{"id":50659,"date":"2023-09-30T10:34:56","date_gmt":"2023-09-30T10:34:56","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=50659"},"modified":"2023-10-07T11:33:02","modified_gmt":"2023-10-07T11:33:02","slug":"assessment-of-anti-biofilm-activity-of-ferns-against-nosocomial-pathogenic-bacteria","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/assessment-of-anti-biofilm-activity-of-ferns-against-nosocomial-pathogenic-bacteria\/","title":{"rendered":"Assessment of Anti-biofilm Activity of Ferns Against Nosocomial Pathogenic Bacteria"},"content":{"rendered":"\n

I<\/strong>ntroduction<\/strong><\/strong><\/p>\n\n\n\n

Nosocomial bacterial infections are a serious health concern around the\nworld, leading to increased morbidity, mortality, treatment costs, and hospital\nadmissions. These nosocomial pathogens are well known to have acquired\nresistance to a large number of antibiotics in current use. The increase of\nmultidrug resistant strains has necessitated the development of novel treatment\nstrategies. Quorum sensing (QS) inhibition is considered one of the attractive\ntargets as many pathogenic bacteria employ QS to modulate their pathogenicity1<\/sup>. Bacterial QS is a mechanism that relies on the release and uptake of\nsmall signaling molecules called auto-inducers (AI),\nthe production of which is dependent on the density of the bacterial population\nin the surrounding medium2<\/sup>.The\nproduced AIs play a major role in regulating the gene expression, which in turn\ncontrols various bacterial responses such as bio-film formation, production of virulence factors,\nmotility, the establishment of genetic competence, and fluorescence3<\/sup>. With\nthe use of QS inhibitors antimicrobial resistance, persistence, and virulence\ncould be addressed in pathogenic bacteria. The major advantage of using the QS\ninhibitors is that they do not kill bacteria but rather control bacterial\nvirulence factor production without encouraging the development of bacterial\nresistance4<\/sup>.<\/p>\n\n\n\n

Synthetic QS inhibitors have been shown to successfully suppress QS in\nbacteria. However, due to the presence of certain compounds, they are\nconsidered to be hazardous to human health and have found limited application5<\/sup>.Because\nof their high reactivity and instability, the majority of the synthetic anti-QS\ncompounds have yet to be qualified as chemotherapeutic agents5<\/sup>. Considering this, there is a compelling need for discovering novel\nanti-QS compounds with low toxicity and high reactivity. Natural sources are\nbeing investigated for potential therapeutic and antipathogenic compounds that\ncould operate as non-toxic QS inhibitors, allowing infections to be controlled\nwithout the development of bacterial resistance. Plants are appraised to\npossess a wide range of phytochemicals exhibiting various kinds of activities\nagainst microorganisms. Ferns, a group of vascular plants found in abundance in\nthe tropics have been used in Ayurveda and other traditional medicine to treat\ndifferent ailments6<\/sup>.\nAlthough a few researchers have worked on the antimicrobial activity of these\nferns, studies on QS modulators are lacking. Considering this, the current\nstudy intends to identify ferns that have anti-QS efficacy against a few\nnosocomial bacterial pathogens.<\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

Ferns used and preparation of crude extracts <\/strong><\/p>\n\n\n\n

Fresh plant material from eight different ferns was collected from Mangaluru and neighbouring districts, situated in South Karnataka. The ferns were washed with distilled water and dried at 40\u00baC for 48-72 h in a hot air oven, pulverized, and subjected to extraction. The list of ferns used for the current study is provided in Table 1.<\/p>\n\n\n\n

Table 1: List of ferns used in the study<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Sl. <\/strong>No.<\/strong><\/p>\n<\/td>\n

\n

Common name <\/strong><\/p>\n<\/td>\n

\n

Scientific name <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1.<\/p>\n<\/td>\n

