Survival of Bacillus spp. SUBB01 at high temperatures and a preliminary assessment of its ability to protect heat-stressed Escherichia coli cells
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The bacterial stressed state upon temperature raise has widely been observed especially in Escherichia coli cells. The current study extended such physiological investigation on Bacillus spp. SUBB01 under aeration at 100 rpm on different culture media along with the high temperature exposure at 48, 50, 52, 53 and 54 °C. Bacterial growth was determined through the enumeration of the viable and culturable cells; i.e., cells capable of producing the colony forming units on Luria–Bertani and nutrient agar plates up to 24 h. Microscopic experiments were conducted to scrutinize the successive physiological changes. Suppression of bacterial growth due to the elevated heat was further confirmed by the observation of non-viability through spot tests.
As expected, a quick drop in both cell turbidity and colony forming units (~104) along with spores were observed after 12–24 h of incubation period, when cells were grown at 54 °C in both Luria–Bertani and nutrient broth and agar. The critical temperature (the temperature above which it is no longer possible to survive) of Bacillus spp. SUBB01 was estimated to be 53 °C. Furthermore, a positive impact was observed on the inhibited E. coli SUBE01 growth at 45 and 47 °C, upon the supplementation of the extracellular fractions of Bacillus species into the growing culture.
Overall the present analysis revealed the conversion of the culturable cells into the viable and nonculturable (VBNC) state as a result of heat shock response in Bacillus spp. SUBB01 and the cellular adaptation at extremely high temperature.
KeywordsHigh temperature stress Bacillus spp. Colony forming units (CFUs) Viable but non culturable (VBNC) cells
Bacillus species are well known spore-forming pathogenic bacteria which are frequently found in the environment. Like Escherchia coli, these bacterial species may encounter a number of growth retarding stress factors, including nutrient depletion, temperature fluctuation, variation in pH and redox potential, limited water activity (aw), elevated level of reactive oxygen species (ROS), osmotic imbalance along with unusual solute concentrations, etc. [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15]. In response to such stress causing stimuli, different bacterial species have been observed to employ various defensive strategies to cope with the stress signals [11, 16, 17]. To deal with the heat stress, a number of reports suggested the expression of the global molecular chaperones and other components (CspB and CspE in Bacillus spp. cells, GroEL and DnaK proteins in Salmonella spp., rpoE, rpoS and rpoH genes in E. coli and Pseudomonas spp.) to combat against the stress as well as to maintain the cellular homeostasis [5, 6, 13, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34].
Our earlier studies unraveled the influence of the temperature up-shift with the generation of oxidative stress retarding the amount of viable and culturable bacterial cells [5, 12, 13, 35]; spontaneous accumulation of the ROS not only at the beginning of the early stationary phase but also by the supplementation of hydrogen peroxide (H2O2) in the growing culture [2, 5, 32, 34] and the effect of different aeration speed on the cellular capability to produce the colony forming units on agar plates . In all instances, the physiological response of E. coli SUBE01, Pseudomonas spp. SUBP01, Salmonella spp. SUBS01 and Bacillus spp. SUBB01 against oxidative stress was observed through their sustainability in retaining the culturable cells [5, 12, 13, 32, 33, 34, 36]. Besides, the information on the defense strategy of these bacteria especially those belonging to Bacillus spp. SUBB01 under the static condition was evidently noticed through their phenotypic behavior . Along these lines of information, current study was conducted to further scrutinize the heat-shock response in Bacillus spp. SUBB01 under the shaking condition at 100 rpm in different culture media.
Demonstration of culturable Bacillus spp. SUBB01 under heat stress
Laboratory stock culture of Bacillus spp. SUBB01 and E. coli SUBE01 were used in this study. Experiments demonstrating the bacterial growth in terms of cell turbidity (optical density at 600 nm) and colony forming units (CFUs) were conducted as described earlier by Nur et al. . Nutrient agar (NA), Luria–Bertani (LB) agar, nutrient broth (NB) and Luria–Bertani broth were used for the assay of culturability . After 24 h of incubation on nutrient agar plates at 37 °C, one loopful of the bacterial culture was introduced into 5 ml nutrient broth followed by incubation at 37 °C for 4–6 h at 100 rpm (pre-culture). After adjusting the optical density of the pre-culture at 600 nm (OD600) to 0.1, 30 µL each was introduced into 2 different sets of 30 ml of nutrient broth and Luria–Bertani broth and incubated at 48, 50, 52, 53 and 54 °C at shaking condition (100 rpm). At the time points of 12 and 24 h, the cell growth was monitored by measuring OD600 and by counting the colony forming units (CFUs) . All the experiments were conducted three times. Statistical analysis regarding bacterial growth was performed by determining P value through t test. Standard deviations for all data have been indicated by error bars.
Assessing cell viability was further confirmed by the spot tests [13, 32, 33, 34]. As described previously, each the culture suspension was serially diluted in 9 ml nutrient broth to obtain up to 10−4 fold dilution. From each dilution, an aliquot of 5 µl was dropped on to the nutrient agar and Luria–Bertani agar, dried off for 15 min, and finally the plates were incubated at 37 °C for 24 h. Spotting on the agar was accomplished at 24 h of growth.
