Alternative sigma factor B (σB) and catalase enzyme contribute to Staphylococcus epidermidis biofilm’s tolerance against physico-chemical disinfection

Staphylococcus epidermidis is the predominant cause of recalcitrant biofilm-associated infections, which are often highly resistant to antibiotics. Thus, the use of physico-chemical agents for disinfection offers a more effective approach to the control of S. epidermidis biofilm infections. However, the underlying tolerance mechanisms employed by S. epidermidis biofilm against these physico-chemical disinfectants remain largely unknown. The expression of a σB-dependent gene, alkaline shock protein 23 (asp23) and catalase activity by S. epidermidis biofilm and planktonic cells exposed to heat (50 °C), 0.8 M sodium chloride (NaCl), 5 mM sodium hypochlorite (NaOCl) or 50 μM hydrogen peroxide (H2O2) for 60 minutes were compared. Significantly higher asp23 expression levels were observed in biofilms exposed to 50 °C, 5 mM NaOCl or 50 μM H2O2 compared to the corresponding planktonic cells (p < 0.05). Conversely, asp23 expression levels in biofilm and planktonic cells exposed to 0.8 M NaCl were not significantly different (p > 0.05). Further, biofilms exposed to 50 °C, 0.8 M NaCl, 5 mM NaOCl or 50 μM H2O2 exhibited significantly higher catalase activity than the planktonic cells (p < 0.05). These results suggest that activities of σB and catalase may be involved in the tolerance of S. epidermidis biofilm against physico-chemical disinfection.

. The growth of S. epidermidis cells exposed to increasing physico-chemical disinfectant concentrations. The growth curves for a single mixture of S. epidermidis biofilm or planktonic culture subjected to increasing temperatures (a) or concentrations of NaCl (b), NaOCl (c) or H 2 O 2 (d) for 60 minutes. The plots at each physico-chemical disinfectant concentration depict the mean ± standard deviation of three independent experiments with three technical replicates. The blue dotted arrow represents the optimal temperature/ concentration at which both S. epidermidis biofilm and planktonic cells were considerably stressed (growth reduced by almost 2-fold with reference to the highest CFU value).

S. aureus ATCC 29213
asp23 Forward TCGCTGCACGTGAAGTTAAA 53 Reverse CAGCAGCTTGTTTTTCACCA 16S rRNA Forward GTAGGTGGCAAGCGTTATCC 54 Reverse CGCACATCAGCGTCAG Table 1. Primers used for qPCR amplification.  Table 2. Effect of physico-chemical disinfectant-exposure on asp23 expressions by S. epidermidis cells. The asp23 expression levels of S. epidermidis biofilm and planktonic cells exposed to 50 °C, 0.8 M NaCl, 5 mM NaOCl or 50 μM H 2 O 2 for 60 minutes and the unexposed controls. For each sample, three repeats of asp23 expression measurements were performed. Boldface represent a statistically significant difference between the physico-chemical disinfectant-exposed cells and the unexposed controls as determined by paired-t test (p < 0.05).
www.nature.com/scientificreports www.nature.com/scientificreports/ expression of a σ B -dependent gene, asp23 compared to the corresponding planktonic cells (p = 0.0259; Fig. 2a). Further, the S. epidermidis biofilm or planktonic cells exposed to 50 °C temperature for 60 minutes exhibited significantly increased asp23 expressions than their respective cells exposed to 25 °C temperature (controls) (p < 0.0001; Table 2). Taken together, these results indicated that 50 °C-exposure enhances σ B activity in both S. epidermidis biofilm and planktonic cells, but with significantly higher activity levels in the biofilm cells.
NaCl exposure enhances asp23 expression in biofilm cells, but not in planktonic cells. When exposed to 0.8 M NaCl for 60 minutes, the expression levels of asp23 between S. epidermidis biofilm and planktonic cells were not significantly different (p = 0.4029; Fig. 2b). However, ap23 expression by 0.8 M NaCl-treated S. epidermidis biofilm cells was significantly higher compared to that of the untreated controls (p = 0.0203; Table 2). These findings indicated that 0.8 M NaCl exposure specifically enhances σ B activity in S. epidermidis biofilm cells, but not in the corresponding planktonic cells.
