore be less pronounced than in the case of exponentially growing cells. In the study of Davis
et al. [
26], on acid tolerance of
Listeria monocytogenes, it was shown that resistance against exposure to acid stress was dependent on the growth phase of the cells, with those being in stationary phase showing increased tolerance to lethal pH (pH 3.0) compared to exponentially growing cells. Additionally, the cells in the present study originated from a continuous cultivation, where their metabolism was stimulated towards maintaining a steady growth rate for a number of generations before they were exposed to NaCl and sorbic acid. It could hence be possible that maintenance of balanced growth and metabolism has induced certain adaptive responses in the cell, which became more resistant towards certain stresses. Another explanation could be that the cells, due to their previous growth in a continuous mode, would need more time to respond or adapt to acid stress, since the latter is known to be harsh for bacterial cells.
3.2. Activation of the SOS Response
The activation of the SOS response mechanism due to the use of 2% NaCl and/or 0.15% sorbic acid was investigated through the transcripts of
recA produced. The
recA mRNA transcripts of each treatment were normalized to those of BHI with no additives and the calculated
recA relative ratios are presented in
Figure 4.
A modest increase in the recA transcript levels was noted for 2% NaCl cultures during the first and fourth hour of incubation, reaching the level of around 1.5 in relative transcript ratio. The observed transcription pattern could indicate activation of the SOS response in two phases, either due to a latter activated subpopulation or due to the increasing intracellular accumulation of solutes over time. An interesting observation was noted for the cultures treated with 0.15% sorbic acid and the combination of sorbic acid and NaCl, where increased recA transcript levels, about two in relative ratio, were observed after 3 h of incubation. Though these treatments had no evident effect on the RF and sea gene levels, it appears that SOS activation did actually occur. The low pH of the culture however that affects enzyme activity and protein synthesis could be the parameter influencing the production of SEA. Increased recA transcript levels were observed for MMC treatment during the first hour of incubation, which was expected considering the mechanism of action of this agent.
Figure 4.
Relative
recA mRNA transcript levels of Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
), 0.15% sorbic acid (
), 2% NaCl + 0.15% sorbic acid (
) and the control of MMC (
), obtained from two independent experiments. The
Y axis represents the
recA relative ratio levels as calculated from Cq values and the
X axis the time in h. Values below/above 1 in relative ratio demonstrate down- or up-regulation of
recA in relation to the control condition of BHI with no additives (value of 1).
Figure 4.
Relative
recA mRNA transcript levels of Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
), 0.15% sorbic acid (
), 2% NaCl + 0.15% sorbic acid (
) and the control of MMC (
), obtained from two independent experiments. The
Y axis represents the
recA relative ratio levels as calculated from Cq values and the
X axis the time in h. Values below/above 1 in relative ratio demonstrate down- or up-regulation of
recA in relation to the control condition of BHI with no additives (value of 1).
3.3. SEA production
The impact of the different treatments on SEA production was also evaluated and is presented in
Figure 5.
For 2% NaCl, the SEA levels produced did not correspond to the observations on RF and
sea gene copy levels, since the expected boost of SEA formation was not seen, as in the case of the control with MMC. Overall SEA formation from cultures with 2% NaCl did not exceed 800 ng·mL
−1 (peak at 3 h of incubation), which was below the level of SEA produced under BHI with no additives at the same point of incubation. This difference between the copies of the gene and the SEA produced could be attributed to the suboptimal NaCl concentration for enterotoxin production as well as the state of the cell under osmotic stress. To survive osmotic stress, bacteria are known to accumulate compatible solutes that help them maintain the cytoplasmic osmotic pressure higher than that of the environment. During this maintenance energy state, it is possible that biosynthesis and secretion of exoproteins are down regulated [
27]. Among the solutes accumulated by
S. aureus under NaCl stress are glycine betaine and proline [
28]. These amino acids are also important for SEA synthesis and their absence has been shown to have a negative effect on the yield (%) of the enterotoxin [
29]. For the treatments of 0.15% sorbic acid and combination of NaCl and sorbic acid, the levels of SEA produced were in agreement with what was detected for RF and
sea gene copies, as they remained below 200 ng·mL
−1, which was lower than the SEA produced under BHI with no additives.
Figure 5.
SEA levels produced by Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
), 0.15% sorbic acid (
), 2% NaCl + 0.15% sorbic acid (
) and the controls of MMC (
) and BHI with no additives (□), obtained from two independent experiments. The left
Y axis represents the SEA in ng·ml
−1 for MMC control, while the right
Y axis represents the SEA in ng·ml
−1 for NaCl, sorbic acid, NaCl + sorbic acid and BHI with no additives. The
X axis represents the time in h.
Figure 5.
SEA levels produced by Sa17 steady state cells under exposure in batch conditions to 2% NaCl (
), 0.15% sorbic acid (
), 2% NaCl + 0.15% sorbic acid (
) and the controls of MMC (
) and BHI with no additives (□), obtained from two independent experiments. The left
Y axis represents the SEA in ng·ml
−1 for MMC control, while the right
Y axis represents the SEA in ng·ml
−1 for NaCl, sorbic acid, NaCl + sorbic acid and BHI with no additives. The
X axis represents the time in h.