\n

Black maidenhair<\/p>\n<\/td>\n

\n

Adiantumphilipense<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

2.<\/p>\n<\/td>\n

\n

Whisk fern<\/p>\n<\/td>\n

\n

Psilotumnudum<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

3.<\/p>\n<\/td>\n

\n

Oak leaf fern (leaf and rhizome)<\/p>\n<\/td>\n

\n

Drynariaquercifolia<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

4.<\/p>\n<\/td>\n

\n

Sheevothi<\/p>\n<\/td>\n

\n

Sellaginelladelicatula<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

5.<\/p>\n<\/td>\n

\n

Rock fern<\/p>\n<\/td>\n

\n

Cheilanthestenuifolia<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

6.<\/p>\n<\/td>\n

\n

Lady fern<\/p>\n<\/td>\n

\n

Athyriumhohenackerianum<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

7.<\/p>\n<\/td>\n

\n

Starry spike moss<\/p>\n<\/td>\n

\n

Sellaginella<\/em>wildtype<\/p>\n<\/td>\n<\/tr>\n

\n

8.<\/p>\n<\/td>\n

\n

Dixie silverback fern <\/p>\n<\/td>\n

\n

Pityrogrammacalomelanos<\/em><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Sequential extraction of pulverized fern material\nwas performed according to a\npreviously described method by Dharajiyaet al<\/em>. (2017)7<\/sup> with\nminor modifications. To 100 ml of hexane, 10g of the pulverized\nmaterial was mixed and incubated in a shaker for 24 h at 37\u00baC. Filtration of\nthe obtained extract was done using a clean muslin cloth. Second and third\nextractions were carried out using an additional 100ml of solvent. A rotary\nflash evaporator was used to concentrate the extracts under reduced pressure.\nThe plant material used for the extraction was allowed to dry and once\ncompletely dried was further used for extraction with other solvents such as\nacetone, ethanol, and water. The extracts thus obtained were weighed and stored\nat 4\u00baC until further use.<\/p>\n\n\n\n

Bacterial strains<\/strong><\/p>\n\n\n\n

Chromobacteriumviolaceum<\/em>\nMTCC 2656, Chromobacteriumviolaceum<\/em>MCC 2216 Acinetobacter baumannii<\/em>\nATCC 19606, Escherichia coli <\/em>ATCC 25922, Klebsiella pneumoniae <\/em>ATCC 700603, Staphylococcus aureus <\/em>ATCC 25923,\nand Pseudomonas aeruginosa<\/em> ATCC 15692. All the ATCC strains were procured from Himedia, the authorized\ndistributors of ATCC products and C.\nviolaceum<\/em> cultures were purchased from MTCC, Chandigarh.<\/p>\n\n\n\n

Assessment\nof antibacterial activity <\/strong><\/p>\n\n\n\n

The antibacterial activity of different\nsolvent extracts was assessed against the test bacteria by the disc diffusion\nmethod as per the method of Alva\net al. (2019)8<\/sup>.1ml of DMSO was used for dissolving 50mg of\nthe dried extracts. Approximately 1×106<\/sup> CFU\/ml cultures of A.\nbaumannii, E.  coli, K. pneumoniae, S. aureus<\/em>, and P. aeruginosa<\/em>\nwere used to lawn nutrient agar plates. The plates were allowed to dry and\nsterile discs impregnated with 50\u03bcl of extract were placed. 50\u03bcl of DMSO served\nas the negative control and ciprofloxacin at a concentration of 50\u03bcg\/disc\nserved as the drug control. The plates were incubated for 24 h at 37\u00baC. The\nzone of inhibition around the sample extract in comparison to the control was\nconsidered to be positive.<\/p>\n\n\n\n

Determination of minimum inhibitory\nconcentration (MIC)<\/strong><\/p>\n\n\n\n

Various concentrations of extracts ranging from 0.25mg\/mL \u2013\n40mg\/mL in DMSO were freshly prepared. Approximately 1×106<\/sup> CFU of 24h culture\nof A. baumannii, E.  coli, K. pneumoniae, S. aureus,<\/em> P.\naeruginosa, <\/em>and C. violaceum <\/em>were used to lawn the\nagar plates onto which discs impregnated with different concentrations of the\nextracts were positioned. 50\u03bcl of the solvent served as the negative control.\nThe plates were incubated overnight and a clear zone of inhibition around the\nsample extract at different concentrations was noted down. The MIC values were\nascertained to the least concentration where there was no visible growth. <\/strong><\/p>\n\n\n\n