NaOCl or H 2 o 2 exposure elevates asp23 expression to higher extent in biofilm cells than in planktonic cells. The S. epidermidis biofilm or planktonic cells treated with 5 mM NaOCl for 60 minutes exhibited significantly increased asp23 expressions than their respective unexposed controls (p < 0.05; Table 2). Further analysis showed that asp23 expression was significantly higher in S. epidermidis biofilm cells compared to the corresponding planktonic cells upon exposure to 5 mM NaOCl for 60 minutes (p = 0.0109; Fig. 2c). Moreover, S. epidermidis biofilm cells exhibited significantly elevated asp23 expression levels compared to the corresponding planktonic cells upon exposure to 50 μM H 2 O 2 for 60 minutes (p = 0.0020; Fig. 2d). Although the asp23 expressions by S. epidermidis planktonic cells upon 50 μM H 2 O 2 -exposure was not significantly different compared to their respective unexposed controls (p = 0.0725; Table 2), the ap23 expression by 50 μM H 2 O 2 -treated S. epidermidis biofilm cells was significantly higher than in the unexposed controls (p = 0.0002; Table 2). Taken together, these results indicated that 5 mM NaOCl and 50 μM H 2 O 2 -exposure significantly enhances σ B activity in S. epidermidis biofilm cells than in the corresponding planktonic cells.
Heat or NaCl-exposed biofilm cells produce higher amounts of catalase than the planktonic cells. In this study, using a simple visual assay, we quantified catalase activity by measuring the trapped oxygen (O 2 ) gas, which is visualized as foam. The O 2 gas generated during the H 2 O 2 -catalase reaction in the test tubes and how the measurements were done is shown in Fig. 3.
The S. epidermidis biofilm cells exposed to 50 °C for 60 minutes had a significantly higher height of foam compared to the corresponding planktonic cells (p < 0.0001; Fig. 4a). Moreover, a significant increase in height of foam was observed in S. epidermidis biofilm cells exposed to 0.8 M NaCl for 60 minutes compared to the analogous planktonic cells (p < 0.0001; Fig. 4b). Further, S. epidermidis biofilm or planktonic cells exposed to 50 °C or 0.8 M NaCl for 60 minutes exhibited significantly higher heights of foam than their respective unexposed controls (p < 0.0001; Table 3). Taken together, these findings implied that both S. epidermidis biofilm and planktonic cells enhance catalase release in response to 50 °C or 0.8 M NaCl exposure, but with significantly higher release by the biofilm cells.
NaOCl or H 2 o 2 -exposed biofilm cells release more catalase than the planktonic cells. The S. epidermidis biofilm cells subjected to 5 mM NaOCl for 60 minutes had a significantly higher height of foam compared to the corresponding planktonic cells (p < 0.0001; Fig. 4c). Similarly, a significantly higher height of foam was observed in S. epidermidis biofilm cells exposed to 50 μM H 2 O 2 for 60 minutes compared to the planktonic cells (p < 0.0001; Fig. 4d). Further, S. epidermidis biofilm or planktonic cells exposed to 5 mM NaOCl or 50 μM H 2 O 2 for 60 minutes exhibited significantly higher heights of foam than their respective unexposed controls (p < 0.0001; Table 3). Taken together, these results indicated that 5 mM NaOCl or 50 μM H 2 O 2 -exposure stimulates catalase release by S. epidermidis biofilm and planktonic cells, with significantly higher release by the biofilm cells. Test tubes that formed well spread foam as shown in test tubes labeled 1-4 were measured in cm as illustrated in test tube labeled 3 (readings were taken at the bottom of the lower and upper meniscus). Test tubes that did not form a well spread foam as presented in test tube labeled C were not quantified. (2019) 9:5355 | https://doi.org/10.1038/s41598-019-41797-8 www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The mechanisms underlying the tolerance of S. epidermidis biofilm against physico-chemical disinfection remain largely unknown. Therefore, in this study, the activities of σ B and catalase were evaluated as potential tolerance mechanisms employed by S. epidermidis biofilm against heat, NaCl, NaOCl or H 2 O 2 -exposure.
Results presented showed that σ B activity is enhanced in both S. epidermidis biofilm and planktonic cells in response to 50 °C-exposure, but with significantly higher activities in the biofilm cells. The present finding is in agreement with previous reports on B. cereus 15 and B. subtilis 16 . However, the previous studies only focused on the   Table 3. Impact of physico-chemical disinfectant-exposure on catalase activity in S. epidermidis cells. The height of O 2 -forming foam of S. epidermidis biofilm and planktonic cells exposed to 50 °C, 0.8 M NaCl, 5 mM NaOCl or 50 μM H 2 O 2 for 60 minutes and the unexposed controls. Statistical differences between physicochemical disinfectant-exposed cells and the unexposed controls were determined using paired t-test (all the differences were significant; p < 0.05).

