Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

Biofilm examination using compound brightfield and scanning electron microscopes (SEM). In order to study the effect of DSE on the biofilm morphology, the formed biofilm by P. aeruginosa with (treated isolates) and without (untreated isolates) DSE was examined using a BFM and SEM. A remarkable reduction in the biofilms formed by P. aeruginosa was revealed in 15 isolates. A representative example of the visible inhibition of the formed biofilm is shown in Figs. 4 and 5. www.nature.com/scientificreports/ Quantification of inhibition of violacein production. DSE exhibited an inhibitory effect on the pigment produced by the standard isolate Chromobacterium violaceum (ATCC 12472) in a concentration-dependent mode, as shown in Fig. 6. Maximum violacein inhibition of 44.9 ± 0.9% was observed at a DSE concentration of 1000 µg/ml.       In vivo biofilm inhibition assay:. Naked eye examination of the burned skin revealed that the skin area of the control (group I) showed no apparent change. In group II (subgroup IIA), the burned area was partly covered with a brown dissected scab with exudate formation, and the size of the wound did not exhibit an apparent change (Fig. 9A). After 11 days of burn induction (subgroup IIB), the burned area was covered entirely by scab, and some areas showed necrosis. The size of the wound was nearly the same as in the previous subgroup IIA (Fig. 9B).
In group III (subgroup IIIA), the burned area appeared reddish in colour with less redness around it compared to group II. Additionally, it was dry with neither exudate nor necrosis. There was no scab, and the wound size   www.nature.com/scientificreports/ was apparently smaller than that of the burn model group II (Fig. 9C). Eleven days after the treatment with DSE (subgroup IIIB), the burned area was markedly smaller. The edges showed healing as represented by coverage by the epidermis with hair (Fig. 9D). Examination with hematoxylin and eosin (H&E) using a brightfield microscope revealed that the skin sections of the control group had two layers of skin: the epidermis and dermis. The epidermis consisted of squamous keratinized epithelium that was stratified, and the dermis consisted of connective tissue. It was divided into two  www.nature.com/scientificreports/ layers without a sharp demarcation: a superficial thin papillary layer and a deep thick reticular layer. Hair follicles were found in the dermis (Fig. 10A). The H&E examination of thin skin sections of group IIA revealed an exfoliated upper part of the epidermis. The basal layer cells of the epidermis appeared flattened, and the collagen fibers in the upper dermis appeared hypocellular. Moreover, the collagen fibers appeared deeply acidophilic and fused with loss of the distinction between the collagen fibers. Skin appendages such as hair follicles and associated sebaceous glands were damaged, leaving empty spaces (Fig. 10B). The H&E examination of thin skin sections of group IIB showed an epidermal loss, and a scab was formed covering the whole wounded area. The scab showed a deeply acidophilic structure composed of fine fibers and cells. The collagen fibers in the upper dermis appeared hypocellular and deeply acidophilic (Fig. 10C). The deep part of the dermis contained extravasated RBCs, mononuclear inflammatory cellular infiltration, and loose fine collagen fibers (Fig. 10D).
Examination of H&E thin skin sections of group IIIA revealed preservation of a thin epidermis. The epidermis consisted of one layer of flattened keratinocytes with flat nuclei laid on a straight basement membrane. The underlying dermis presented thick and fused collagen fibers (Fig. 10E). H&E staining of group IIIB stained sections revealed epidermal reepithelization, which appeared thick and consisted of stratified squamous epithelium with a thin coating of keratin. The epidermis was relatively thicker than that of the previous subgroup but still thinner than that of the control group, and it showed recesses of the hair follicles. The papillary layer of the dermis was more cellular and had numerous fibroblasts, capillaries, and fine collagen fibers. The reticular dermis revealed large aggregates of collagen bundles that contained thick collagen fibers (Fig. 10F).
SEM examination of thin skin sections from the control group revealed that the epidermis' outer layer (stratum corneum) had a tough coating made up of overlapping layers of dead skin cells (Fig. 11A).