Demonstration of morphological changes
Simple staining (Crystal Violet, Hucker’s Solution) was conducted to assess the viability and the cellular morphology as previously done [32, 33, 34]. Spore staining (malachite green oxalate, safranin O) was conducted to differentiate the bacterial spores from vegetative cells following standard procedures . An aliquot of 10 µl from the bacterial culture suspension was removed at 24 h of growth, and the cellular morphology, shape and organization were observed under the light microscope (Optima Biological Microscope G206, manufactured in Taiwan) at 1000× magnification .
Preparation of organic and inorganic supplements
To prepare the extracellular fractions of bacteria (E. coli and Bacillus species), cells were grown separately in 6 different sets of Durham’s bottle containing 5 ml minimal broth, which were kept in a shaking water bath at 100 rpm for 24 h at 37 °C (optimum growth temperature) . Subsequently, actively growing bacterial cells were centrifuged at 4000 rpm for 15 min, and the resulting pellets were collected. Afterward pellets were centrifuged at 4000 rpm for 15 min for 3 times with 10 % glycerol and 50 mM CaCl2, respectively. The resulting supernatants were collected and used as organic supplements to observe the possible retrieval of E. coli cell viability both at high temperature stress (45 °C) and around the critical temperature (47 °C). Subsequently, a mixture of 20 mM MgSO4 and 5 mM ethylene diamine tetraacetic acid (EDTA) were used as inorganic supplement to conduct a similar experiment .
Results and discussions
Growth retardation of Bacillus spp. SUBB01upon heat shock
Confirmative demonstration of loss of culturability of Bacillus spp. SUBB01cells
As stated earlier, our previous investigations on stress responses in E. coli SUBE01 showed the influence of the temperature up-shift resulting in oxidative stress  which further led us to investigate the stress response against external and internal oxidative stress stimuli within Bacillus spp. SUBB01, E. coli SUBE01, Pseudomonas spp. SUBP01 and Salmonella spp. SUBS01 [5, 12, 13, 32, 33, 34, 36]. Those studies clearly revealed that E. coli SUBE01 lost viability upon heat shock . Moreover, the external and internal oxidative stresses in the early stationary phase of E. coli SUBE01 and Pseudomonas spp. SUBP01 culture were found to influence the formation of culturable cells; i.e., capable of forming colonies [2, 32, 33, 34]. With a succession of those work, the results presented in this study showed that Bacillus spp. SUBB01 is likely to exhibit the alteration in cellular homeostasis and culturability due to heat shock at 48–53 °C, under aeration (100 rpm) condition on different culture media. Notably, cells were found to lose culturablility completely at 54 °C, wherein spores were observed under light microscope (Fig. 2). Previously several studies reported that Bacillus cells exhibits six classes of heat shock genes upon environmental stress and the activation of the heat shock genes of Bacillus species especially depends on the commencement of specific temperature [45, 46].
Demonstration of growth retrieval
As stated earlier, our previous studies unraveled the defensive strategies of various bacterial species against heat shock and oxidative stress [5, 12, 13, 32, 33, 34, 35, 36]. Recently the response within yeast cells against heat stress and osmotic shock has also been observed . While the mechanisms of survival of E. coli cells have clearly been chalked out very recently , the retrieval of a heterogeneous E. coli population consisting of viable cells and defective cells (incapable of forming colonies on agar plates) by the Bacillus extracts as found in the current study is being reported for the first time so far to our knowledge. Such an experimental demonstration could be of significance to understand the cellular survival strategies mediated by different bacterial species.
Despite the lack of molecular investigation as well as an apparent impression of descriptive nature of research stipulation, the data in the current study is quite consistent to the previous findings with the novel projection on the critical growth temperature of Bacillus strain. Moreover, the work clearly illustrated the phenotypic changes in the bacterial cell caused by the heat shock at the optimum speed of aeration which, unlike to that of E. coli, is relatively new in the field of heat shock response in Bacillus cells. Such preliminary findings could be worth incrementing the existing knowledge on bacterial cell biology and signal transduction. Finally, the observation of E. coli growth retrieval upon supplementation of the extracellular fractions from Bacillus spp. have been indeed interesting to ponder on the heat stress resistance mechanisms of Bacillus spp. However, further molecular studies on the genetic makeup of such stress responses as well as the growth retrieval mechanisms by means of exogenous organic factors (Bacillus extracts) would be of greater effectiveness.
This work was carried out in collaboration between all authors. Author MSM managed the analyses of the study and wrote the first draft of the manuscript. Authors JT and MMHA performed the experiments. Author ITN managed the literature searches. Author RN designed the study and critically revised the manuscript. All authors read and approved the final manuscript.
We thank Stamford University Bangladesh for providing us the facilities to carry out the experiments. However, the authors received no specific funding for this work.
All the authors are from Department of Microbiology, Stamford University Bangladesh. Authors MSM, JT and ITN are thesis students of MS program and MMHA is thesis students of BSc. (Hons) program of the department. Author RN is corresponding author of the manuscript, has been working as Associate Professor and Chairman of the Department of Microbiology, Stamford University Bangladesh.
The authors declare that they have no competing interest.
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