Introduction

Bacterial infections linked to biofilm pose a serious threat to mankind as they show considerable drug resistance against a variety of antibiotics (Liu et al. 2020; Yu and Chua 2020). In this regard, the literature reports have stated that nearly 80% of the microbial infections are linked to biofilm (Chakraborty et al. 2021). Biofilm happens to be a conglomeration of microbial population often adhering to various biotic or abiotic surfaces by producing a self-secreted matrix commonly termed as the extracellular polymeric substance or EPS (Gupta et al. 2016). This matrix has been reported to play a pivotal role in the survival of the bacteria inside the biofilm. In addition, they have also been found to protect the bacteria by preventing the diffusion of the antimicrobials through EPS (Cortes et al. 2011). Several organisms have been found to form biofilm to ensure protection from external stress (Anderson and O’Toole 2008; Hall-Stoodley and Stoodley 2009). One such biofilm-forming organism is Staphylococcus aureus which is a Gram-positive, spherical-shaped opportunistic pathogen. It has been found to cause various infections in the human host including skin and soft tissue infections (Azmi et al. 2019). Moreover, they can penetrate the barriers of skin by either wound or incision and form biofilm on tissues and/or medical implants (Archer et al. 2011). Staphylococcal biofilm often causes diverse chronic infections such as endocarditis, osteomyelitis, cystic fibrosis lung infections, medical device implants, and so on (Paharik and Horswill 2016). Hence, efficient strategies need to be adopted urgently to manage the biofilm threats to weaken the infections (Römling and Balsalobre 2012; Vuotto and Donelli 2019). The existing literature reported that biofilm-linked challenges could be controlled by either obstructing the development of biofilm or disintegrating the pre-existing biofilm (Gupta et al. 2016; Liu et al. 2020). It has been mentioned that S. aureus can develop biofilm in three steps namely microbial adherence, maturation followed by dispersion (Otto 2013). Thus, agents interfering with the mentioned steps could hold the prospect to inhibit the formation of microbial biofilm. Towards this direction, we had already reported that 1,4-naphthoquinone could efficiently inhibit the formation of microbial biofilm by S. aureus (Paul et al. 2021). However, the effect of the same compound on the disintegration of pre-existing biofilm was not tested so far. Moreover, the disintegration of preformed biofilm remains a major area of concern as effective solutions are yet to be explored in this direction. Thus, our present study targets to examine whether the same compound (1,4-naphthoquinone) can exhibit any biofilm disintegration properties against S. aureus. Considering all our experimental results, it can be concluded that 1,4-naphthoquinone was found to efficiently disintegrate the pre-existing biofilm of S. aureus.

Materials and methods

Microbial strain, growth media, and culture conditions

The test organism that we have considered for our present study is S. aureus (MTCC 96). We have received this organism as a kind gift from Dr. Surajit Bhattacharjee (Department of Molecular Biology and Bioinformatics, Tripura University, India). The test organism was cultivated in Tryptone soya broth (TSB) medium (Himedia, India) at 37 °C for 24 h to get the optimum growth of the organism. In the present study, 1,4-naphthoquinone (purchased from Sigma) was used to examine whether the molecule could disintegrate the pre-existing biofilm.

Analysis of biofilm formation by S. aureus

To examine the time-dependent biofilm formation by S. aureus, an equal number of cells (1 × 105 CFU/mL) was inoculated in several tubes containing 5 mL of sterile TSB media. To it, autoclaved coverslips were separately added to allow the formation of microbial biofilm over it. All the tubes were then incubated at 37 °C for varying periods (6 h, 12 h, 18 h, and 24 h). Crystal violet (CV) assay was performed to measure the degree of biofilm formation of S. aureus by following the protocol described by Chakraborty et al. (2018a). Post-incubation, the planktonic cells were discarded from the tubes. After that, sterile double-distilled water was added to the tubes to wash them properly. Then, 0.4% CV solution was added and incubated at room temperature for 30 min to stain the biofilm cells (if any). Later, the tubes were washed with sterile double-distilled water and dried adequately. After that, 33% glacial acetic acid (5 mL) was added to the respective tubes and the intensity of the color generated in each tube was estimated by measuring its optical density (OD) at 630 nm. For further confirmation, microscopic analysis was carried out to examine the degree of biofilm formation by S. aureus. To do this experiment, the coverslips were recovered from the respective test tubes at different time intervals (6 h, 12 h, 18 h, and 24 h) and further stained with acridine orange (4 μg/mL) for 15 min. Following this, sterile double-distilled water was used to wash the stained coverslips. Thereafter, these coverslips were dried and viewed under a fluorescence microscope (Fluorescein isothiocyanate or FITC filter) at excitation and emission wavelength of 491 nm and 516 nm, respectively.