Determination\nof anti-QS activity using C. violaceum<\/em>MTCC2656<\/strong><\/p>\n\n\n\n

The method of Alva et\nal. (2019)8<\/sup> was used to determine the anti-QS activity of essential\noils.Sterile\ndiscs were soaked in different concentrations of the extracts (50 \u00b5l) at\ntheir sub-MIC levels. Such discs were placed on previously inoculated nutrient\nagar plates. After overnight incubation, the plates were examined for the\npigment inhibition surrounding the disc, and the diameter of the pigment\ninhibition was measured (mm). C. violaceum <\/em>MCC 2216 a mutant strain for violacein production was\nmaintained as the negative control.<\/p>\n\n\n\n

Biofilm\nquantification<\/strong><\/p>\n\n\n\n

Biofilm formed by\nthe bacterial strains was quantified by the microtitre plate method according\nto the method of O’Toole (1998)9<\/sup>\nwith minor modifications. 1×106<\/sup>CFU\/mL of the test bacteria grown in\nLuria Bertani broth was diluted with fresh medium in the ratio of 1:100. In\neach well of the 96 well microtitre plate 100\u03bcl of the diluted culture was\ntaken and incubated for 24 h at 37\u00b0C. After incubation, the plate was inverted,\nunattached cells were discarded, and adherent cells in the medium were washed\nthrice with PBS of pH 7.4. The pellet was mixed with 100\u00b5l of 0.1% freshly\nprepared crystal violet and incubated for 10 min. The stain removal was carried\nout by washing the pellet with PBS three times. For stain solubilization, 125\u00b5l\nof 30% acetic acid was added to the stained and rinsed pellet and incubated for\n15 minutes. From each well 100\u00b5l was transferred to a fresh plate and optical\ndensity was measured at 550 nm in an ELISA reader. The percent reduction in\nbiofilm formation in the presence of plant extracts was measured as (OD of\ncontrol – OD of treated) \/ (OD of control) x100<\/p>\n\n\n\n

Statistical analyses <\/strong><\/strong><\/p>\n\n\n\n

The data were subjected to one sample t-test using SPSS, 16.0 software\nand was considered to be significant at P \u2264 0.05.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Antibacterial\nactivity<\/strong><\/p>\n\n\n\n

Variation in the zones of inhibition was observed against the selected bacterial pathogens. The zones of inhibition were in the range of 8-17mm with different extracts. None of the bacterial strains showed susceptibility to water extracts. The solvent control (DMSO) exhibited a zone of 0.7\u00b10.1 mm. Of the selected pathogens used in the study, S. aureus<\/em> was found to be more susceptible to extracts of different ferns and K. pneumoniae<\/em> showed the highest resistance.  The highest activity was observed with hexane extract of C. tenuifolia<\/em>against K. pneumoniae<\/em> followed by hexane extract of S. delicatula<\/em>against E. coli<\/em>. A. hohenackerianum, Selaginella<\/em>wildtype, P. calomelanos, <\/em>and D. quercifolia<\/em>(rhizome) were excluded from the further investigation as they did not exhibit any antibacterial activity. The remaining 5 fern extracts were considered for MIC determination. Table 2 illustrates the zones of inhibition of different fern extracts against the selected pathogenic bacteria.<\/p>\n\n\n\n

Table 2: Zones of inhibition (mm) against the selected bacterial pathogens<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Extracts<\/p>\n<\/td>\n

\n

Fern<\/p>\n<\/td>\n

\n

E. coli<\/em><\/p>\n<\/td>\n

\n

A. baumannii<\/em><\/p>\n<\/td>\n

\n

K. pneumoniae<\/em><\/p>\n<\/td>\n

\n

S. aureus<\/em><\/p>\n<\/td>\n

\n

P. aeruginosa<\/em><\/p>\n<\/td>\n<\/tr>\n

\n

Hexane<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

8\u00b10<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

12.5\u00b10.7<\/p>\n<\/td>\n

\n

11\u00b10.1<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

12.5\u00b10.7<\/p>\n<\/td>\n

\n

10\u00b10<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em>leaf<\/p>\n<\/td>\n