3.4. The Impact of Physiological Cell History on Prophage Induction
To investigate the impact of the previous physiological state of cells upon subsequent exposure to stress in respect to prophage induction and
sea gene regulation, Sa17 cells were grown overnight (16–18 h) in BHI under optimal conditions (control pre-culture) and BHI + 2% NaCl (NaCl pre-culture), and the resulting pre-cultures were used to inoculate flasks with BHI + 2% NaCl and BHI with no additives (
Figure 1). The effect on growth, RF and
sea gene copy levels, the
recA transcript levels and the SEA produced was investigated under batch cultivation conditions. The results presented derive from one experiment, and more replicates are required to further support the obtained observations.
Regarding growth of Sa17 and irrespective to the pre-culture conditions, when cells were exposed to BHI + 2% NaCl (Co–Na, Na–Na), slightly slower growth was observed compared to the cultures growing in BHI with no further additives (Co–Co, Na–Co) (
Figure 6). The difference in the growth pattern was noted after the fourth hour of incubation. Yet, Sa17 cells originating from the control pre-culture and further grown in BHI + 2% NaCl (Co–Na), were those exhibiting the lowest growth. It can be therefore presumed that pre-cultivation in the presence of NaCl triggered an adaptive response of the
S. aureus cells which were less affected by subsequent exposure to stress compared to optimally pre-grown cells.
Figure 6.
Growth in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal or NaCl stress. The
Y axis represents OD measured at 620 nm and the
X axis the time in hours. The different treatments investigated are designated with different symbols. Co–Co is presented with (●), Co–Na with (
), Na–Co with (
) and Na–Na with (
×). The results are obtained from one experiment.
Figure 6.
Growth in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal or NaCl stress. The
Y axis represents OD measured at 620 nm and the
X axis the time in hours. The different treatments investigated are designated with different symbols. Co–Co is presented with (●), Co–Na with (
), Na–Co with (
) and Na–Na with (
×). The results are obtained from one experiment.
Investigation of the RF and
sea gene copy levels showed a relatively similar increase, for the studied conditions, at the fourth hour of growth, which then dropped for all treatments apart from that of Co–Na (
Figure 7). Interestingly, the latter exhibited a slight increase on both RF and
sea gene copy levels between the sixth and eighth hour of incubation. Considering the observations on growth of Sa17 under the condition of Co–Na, it can be concluded that the impact of osmotic stress, regarding prophage induction, is greater on previously optimally grown cells than in cells pre-exposed to NaCl.
Quantification of the SEA levels formed under the four tested conditions, did not reveal a clear enough pattern for any comparative conclusions to be drawn (data not shown). Nevertheless, of interest was the observation on the SEA levels formed under the Na–Na culture condition, where increased enterotoxin levels were observed at the fourth hour of incubation, compared to the other treatments.
The impact of the previous physiological state of cells on prophage induction was investigated on the
recA transcripts to examine possible activation of the SOS response. The detected
recA transcripts from each treatment were normalized to those of Co–Co (control conditions for both pre-culture and main culture growth) and the calculated relative ratios are demonstrated in
Figure 8. As observed, pre-exposure of cells to NaCl resulted in increased
recA transcript levels from the beginning of incubation; an observation that implies already enhanced
recA transcription from the pre-culture environment. However, in the progression of growth only the Na–Na cultures maintained the enhanced
recA transcription levels, indicating that the cells managed to recover when the conditions of the environment became favorable. For the Co–Na cultures, an increase on the levels of
recA transcripts was observed at the sixth h of incubation, which corresponds time wise with the increase on the levels of RF and
sea gene copies.
Figure 7.
RF (bars) and
sea gene (symbols) copies formed in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal or NaCl stress. The
Y axis represents the relative ratio levels of RF and
sea gene as calculated from Cq values obtained from one independent experiment and the
X axis represents the time in h. The investigated treatments are designated: Co–Co with (
) and (
), Co–Na with (
) and (
), Na–Co with (
) and (
×) and Na–Na with (
) and (
).
Figure 7.
RF (bars) and
sea gene (symbols) copies formed in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal or NaCl stress. The
Y axis represents the relative ratio levels of RF and
sea gene as calculated from Cq values obtained from one independent experiment and the
X axis represents the time in h. The investigated treatments are designated: Co–Co with (
) and (
), Co–Na with (
) and (
), Na–Co with (
) and (
×) and Na–Na with (
) and (
).
Figure 8.
Relative
recA mRNA transcript levels formed in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal and NaCl stress. The
Y axis represents the
recA relative ratio levels in reference to the condition Co–Co, as calculated from Cq values obtained from one independent experiment, and the
X axis represents the time in h. The different growth conditions are designated: Co–Na (
), Na–Co (
) and Na–Na (
).
Figure 8.
Relative
recA mRNA transcript levels formed in batch cultivation of Sa17 cells under exposure to either NaCl stress or optimal conditions. The cells were pre-grown under optimal and NaCl stress. The
Y axis represents the
recA relative ratio levels in reference to the condition Co–Co, as calculated from Cq values obtained from one independent experiment, and the
X axis represents the time in h. The different growth conditions are designated: Co–Na (
), Na–Co (
) and Na–Na (
).
From the results obtained we can conclude that cells pre-exposed to stress were less affected by subsequent exposure to similar stress regarding sea regulation and SEA production, compared to cells previously grown under optimal conditions. A more extensive study would provide more insights on the impact of cell history on subsequent exposure to stress and response in terms of for example SEA production. Investigation of not only osmotic but also other stresses encountered in food production should be included for a complete factorial design construction that will greatly implement the presented observations.