Examination of SEM of thin skin sections of group IIB showed a marked increase in the aggregates like structures formed by P. aeruginosa bacteria to a degree higher than that of group IIA. These aggregates, like structures, masked the skin's outer surface and extended deeply into the dermis (Fig. 11B,C). www.nature.com/scientificreports/ Examination of SEM of thin skin sections of group IIIB showed a marked reduction of P. aeruginosa bacteria on the skin's outer surface, with the appearance of nearly normal stratum corneum to a degree higher than that of group IIIA. The dermis also showed a few aggregates of P. aeruginosa bacteria in between collagen fibers (Fig. 11D,E). Figure Table 3).

Phytochemical constituents of DSE.
Cytotoxicity assay. The sulforhodamine B (SRB) assay was used to determine if DSE was cytotoxic to the human skin fibroblast (HSF) cell line 8,9 . DSE was compared to doxorubicin (a positive control), which had a halfmaximal inhibitory concentration (IC 50 ) of 4.36 ± 0.52 µg/ml, and DSE had an IC 50 of 9.4 ± 3.3 µg/ml against the HSF cell line as shown in Fig. 13.

Discussion
Recently, natural products have been regarded as a vital source for detecting many new therapeutic compounds 10 . D. spinulosum exhibited a variety of promising biological activities, including cytotoxic, antioxidant, and antimicrobial potential. This motivated us to undertake this study to search for an alternative effective natural source. Bacteria that are capable of forming biofilms are resistant to many antibiotics. This issue resulted in difficulty in curing biofilm-related infections 11 . It has been established that such bacteria depend mainly on two related phenomena: QS and biofilm formation 12 . The formation of biofilms by pathogenic P. aeruginosa isolates is mainly controlled by QS regulatory genes. Thus, anti-quorum sensing substances are usually investigated for inhibition of biofilm formation 13 . It is found that the commercially available anti-quorum sensing compounds could increase the in vitro and in vivo susceptibility of bacteria embedded in biofilms to antibiotics 14 .
In the current study, the anti-quorum sensing and anti-biofilm activities of DSE were investigated against P. aeruginosa clinical isolates. The MIC values of DSE were determined, and they ranged from 250 to 1000 µg/ml. The impact of DSE on CSH, EPS quantity, biofilm formation, metabolic activity using MTT, biofilm morphology, and pigment production by C. violaceum (ATCC 12472) was assessed at sub-inhibitory concentrations (0.5 MIC values, i.e., at concentrations ranging from 125 to 500 µg/ml) to exclude the effect of DSE on bacterial growth. In addition, its impact on the relative gene expression of the QS and biofilm genes was investigated.   www.nature.com/scientificreports/ Polysaccharides present in the biofilm matrix could provide various benefits to the bacterial cells embedded in biofilms, like adhesion and protection. They can act like glue, which permits the bacterial cells to adhere to each other as well as different surfaces. Moreover, polysaccharides could protect the cells in biofilms from desiccation and immune effectors 16 . Herein, we found that DSE exhibited a significant reduction (p < 0.05) in the extracellular polysaccharides' quantity in 51.4% of P. aeruginosa isolates. The primary mechanism for such a reduction in EPS production after treatment with DSE needs further investigation.
There are two critical steps in biofilm production. The first step involves bacterial adhesion to different surfaces, which provides the groundwork for further biofilm development.
The second step involves the interactions between cells. At this stage, bacteria form microcolonies to construct a multilayer structure for biofilm development 17 . In the current study, the biofilm inhibition by DSE was evaluated using the crystal violet test. DSE decreased the percentage of P. aeruginosa isolates strongly and moderately producing biofilms from 77.1 to 34.3%. Determination of CFU by agar plating is considered as the gold standard method for quantification of biofilm 18 . The main principle of this test is to quantify the living bacterial cells using an agar plate as they grow forming colonies that could be counted 19 . There was a significant reduction in the number of CFU/ml in 34.3 % of the isolates after treatment with DSE.