Type of cell
www.nature.com/scientificreports www.nature.com/scientificreports/ planktonic forms of the Bacillus species. Microbial metabolic activities are temperature-dependent 30 . Considering that σ B regulates bacterial metabolism 31 and that planktonic cells have higher metabolic activity than biofilm 32 , it was expected that planktonic cells would have a higher σ B activity than the corresponding biofilm cells. However, the observed nearly 2-fold higher σ B activity by S. epidermidis biofilm compared to the planktonic cells suggests that the tolerance of biofilm against heat exposure might be more dependent on σ B activity.
Further, we showed that 0.8 M NaCl-exposure enhances σ B activity in the S. epidermidis biofilm cells, but not in the planktonic cells. Our finding that σ B activities between 0.8 M NaCl-treated planktonic cells and their untreated controls were not statistically different contradicts previous reports on different bacterial species 14,15,17,18 . Considering that NaCl-exposure response regulatory mechanisms in different bacterial species may not follow common patterns 33 , the discrepancy between the present and previous outcomes could be attributed to the different regulatory patterns in planktonic cells of S. epidermidis and the previously studied bacterial species. Although we did not observe a significant difference in σ B activity between NaCl-exposed S. epidermidis planktonic cells and the unexposed controls, some low level of σ B activity was detected. Perhaps the low levels of σ B activity detected in the planktonic cells could be as a result of the basal σ B activity 22 , and not necessarily as a NaCl-exposure response mechanism. A possible explanation for the observed significantly elevated σ B activity levels in biofilm cells, but not in planktonic cells is that tolerance of S. epidermidis biofilm against NaCl-exposure might be more specifically dependent on σ B activity.
Further, our data showed that 5 mM NaOCl-exposure upregulates σ B activity in both S. epidermidis biofilm and planktonic cells, but with significantly higher activities in the biofilm cells. So far, there are no direct reports on the effect of NaOCl exposure on σ B activity. NaOCl-exposure exerts its anti-bacterial effects by targeting several metabolic processes, such as DNA synthesis, adenosine triphosphate synthesis etc. 34 . The same metabolic processes are regulated by σ B activity 31 . Considering that planktonic cells have higher metabolic activity than the corresponding biofilm 32 , planktonic cells should have exhibited higher σ B activity in response to NaOCl exposure. However, we observed the opposite, suggesting that tolerance of S. epidermidis biofilm against NaOCl exposure is more dependent on the σ B activity.
Furthermore, we revealed that 50 μM H 2 O 2 exposure enhances σ B activity in S. epidermidis biofilm cells, but not in the planktonic cells. The present findings concur with a previous report, showing that 50 μM H 2 O 2 has a limited effect on σ B expression by B. cereus cells 15 . Moreover, the present finding agrees with a previous report in which 60 μM H 2 O 2 was shown not to affect σ B expression in S. aureus cells 14 . However, the previous reports only focused on the planktonic forms of B. cereus and S. aureus. It is probable that no σ B expressions were detected in the 60 μM H 2 O 2 -exposed to S. aureus 14 because the study employed northern blot analysis, which is insensitive and only detects large gene expressions 35 . Taking into the findings of the present and previous studies, it appears that σ B activity by planktonic cells is largely independent of H 2 O 2 -exposure. The low levels of σ B activity detactable in planktonic cells in the present and previous studies could be probably be related to the basal σ B activity 22 , but not necessarily as a H 2 O 2 -stress response mechanism. A more plausible explanation for the observed significantly higher σ B activities in the biofilm cells, but not in planktonic cells is that σ B has a more significant role in the S. epidermidis biofilm's tolerance against H 2 O 2 -exposure.