Estimation of biofilm disintegration by CV assay

To examine the biofilm disintegration ability of 1,4-naphthoquinone against S. aureus, the organism was first allowed to develop biofilm and subsequently challenged with this compound. To elaborate, 50 µL (1 × 105 CFU/mL) microbial culture was separately inoculated into several test tubes containing 5 mL of sterile TSB media. After the incubation for 24 h at 37 °C, the developed biofilm was exposed to 1,4-naphthoquinone (10 µg/mL). However, in the control set, the developed biofilm was not challenged with 1,4-naphthoquinone. All the tubes were then incubated at 37 °C for different length of time (0 h, 2 h, 4 h, and 6 h). Thereafter, the CV assay was followed to determine the disintegration of pre-existing biofilm of S. aureus under the influence of 1,4-naphthoquinone (Chakraborty et al. 2018a). The non-biofilm cells were discarded from all the experimental sets and tubes were subsequently washed with sterile double-distilled water. Then, 5 mL CV solution (0.4%) was added to each tube and incubated for another 30 min to stain the degree of residual biofilm on the glass surface. After that, the CV solution was discarded from all the tubes. Later, 33% acetic acid was added to dissolve the CV adsorbed to the biofilm. The intensity of the color generated in the tubes was quantified by measuring its OD at 630 nm.

Estimation of total biofilm protein

Total biofilm protein estimation appears to be a common method to determine the degree of microbial associations in a biofilm (Chakraborty et al. 2021). The degree of protein recovery has been found to be directly proportional to the extent of microbial associations in biofilm (Paul et al. 2021). Thus, the total biofilm protein was estimated to determine the extent of biofilm disintegration of S. aureus under the influence of 1,4-naphthoquinone. To perform the experiment, like CV assay, 50 µL of S. aureus cells (1 × 105 CFU/mL) was allowed to grow in sterile test tubes containing autoclaved TSB media for 24 h at 37 °C for efficient biofilm formation. Post-incubation, the developed biofilm on the test tubes was challenged with 1,4-naphthoquinone (10 µg/mL). In the control set, the pre-existing biofilm cells were incubated in the absence of 1,4-naphthoquinone. Thereafter, all the prepared experimental sets (control and treated) were incubated at 37 °C for different length of time (0 h, 2 h, 4 h, and 6 h). After the desired incubation, non-biofilm cells were removed and tubes were subsequently rinsed with sterile double-distilled water. Thereafter, 5 mL of 0.3 (N) NaOH was added to each tube and kept in a water bath for 30 min at 100 °C. After the incubation, the boiled microbial suspension was centrifuged at 8000 rpm for 10 min. Then, the supernatant was collected and the protein concentration of the supernatant was estimated by following the Lowry method (Lowry et al. 1951).

Microscopic analysis

To examine the effect of 1,4-naphthoquinone on the disintegration of pre-existing biofilm of S. aureus, cells (1 × 105 CFU/mL) were added to various tubes containing 5 mL of TSB growth media. Sterile coverslips were incorporated in each tube for the effective development of microbial biofilm over it. After the incubation at 37 °C for 24 h, the biofilm developed in tubes was incubated with 1,4-naphthoquinone (10 µg/mL) except the control set. After an incubation of 6 h, coverslips were meticulously withdrawn from each tube and the adhered microbial population over it was stained with acridine orange (4 μg/mL) for 15 min (Tribedi et al. 2015; Chakraborty et al. 2018a). The coverslips were rinsed with sterile double-distilled water and subsequently observed under a fluorescence microscope (FITC filter) at excitation and emission wavelength of 491 nm and 516 nm, respectively.