MTT assay was used to detect the effect of DSE on the viability of bacteria embedded in the biofilm. Viable bacterial cells with active metabolism will have the capability to convert MTT into formazan (purple colour) with an absorbance of 550 nm. On the other hand, dead bacterial cells lose this capability. So, purple colour formation is a marker for viable cells only 20 . A non-significant change (p > 0.05) in the bacterial cells' viability was observed after treatment with DSE. To better understand the effect of DSE on biofilm formation by P. aeruginosa isolates, biofilm morphology was inspected using a compound bright field microscope and SEM. Microscopical examination confirmed DSE's biofilm inhibitory action against the tested isolates, as a significant reduction in the biofilms formed by P. aeruginosa isolates was observed after treatment with DSE.
Violacein is a dark purple indole derivative agent. Its production is essential for QS and hence biofilm formation. A reference strain, C. violaceum (ATCC 12472), is known for its ability to produce violacein, and thus, it is widely utilized for QS studies 21 . DES showed an anti-quorum sensing activity against C. violaceum (ATCC 12472) as it inhibited violacein production in a concentration-dependent manner.
qRT-PCR was utilized to inspect the impact of DSE on QS and biofilm formation genes in 15 P. aeruginosa isolates. Our results revealed that treatment with DSE decreased the expression of lasI, lasR, rhlI, rhlR, and ndvB genes in 46.67%, 40%, 60%, 53.33%, and 66.67% of the selected P. aeruginosa isolates, respectively.
The current study produced a partial thickness skin burn injury in rat groups II and III by scalding with hot water (99 °C) for 3 s 22 . Moreover, a mould was created to restrict the burn injury to a small uniform area of 1.1% of the total body surface area in the back to avoid shock and the death of rats 23 . Examination of group II (burn model) sections stained by H&E showed a range of epidermal thinning, exfoliation, and necrosis. This came per Tanaka et al. 24 . In addition, Yang et al. 25 found that the skin epidermis in deep second-degree burns was almost entirely dissected from the underlying dermis.
Meanwhile, the underlying dermis in the present study showed collagen denaturation in the form of swollen, fused, and closely packed collagen fibers. Similarly, Park et al. 26 noticed that when the skin was thermally damaged, the fibers had a glass-like appearance from the dense coagulation of collagen. On the other hand, Tanaka et al. 24 detected a loss of boundaries between collagen fibers and attributed this to water leakage from capillaries, causing edema and fusion of collagen fibers. The hair follicles with their associated glands were completely damaged in group II. Younan et al. 27 detected the same finding.
SEM examination of group II showed biofilm formation. Chu et al. 26 stated that owing to the ability of P. aeruginosa to create biofilms on both biotic and abiotic surfaces within hours, and it could form biofilms within the burn eschar (a collection of dead tissues). These biofilms could serve as a nidus for long-term wound infection and transplant failure. In addition, P. aeruginosa also expresses various proteases, including collagenase, which may aid it in entering burned skin 22 . Furthermore, the biofilm infection causes an inflammatory response in the local burn skin infected with P. aeruginosa, as evidenced by increased circulating neutrophils and increased myeloperoxidase activity. The inflammatory cells seen in H&E -stained tissue sections mainly were neutrophils. www.nature.com/scientificreports/ Meanwhile, in group III, treatment with DSE resulted in epidermal regeneration, which was started by the migration of cells from the basal layer of adjacent healthy skin. DSE significantly improved wound contracture and healing. Furthermore, it appeared to improve the healing process by regenerating the epidermis and reducing inflammatory cell infiltration associated with collagen fiber deposition compared to the infected group. It also showed nearly intact hair follicles. This comes in agreement with Negm et al. 7 , who stated that DSE showed higher activity against Gram-negative bacteria, like E. coli and P. aeruginosa, than Gram-positive bacteria.

Materials and methods
Bacteria. Thirty-five P. aeruginosa isolates were collected from Tanta University hospitals. These clinical isolates were subjected to microscopical examination, and they were identified using standard biochemical tests according to MacFaddin 28 . Pseudomonas aeruginosa ATCC 27853 was utilized as the standard isolate.
Plant material. Dioon spinulosum Dyer Ex Eichler leaves were selected from El Abd Garden in Giza city on January 14th, 2017. The permission for plant collection was obtained from the landowner, Mr. Rabea Sharawy. Then, Dr. Esraa Ammar (a plant ecology lecturer at Botany Department, Faculty of Science, Tanta University) and Mr. Rabea Sharawy (an agronomist and palm researcher were) kindly identified the plant. A voucher specimen number: PGG-002 was deposited at Pharmacognosy Department herbarium, Faculty of Pharmacy, Tanta University.