\n

13.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

12\u00b11.4<\/p>\n<\/td>\n

\n

12\u00b10.2<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

16.5\u00b10.7<\/p>\n<\/td>\n

\n

13.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

12\u00b10<\/p>\n<\/td>\n

\n

11\u00b10.4<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

12.5\u00b10.7<\/p>\n<\/td>\n

\n

8\u00b10<\/p>\n<\/td>\n

\n

17\u00b11.4<\/p>\n<\/td>\n

\n

11\u00b11.4<\/p>\n<\/td>\n

\n

10\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

A.  hohenackerianum<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Sellaginella wildtype<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

P. calomelanos<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em> rhizome<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Acetone<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

11.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

10.5\u00b10.7<\/p>\n<\/td>\n

\n

15.5\u00b12.12<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

10\u00b12.8<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

10.5\u00b12.1<\/p>\n<\/td>\n

\n

14.5\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em>leaf<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

10.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

11\u00b11.4<\/p>\n<\/td>\n

\n

8.5\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

9\u00b10<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

11.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

12.5\u00b10.7<\/p>\n<\/td>\n

\n

9.5\u00b10.7<\/p>\n<\/td>\n

\n

12\u00b11.4<\/p>\n<\/td>\n

\n

11.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

A.  hohenackerianum<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Sellaginella wildtype<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

P. calomelanos<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em> rhizome<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Ethanol<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

10\u00b10<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

11\u00b12.8<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

9\u00b11.4<\/p>\n<\/td>\n

\n

9.5\u00b10.7<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

10.5\u00b13.5<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em>leaf<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

9\u00b10<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

10\u00b14.2<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

12\u00b12.8<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

13\u00b11.4<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

9\u00b11.4<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

A.  hohenackerianum<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Sellaginella wildtype<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

P. calomelanos<\/em><\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em> rhizome<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Drug Control (Ciprofloxacin)<\/p>\n<\/td>\n

\n

22\u00b11.4<\/p>\n<\/td>\n

\n

24\u00b11.4<\/p>\n<\/td>\n

\n

27.5\u00b13.5<\/p>\n<\/td>\n

\n

23.5\u00b10.7<\/p>\n<\/td>\n

\n

26\u00b12.8<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

MIC\ndetermination<\/strong><\/p>\n\n\n\n

Fernextracts from the initial antibacterial screening that showed activity against selected pathogens were assessed for their MIC values. The concentration of the extracts in the range of 0.25-40 mg\/mL was used (Table 3). The least MIC values for the test bacteria ranging from 3.0-10.0 mg\/mL and 0.50-2 mg\/ml for C. violaceum<\/em> were considered for further anti-QS activity studies. <\/p>\n\n\n\n

Table 3: MIC determination of fern extracts against selected pathogens<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Extract<\/strong><\/p>\n<\/td>\n

\n

Ferns<\/strong><\/p>\n<\/td>\n

\n

E. coli<\/em><\/strong><\/p>\n<\/td>\n

\n

K. pneumoniae<\/em><\/strong><\/p>\n<\/td>\n

\n

S. aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

A. baumannii<\/em><\/strong><\/p>\n<\/td>\n

\n

P. aeruginosa<\/em><\/strong><\/p>\n<\/td>\n

\n

C. violaceum<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Hexane<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

– <\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

  – <\/p>\n<\/td>\n

\n

20mg<\/p>\n<\/td>\n

\n

0.50mg<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

3mg<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

20mg<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

20mg<\/p>\n<\/td>\n

\n

8mg<\/p>\n<\/td>\n

\n

6mg<\/p>\n<\/td>\n

\n

0.50mg<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

4mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

8mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

1mg<\/p>\n<\/td>\n<\/tr>\n

\n

Acetone<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.50mg<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

4mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

20mg<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

2mg<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

30mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

4mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

Ethanol<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

  – <\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

0.75mg<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

10mg<\/p>\n<\/td>\n

\n

  – <\/p>\n<\/td>\n

\n

 –<\/p>\n<\/td>\n

\n

2mg<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Determination\nof anti-QS activity<\/strong><\/p>\n\n\n\n

Fern extracts with concentrations lower than their MIC values were used for assessing the anti-QS activity using C. violaceum<\/em>. The diameter of the zone of pigment inhibition surrounding the discs was measured. Hexane extract of S. delicatula<\/em> at a concentration of 0.25mg, acetone extract (0.25mg) of A. philippense, <\/em>and ethanol extract of C. teneifolia<\/em> (0.75mg) produced the highest pigment inhibition (Table 4).<\/p>\n\n\n\n

Table 4: Anti-QS activity of the extracts against C. violaceum<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Extract<\/strong><\/p>\n<\/td>\n

\n

Fern<\/strong><\/p>\n<\/td>\n

\n

Concentration (mg\/mL)<\/strong><\/p>\n<\/td>\n

\n

Zone of pigment Inhibition (mm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Hexane<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

10\u00b11.4<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

0.50<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

0.50<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

11.5\u00b12.1<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

0.75<\/p>\n<\/td>\n

\n

9.5\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

Acetone<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

11.5\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

0.50<\/p>\n<\/td>\n

\n

9\u00b11.4<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

1.00<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

9.5\u00b10.7<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n<\/tr>\n

\n

Ethanol<\/p>\n<\/td>\n

\n

A. philippense<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

9\u00b11.4<\/p>\n<\/td>\n<\/tr>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

0.25<\/p>\n<\/td>\n

\n

10.5\u00b12.1<\/p>\n<\/td>\n<\/tr>\n

\n

D. quercifolia<\/em><\/p>\n<\/td>\n

\n

0.50<\/p>\n<\/td>\n

\n

10\u00b11.4<\/p>\n<\/td>\n<\/tr>\n

\n

S. delicatula<\/em><\/p>\n<\/td>\n

\n

0.50<\/p>\n<\/td>\n

\n

10\u00b11.4<\/p>\n<\/td>\n<\/tr>\n

\n

C. teneifolia<\/em><\/p>\n<\/td>\n

\n

0.75<\/p>\n<\/td>\n

\n

11\u00b11.4<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Biofilm\nquantification<\/strong><\/p>\n\n\n\n

Although all 5 ferns used for assessing the anti-QS activity against C. violaceum<\/em> were found to be effective in inhibiting violacein production at their sub-MIC values, only P. nudum<\/em> and C. teneifolia<\/em> were selected for evaluating the biofilm inhibition. The wide spectrum of activity of these two ferns against the selected pathogens made us select them for biofilm assay. There was a significant variation in the biofilm formed by bacteria in the presence of fern extracts at their sub-MIC values. The highest biofilm inhibition was found with hexane extract of P. nudum<\/em> against S. aureus<\/em> followed by ethanol extract of C. tenuifolia<\/em>against E. coli<\/em>at concentrations of 9.75mg\/mL. The percent reduction in biofilm formation by the test bacteria in the presence of plant extracts is shown in table 5. <\/p>\n\n\n\n

Table 5: Percent reduction in biofilm formation in the presence of plant extracts<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Organism<\/strong><\/p>\n<\/td>\n

\n

Fern<\/strong><\/p>\n<\/td>\n

\n

Extract<\/strong><\/p>\n<\/td>\n

\n

% reduction<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

3.75mg\/mL<\/strong><\/p>\n<\/td>\n

\n

9.75 mg\/mL<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

E. coli<\/em><\/p>\n<\/td>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

Hexane<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

33.89<\/p>\n<\/td>\n<\/tr>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Acetone<\/p>\n<\/td>\n

\n

27.45<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Ethanol<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