Further results of this study demonstrated that 50 °C-exposure enhances catalase release by S. epidermidis biofilm and planktonic cells, but with higher release by the biofilm cells. The present finding is consistent with two previous reports on a fungus Aspergillus nidulans 36 and Rhodobacter sphaeroides 37 . However, the previous reports only focused on the planktonic forms of the two organisms. Taking into account that temperatures above 37 °C increases the porosity of S. epidermidis biofilm 38 , a probable explanation for the higher catalase release by the biofilms could be related to the increased porosity of the S. epidermidis biofilm by the 50 °C-exposure thus availing more catalase for quantification. Alternatively, catalase release is a metabolic process, which is expected to be higher in planktonic cells compared to the biofilm 32 ; however, S. epidermidis biofilm exhibited higher catalase activity in response to 50 °C-exposure suggesting a more important role of catalase in biofilm's tolerance against heat-exposure.
Further, we showed that 0.8 M NaCl exposure stimulates catalase release by both S. epidermidis biofilm and planktonic cells, but with significantly higher release by the biofilm cells. Our observation that 0.8 M NaCl-treated planktonic cells released higher catalase than the untreated controls is in line with previous reports on different bacterial species 25,26 . Additional analyses demonstrated that 5 mM NaOCl or 50 μM H 2 O 2 -exposure enhances catalase release by S. epidermidis biofilm and planktonic cells, but with significantly more production by the biofilm cells. Our observations that NaOCl or H 2 O 2 -exposed planktonic cells produce significantly higher catalase than the unexposed controls are in agreement with previous reports on different bacterial species 37,39 . However, a contradicting observation has been reported for P. aeruginosa exposed to H 2 O 2 40,41 . The differences in the exposure durations prior to catalase measurements could be responsible for the observed discrepancy between the results of the present and the two previous studies. From the present findings, it appears that catalase activity is involved in both S. epidermidis biofilm and planktonic cells' tolerance against NaCl, NaOCl or H 2 O 2 -exposure. However, considering that catalase production is a metabolic activity, we expected planktonic cells to show significantly higher catalase activity than the biofilm 32 per unit viable cells. We, instead, observed the opposite, suggesting that catalase might be involved to a greater extent in the tolerance of S. epidermidis biofilm against NaCl, NaOCl or H 2 O 2 -exposure.
In conclusion, we observed that 50 °C, 0.8 M NaCl, 5 mM NaOCl or 50 μM H 2 O 2 -exposed S. epidermidis biofilm cells significantly enhance σ B and catalase activities more than the corresponding planktonic cells, suggesting that both σ B and catalase activities might be having a greater contribution in the tolerance of S. epidermidis biofilm against the physico-chemical disinfectants than in the planktonic cells. Therefore, σ B and /or catalase could be explored further as promising targets for the development of more potent anti-staphylococcal biofilm eradication approaches. Nevertheless, further studies incorporating σ B and catalase mutants or promoter reporters are www.nature.com/scientificreports www.nature.com/scientificreports/ required to reach a more definite conclusion regarding the dependence of S. epidermidis biofilm on the activities of σ B and /or catalase enzyme for survival against the physico-chemical disinfectants.

Methods
Sample collection, bacterial isolates and growth conditions. Skin swab samples were collected from outpatients at Kisumu County Referral Hospital (KCRH), Kenya in accordance with relevant guidelines and regulations and research approved by the Maseno University Ethics Review Committee (Reference number: MSU/DRPI/MUERC/000187/15). Further, permission to recruit outpatients was granted by the KCRH. Written informed consent was obtained from participants before recruitment. Collection of the skin swabs and S. epidermidis isolation on mannitol salt agar (HiMedia Laboratories Pvt. Limited, Nashik, India) were conducted as described elsewhere 42 . The S. epidermidis isolates were further identified using routine microbiologic methods namely, Gram staining, catalase, coagulase and novobiocin sensitivity tests and grown on tryptic soy agar (TSA; HiMedia Laboratories Pvt. Limited, Mumbai, India) at 37 °C overnight. Staphylococcus aureus American Type Culture Collection (ATCC) 29213, a good former of mature biofilm within 24 hours 43 , was used as a reference control strain. In this study, analogous planktonic cells served as control samples.