One kilogram of shade-dried powdered leaves of D. spinulosum was extracted by cold maceration with MeOH (3L×3, 48 h each) then concentrated under vacuum using a rotary evaporator at 45 °C to yield the crude extract (87.2 g) that were used for biological investigations.

Determination of MICs of DSE against the tested isolates.
The MICs of DSE were determined by the broth microdilution method 29 . Each plate contained a positive control (untreated bacteria) and negative control (non-inoculated well).
All the following experiments were performed before and after treatment of the tested isolates with subinhibitory concentrations of DSE (0.5 MIC values).

Determination of CSH.
It was determined in the tested isolates before and after treatment with DSE (with concentrations of 0.5 MIC values that ranged from 125 to 500 µg/ml) according to the method previously explained 30 . After centrifugation of the bacterial suspensions, the pellets were resuspended in phosphate urea magnesium sulfate buffer (PUM buffer, pH 6.9). Then, several volumes of n-hexane (0.3, 0.9, 1.2, and 1.8 ml) were added to 4.8 ml of each bacterial suspension in the PUM buffer and vortexed. The suspension was allowed to stand for complete phase separation. Finally, the absorbance of the bacterial isolates that remained in the aqueous phase was measured at 540 nm. The HI was determined as follow: Quantification of EPS. EPS extraction and quantification were carried out as previously described 31 .
Briefly, the tested isolates were overnight grown at 37°C in Luria-Bertani (LB) broth with (treated isolates) and without (untreated isolates) DSE (using concentrations ranging from 125 to 500 µg/ml). After incubation, they were centrifuged, and the pellets were resuspended in phosphate-buffered saline (PBS) buffer and centrifuged again. Then, ethyl alcohol was added to the supernatant with an equal volume and centrifuged. Finally, 1 ml of the EPS solution was thoroughly mixed with an equal volume of cold 5% phenol plus 5 ml concentrated sulfuric acid. The optical intensity (OD) of the produced red colour was measured at 490 nm, and the percentages of inhibition of EPS were calculated.
In vitro biofilm inhibition assay. Biofilm formation was examined in the tested isolates before and after treatment with DSE (with concentrations ranging from 125 to 500 µg/ml) using the crystal violet test 32,33 . The OD at 490 nm was measured using an ELISA reader (Sunrise Tecan, Grodig, Austria). The tested isolates were characterized into four groups based on their ODs as follows: i. NBP: non-biofilm producer (ODc < OD < 2 ODc) ii. WBP: weak biofilm producer (2 ODc < OD < 4 ODc) iii. MBP: moderate biofilm producer (4 ODc < OD < 6 ODc) iv. SBP: strong biofilm producer (6 ODc < OD) The cut-off OD (ODc) is the mean OD plus 3 SD of the negative control. with PBS so that the loosely attached cells were removed. Then, the biofilms were scraped off the wells through vigorous scraping using a pipette tip after adding 200 μl PBS. They were then vortexed in order to homogenize the biofilm. After that, they were serially diluted, and 100 μl from each dilution was plated on Muller-Hinton agar (MHA) plates. After overnight incubation, the CFU were counted 18 .
Estimation of the metabolic activity of biofilm by MTT assay. The MTT colorimetric assay was performed as previously described 30,34 . Briefly, the bacterial suspensions were overnight incubated with (treated isolates) and without (untreated isolates) DSE (using concentrations ranging from 125 to 500 µg/ml). To each well of the microtitration plate, 150 µl of PBS and 50 µl of MTT solution were added. Then, the MTT solution was withdrawn after 2 h, and 150 µl of dimethyl sulphoxide (DMSO) was added instantly as a solubilizing agent. Finally, the absorbance was measured at a wavelength of 550 nm by an ELISA reader (Sunrise Tecan, Grodig, Austria).