80.39<\/p>\n<\/td>\n<\/tr>\n

\n

S. aureus<\/em><\/p>\n<\/td>\n

\n

P. nudum<\/em><\/p>\n<\/td>\n

\n

Hexane<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

82.07<\/p>\n<\/td>\n<\/tr>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Acetone<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

37.25<\/p>\n<\/td>\n<\/tr>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Ethanol<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

75.35<\/p>\n<\/td>\n<\/tr>\n

\n

P. aeruginosa<\/em><\/p>\n<\/td>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Hexane<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

79<\/p>\n<\/td>\n<\/tr>\n

\n

K. pneumoniae<\/em><\/p>\n<\/td>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Hexane<\/p>\n<\/td>\n

\n

28.29<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

A. baumannii<\/em><\/p>\n<\/td>\n

\n

C. tenuifolia<\/em><\/p>\n<\/td>\n

\n

Acetone<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

57.61<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 1: <\/strong>Representative images for MIC determination against A. baumannii <\/em>using acetone extract of Drynaria quercifolia <\/em>(A) and anti-QS activity against C. violaceum <\/em>using different extracts (B).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Over the last two decades, drug resistance has tremendously increased\nand resistant bacteria are considered one of the critical threats to human\nhealth. The development of novel approaches to tackle drug resistance is the\nneed of the hour. Inhibition of QS is one such alternative strategy that can be\neffectively employed for managing illnesses caused by resistant bacteria.\nAccording to the World Health\nOrganization, plants are regarded as the best source of antimicrobial compounds10<\/sup>. In India, herbal medicines\nhave long been used to treat a number of ailments. The use of pteridophytes has\nbeen mentioned in ancient Indian systems of medicine such as Ayurveda, Unani, and Siddha11<\/sup>. Although very few researchers have worked on\nits antibacterial activity, to the best of our knowledge, there are no reports\non the anti-QS activity of ferns. The lacuna of studies in this area prompted\nus to undertake this investigation with the aim of screening ferns for their\nanti-QS activity against multidrug resistant bacteria.  <\/p>\n\n\n\n

Initially, sequential extraction of the dried powder of ferns with\nsolvents of increasing polarity was carried out. Dried powder of the extracts\nwas used to prevent the interference of water during extraction. Researchers\nusually employ different solvents for initial extraction as the target compound\nremains to be unknown and different polarities of solvent help in extracting\ndifferent classes of phytochemicals12<\/sup>. Similarly in the present study, different\nsolvents varying in their polarities were used for sequentially extracting the\nanti-QS compounds. The study utilized five strains of bacteria that are reported as the\ncausative agents of serious nosocomial infection13,14<\/sup>. The difference in the antibacterial\nactivity in fern extracts might be ascribed to the phytochemicals found in the\nferns and the bacteria tested. It can also be attributed to the polarity of the\nextractable compounds found in ferns, polarity of solvents used, and propensity\nof the extracted compound to diffuse in the media used for the assay15<\/sup>. In the current investigation, none of the\naqueous extracts showed antibacterial activity indicating that the compounds of\ninterest have a different polarity than water.<\/p>\n\n\n\n

The study employed C. violaceum<\/em> as the biomonitor strain\nfor screening the anti-QS compounds from ferns as demonstrated by the reduction\nin the violacein production. Earlier reports on medicinal plants have indicated\na positive association between antibacterial and anti-QS activity 16,17<\/sup>. Contemplating this\nhypothesis, the current study selected the ferns with antibacterial activity to\nassess the anti-QS potential. We could establish a positive correlation between\nthe antibacterial and anti-QS activity as reported in the earlier studies. <\/p>\n\n\n\n