Biofilm was formed as previously described 44 with few modifications. Briefly, a single S. epidermidis colony, from a TSA plate, was inoculated into 2 mL of tryptic soy broth (TSB; HiMedia Laboratories Pvt. Limited, Mumbai, India) and incubated at 37 °C with shaking at 120 revolutions per min (rpm) overnight. To form biofilm culture, 100 µL of the overnight culture adjusted to ~1 × 10 9 colony-forming units (CFU)/mL was simultaneously inoculated into two polystyrene tubes containing 10 mL of fresh TSB supplemented with 1% glucose (Unilab Limited, Nairobi, Kenya), to enhance biofilm formation, and incubated at 37 °C with shaking at 120 rpm for 24 hours. Then, the spent medium in one of the tubes was removed and the biofilm was rinsed twice with 200 μL of 0.9% NaCl. Biofilm formation on the tube was verified by a qualitative tube method biofilm assay with 0.1% crystal violet staining as described in details elsewhere 45 . In case of strong biofilm formation in the first tube, the spent medium in the parallel second tube was carefully removed, and the biofilm was washed twice with 200 μL of 0.9% NaCl. Then, 1 mL of 0.9% NaCl was added to the tube and vortexed for 2 minutes to detach the biofilm cells (vortexing detaches S. epidermidis biofilm from surfaces 46,47 ). The detached biofilm cell suspension was centrifuged at 10,000 rpm, 4 °C for 10 minutes. To grow planktonic cells, 100 µL of the overnight culture adjusted to ~1 × 10 9 CFU/mL was inoculated into 10 mL of fresh TSB in a polystyrene tube and incubated at 37 °C with shaking at 120 rpm for 18 hours. Then, the bacterial cells in suspension were centrifuged at 10,000 rpm, 4 °C for 10 minutes. The biofilm or planktonic cell pellets were suspended in 0.9% NaCl and adjusted to ~1 × 10 9 CFU/mL.

Optimal concentrations of physico-chemical disinfectants for analysis of the tolerance mechanisms.
To minimize variability between samples, the optimal temperature/concentration of each of the physico-chemical disinfectants was determined using a single mixture of S. epidermidis biofilm or planktonic culture as previously described 42 with some modifications. Briefly, 150 μL of ~1 × 10 9 CFU/mL of a single mixture of S. epidermidis biofilm or planktonic culture (prepared by mixing equivalent amount i.e. 150 μL of ~1 × 10 9 CFU/mL of S. epidermidis biofilm or planktonic culture drawn from six random biofilm samples or corresponding planktonic samples) was inoculated into 1.5 mL of increasing concentrations of NaCl, NaOCl (Supersleek, Nairobi, Kenya) or H 2 O 2 (RFCL Limited, New Delhi, India) and exposed for 60 minutes. For heat exposure, tubes containing 1.5 mL of sterile distilled water were inoculated with 150 μL of ~1 × 10 9 CFU/mL of the single mixture of S. epidermidis biofilm or planktonic culture and exposed to increasing temperatures in a water bath for 60 minutes. The NaOCl and H 2 O 2 -exposed cultures were neutralized by 200 μL of 0.1% sodium thiosulphate. Whereas, NaCl and heat-exposed cultures were neutralized by 200 μL of sterile distilled water at 4 °C. To collect biofilm cells, the bacteria in suspension were discarded and the biofilm was gently rinsed once with 200 μL of 0.9% NaCl. One mL of 0.9% NaCl was added to the biofilm, vortexed for 2 minutes then centrifuged at 9,000 rpm for 8 minutes. Planktonic cells were collected by centrifuging the bacteria in suspension at 9,000 rpm for 8 minutes. The biofilm or planktonic cell pellets were suspended in 1 mL of sterile distilled water and CFUs enumerated on TSA as described elsewhere 42 . At each temperature/concentration of the physico-chemical disinfectant, three experiments were performed with three technical replicates. In this study, 50 °C, 0.8 M NaCl, 5 mM NaOCl or 50 μM H 2 O 2 induced considerable stress to the biofilm and planktonic cells hence, used in the subsequent procedure.