Examination of biofilm morphology. The effect of DSE was tested against biofilm formation by P. aeruginosa isolates on glass surfaces as previously described 35 . Biofilm formed by P. aeruginosa with (treated isolates) and without (untreated isolates) DSE (using concentrations ranging from 125 to 500 µg/ml) was visualized by a compound bright field microscope (Labomed, California, America) using 40× magnification after staining with crystal violet.
Scanning electron microscope (SEM). SEM was used to examine the bacterial biofilms formed on the surfaces of cover glass with (treated isolates) and without (untreated isolates) DSE (using concentrations ranging from 125 to 500 µg/ml) as described previously 36,37 , and examined using an S-3400N scanning electron microscope (Hitachi, Tokyo, Japan).

Quantification of inhibition of violacein production. It was performed as described by Ganesh and
Rai 31 . DSE (with concentrations ranging from 62.5 to 1000 µg/ml) was added to a 10 µl suspension of C. violaceum (ATCC 12472) in LB broth and incubated overnight at 30 °C. Centrifugation was performed after incubation, and 500 µl of DMSO was added to each pellet and vortexed vigorously till the extraction of violacein (purple colour). The extracted violacein (200 µl) was transferred into a 96 well microtitration plate and quantified using an ELISA reader (Sunrise Tecan, Grodig, Austria) at 585 nm. The control was the bacterial culture of C. violaceum in LB broth without the DSE.
qRT-PCR. The expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) 38 , and the biofilm gene (ndvB) were identified using qRT-PCR, and the 16S rRNA gene was utilized as the housekeeping gene 39 . All the experiments were conducted in triplicate, and the results were expressed as mean± SD values. According to the manufacturer's instructions, after extraction of the total RNA, using the GeneJET RNA purification kit, K0731 (Thermo Scientific, Waltham, USA), it was retrotranscribed into cDNA by using a power cDNA synthesis kit (iNtRON Biotechnology, Seongnam, Korea). The utilized primers are shown in Table S1. The amplification was performed using the Power SYBR ® Green master mix (Thermo SCIENTIFIC, Waltham, USA) and the Rotor-Gene Q 5plex apparatus (Qiagen, Hilden, Germany). The relative gene expression was calculated by using the 2 −ΔΔCt method 40 . Non-treated isolates were used as control samples (i.e., expression set to 1).
In vivo biofilm inhibition assay. All  Cytotoxicity assay. The SRB test was used to measure HSF cell viability 8,9 . Aliquots of 100 µl cell suspension (5×10 3 cells) were incubated in full medium for 24 h in 96-well plates. A different aliquot of 100 µl medium and the DSE at varied doses was utilized to treat the cells. The cells were fixed by replacing the medium with 150 µl of 10% trichloroacetic acid (TCA) and incubated for 60 min at 4 °C after 72 h of exposure to DSE. The HSF cells were rinsed five times with distilled water, and liquors of 70 μl SRB solution (0.4% w/v) were added and kept in a dark place for 10 min at room temperature. Plates were washed three times with 1% acetic acid and left to dry overnight in the air. Finally, 150 µl of Tris base solution (10 mM) was added to dissolve the protein-bound SRB stain, and the absorbance was measured at 540 nm using an ELISA reader.
Statistical analysis. All the performed assays were carried out in triplicates, and the results were presented as mean ± SD. The statistical significance of the results was determined by one-way ANOVA using SPSS software (IBM, USA), and only results with a p < 0.05 were considered significant. Statistical analysis: Ethical approval. All experiments were performed in accordance with the relevant guidelines and regula-

Conclusion
The current study revealed the potential in vitro and in vivo anti-biofilm and anti-quorum sensing activities of DSE as it exhibited efficient activity against P. aeruginosa clinical isolates. To our knowledge, this is the first time to report the ability of DSE to reduce biofilm formation by P. aeruginosa via downregulation of quorum sensing genes. This finding could help in the reduction of our significant dependence on antibiotics. Moreover, this could help us efficiently handle the biofilm-related infections caused by such opportunistic pathogens. Thus, further studies are necessary to examine the possible inhibitory effects of DSE on biofilms formed by other pathogens. In addition, preclinical and clinical studies are required to enable its practical implementation in managing P. aeruginosa biofilm-related infections.