Not many studies have\nbeen carried out to evaluate the antibacterial and anti-QS activity of ferns\nagainst pathogenic bacteria. In a study byAdnan et al<\/em>. (2020)18<\/sup>, the crude extract of A. philippense<\/em>showed\nthe antibacterial activity against selected strains of E. coli, S.\naureus, <\/em>and P. aeruginosa<\/em> at concentrations ranging\nfrom 31-1000 \u03bcg\/mL. Although, A. philippense<\/em>in our investigation\ndid show the antibacterial activity against E.\ncoli, S. aureus, <\/em>and P. aeruginosa<\/em>,\nthe concentration at which the activity was seen was higher as compared to the\nstudy by Adnan. In the current investigation, none of the D. quercifolia<\/em> rhizome extracts showed antibacterial activity\nagainst the tested pathogens. On the contrary, studies by Irudayaraj and\nSenthamarai (2004) and Kandhasamyet al<\/em>. (2008)19,20<\/sup> observed\nantibacterial activity of D. quercifolia<\/em>\nrhizome against E. coli, P. aeruginosa,\nS. aureus, <\/em>and K. pneumoniae<\/em>. The\nreason for this difference in the activity could be ascribed to the bacterial\nstrains used and the extraction procedures employed 21<\/sup>.<\/p>\n\n\n\n

Microbial biofilms are\nregarded as one of the important virulence factors of pathogenic bacteria by\nthe virtue of which they resist antimicrobial agents. Such biofilms formed on\nbiotic and abiotic surfaces comprises of microbial assemblages with\nconsiderable changes in their gene expression along with the metabolic activity\nconferring them resistance to antimicrobial treatment22<\/sup>. More importantly, in a\nnumber of pathogenic bacteria causing chronic infection, biofilm formation is\nindicated as an important factor that aids in their survival2<\/sup>3<\/sup>. The hexane extract of P. nudum<\/em> and ethanol extract of C. tenuifolia<\/em> showed\na remarkable reduction in QS activity as indicated by the reduction in biofilm\nformation at a concentration of less than 10mg\/mL in S. aureus<\/em> and E.\ncoli<\/em>. Notably, the concentrations used represent that of the crude\nextracts, and hence, the concentration would be much lower when the presumed\nanti-QS compound is isolated and purified from these ferns. Although, studies\ndo not exist revealing the biofilm inhibition ability of P. nudum <\/em>and C.\ntenuifolia<\/em> extracts, a study by Adnan et al<\/em>. (2020)1<\/sup>8<\/sup> has disclosed the anti-biofilm activity of a\nfern, A. philippense<\/em> at a much lesser concentration.The anti-biofilm activity of the P.\nnudum <\/em>and C. tenuifolia<\/em> extracts could be attributed\nto the presence of tannins, phenolics, saponins, flavonoids, and terpenoids\neither alone or in combinations23<\/sup>.  The mechanism of anti-biofilm\nactivity of the extracts can be speculated due to the degradation or\nmodification of the signaling molecules or a decrease in the synthesis of the\nsignaling molecules24<\/sup>. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The quest for alternative therapeutic resources\nis the resultant effect of drug resistance in pathogenic bacteria. The use of\nQS inhibitors could prove to be an effective and appealing antipathogenic\ntherapy to combat the emergence of antibiotic resistance in bacteria. The study\ndraws special attention to investigate the uncharted anti-QS potential of ferns\nbesides the usual assessment of traditional medicinal plants. The present\ninvestigation examines the use of ferns, the compounds of which could be successfully\nused as anti-QS inhibitors in future in treating infections caused by multidrug\nresistant strains of bacteria. The presence of QS inhibitory compounds in the\nleaves of P. nudum<\/em> and C. tenuifolia<\/em>effectively\ninhibit the biofilm formation in S. aureus<\/em> and E. coli<\/em>\nrespectively thus reducing the virulence of these resistant pathogens. Further\nstudies on these plants are required to isolate and identify the anti-QS\ncompounds which could serve as a promising therapeutic option in controlling\nthe virulence of pathogens.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The authors are thankful to Nitte (Deemed to be\nUniversity) for all the facilities provided.<\/p>\n\n\n\n

Conflict of interest<\/strong><\/p>\n\n\n\n

There is no\nconflict of interest by all the authors.<\/p>\n\n\n\n

Funding sources<\/strong><\/p>\n\n\n\n

The work was supported by the Nitte University grant (N\/RG\/NUSR2\/NUCSER\/ 2020\/12).<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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