Exposure of biofilm and planktonic cells to optimal physico-chemical disinfectants. S. epidermidis biofilm (n = 62) and planktonic (n = 62) samples were exposed to the optimal temperature/concentration of the disinfectants as previously described 48 with some modifications. Briefly, 200 μL of ~1 × 10 9 CFU/mL of biofilm or planktonic suspension were inoculated into 1100 μL of TSB adjusted to 0.8 M NaCl, 5 mM NaOCl or 50 μM H 2 O 2 , vortexed for 2 minutes and incubated at 37 °C with shaking at 80 rpm for 60 minutes. For heat exposure, 200 μL of biofilm or planktonic suspension adjusted to ~1 × 10 9 CFU/mL were inoculated into 1100 μL of TSB alone and transferred to a water bath at 50 °C for 60 minutes. The effects of the disinfectants were neutralized as in the preceding section. Untreated controls were set up by inoculating 200 μL of biofilm or planktonic suspension adjusted to ~1 × 10 9 CFU/mL into 1100 μL of TSB alone and incubated at 37 °C for 60 minutes. A similar set up incubated at 25 °C for 60 minutes served as a control for heat exposure. After incubation, the biofilm or planktonic cells were collected as described in the preceding section. For gene expression analysis, the biofilm or planktonic cells were suspended in 0.9% NaCl, adjusted to ~1 × 10 9 CFU/mL and immediately transferred into an equal volume of a 1:1 mixture of ice-cold acetone and ethanol, then kept at −80 °C for at least 20 minutes. For catalase activity analysis, viable biofilm and planktonic cells were enumerated in duplicate on TSA as described elsewhere 46 and adjusted to 1 × 10 7 cells/mL. (2019) 9:5355 | https://doi.org/10.1038/s41598-019-41797-8 www.nature.com/scientificreports www.nature.com/scientificreports/ Quantification of asp23 expression. Total RNA was isolated from the disinfectant-exposed S. epidermidis biofilm (n = 10) and planktonic (n = 10) samples and the unexposed controls as previously described 49 . Genomic DNA was degraded using DNase I (New England Biolabs, Ipswich, England) following the manufacturer's instructions. The purity and concentration of RNA were determined as previously described 50 . RNA samples with an OD 280 /OD 260 ratio between 1.8 and 2.2 were used for complementary DNA (cDNA) synthesis. Two μg of RNA was reverse transcribed into cDNA using ProtoScript ® First Strand cDNA Synthesis kit (New England Biolabs, Ipswich, England) following the manufacturer's instructions. Quantitative-PCR (qPCR) was performed on a Rotor-Gene Q real-time thermal cycler (Qiagen, Hilden, Germany) as previously described 51 with few modifications. Briefly, each 20 μL of qPCR reaction mixture contained 10 μL of Lunar Universal qPCR mastermix (New England Biolabs, Ipswich, England), 0.5 μL each of the forward and reverse primers (Inqaba Biotechnical, Pretoria, South Africa) specific for 16S rRNA (reference) and asp23 (target) genes (Table 1), 7 μL of nuclease-free water and 2 μL of cDNA template. The thermal cycling conditions were as follows: 1 minute at 95 °C, followed by 45 cycles of 15 seconds at 95 °C, 30 seconds at 60 °C. A control lacking the M-MuLV enzyme mix was included in each reaction. The asp23 expression level was determined using the Efficiency ΔCt method, where, ΔCt = Ct (reference gene)−Ct (target gene) 50 for the disinfectant-exposed samples and their unexposed controls. All primers had an efficiency of ~100%; hence, the real efficiency i.e. 2 was substituted in the Efficiency ΔCt formula. For each isolated RNA, three repeats of asp23 expression measurements were performed. For each sample, asp23 expression level was expressed as percentage change by 100 × [{asp23 expression (exposed cells) − asp23 expression (unexposed control) }/ asp23 expression (unexposed control) ].
Quantification of catalase activity. Catalase activities of the disinfectant-exposed S. epidermidis biofilm (n = 62) and planktonic (n = 62) samples and the unexposed controls were determined by a simple visual assay as previously described 52 with few modifications. Briefly, 100 μL of ~1 × 10 7 of viable biofilm or planktonic cells/ mL was transferred to a Pyrex test tube (1.3 centimetre (cm) diameter by 10 cm height). Then, 100 μL each of 1% Triton X-100 (Rohm and Haas, Philadelphia, USA) and 30% H 2 O 2 were added, mixed thoroughly and incubated at 25 °C. Once reaction stopped, the height of foam that remained constant for 15 minutes was measured in cm using a ruler. For each sample, three repeats of catalase activity measurements were performed. For each sample, height of foam was expressed as percentage change by 100 × [{height of foam (exposed cells) − height of foam (unexposed control) } ⁄ height of foam (unexposed control) ].

Statistical analysis.
Statistical analysis was performed with GraphPad Prism version 5.03. Data normality was verified by D' Agostino and Pearson omnibus test. Differences between groups were determined using two-tailed paired t-test. Data are presented as the mean ± standard error of mean (SEM). A p-value less than 0.05 was considered significant.

Data Availability
All data generated or analyzed during this study are included within this article and its Supplementary Information files.