Non-biofilm cell count analysis

To determine the non-biofilm cells of S. aureus under the influence of 1,4-naphthoquinone, the biofilm was developed first by growing cells in TSB media as mentioned previously. After the incubation, the developed biofilm was exposed to 1,4-naphthoquinone (10 µg/mL). One control set was also kept in which the biofilm cells were not exposed to the compound. Later, this control and the treated sets were incubated at 37 °C for different period of time (0 h, 2 h, 4 h and 6 h). After the desired period of incubation, the culture broth was recovered from all the experimental sets and the number of non-biofilm cells was determined by counting the colony-forming units (CFU) as mentioned by Paul et al. (2021).

Staphylococcal biofilm viability assay

To determine the effect of 1,4-naphthoquinone on the viability of the pre-existing biofilm cells of S. aureus, cells (1 × 105 CFU/mL) were inoculated in different test tubes carrying sterile coverslips and autoclaved TSB growth media (5 mL). After that, all the experimental sets were incubated at 37 °C for 24 h so that the cells could develop efficient biofilm over the coverslips. Then, the formed biofilm cells were exposed to 1,4-naphthoquinone (10 µg/mL). In this study, a control set was also made in which the pre-existing biofilm cells were incubated in the absence of 1,4-naphthoquinone. After an incubation of 6 h at 37 °C, coverslips were withdrawn from both 1,4-naphthoquinone treated and untreated growth media. The microbial biofilm over the coverslip was subsequently stained with acridine orange (4 μg/mL) for 15 min (Tribedi et al. 2015). After the incubation, the acridine orange-stained coverslips were viewed under a fluorescence microscope (FITC filter) at excitation wavelength of 491 nm and emission wavelength of 516 nm. Thereafter, the coverslips were gently washed with sterile double-distilled water and stained with ethidium bromide (4 μg/mL) for another 15 min (Tribedi et al. 2015). Post-incubation, the coverslips were gently washed with sterile double-distilled water and air-dried. After that, ethidium bromide-stained coverslips were observed under a fluorescence microscope (Tetramethylrhodamine or TRITC filter) at excitation and emission wavelength at 526 nm and 605 nm, respectively. Finally, acridine orange and ethidium bromide-stained images were merged together to see whether any yellow colored spot appeared.

Estimation of the metabolic activity of biofilm

To examine the effect of 1,4-naphthoquinone on the metabolic activity of biofilm, fluorescein diacetate (FDA) hydrolysis assay was carried out by adhering to the protocol mentioned previously (Adam and Duncan 2001). In this regard, biofilm cells either treated or untreated with 1,4-naphthoquinone were incubated at 37 °C for 6 h. At regular time (0 h, 2 h, 4 h and 6 h) intervals, the metabolic activity of the biofilm cells was determined by exposing them to 5 mL of FDA solution (10 µg/mL) for 2 h at 37 °C. FDA solution was prepared by dissolving the FDA powder in phosphate buffer solution (60 mM sodium phosphate buffer, pH 7.6). After the incubation, the tubes were vortexed gently and centrifuged at 8000 rpm for 10 min. The supernatant was collected and the absorbance of the same was measured at 494 nm.

Determination of the cellular accumulation of reactive oxygen species (ROS)

The production of ROS by S. aureus under different conditions was measured using an oxidation-sensitive fluorescent probe 2’,7’-dichlorofluorescein diacetate (DCFDA) (Dwivedi et al. 2014; Ong et al. 2017). ROS detection assay kit (ab113851) purchased from Abcam was used for the present study for the estimation of cellular accumulation of ROS. Microbial esterase deacetylates DCFDA which immediately gets oxidized to a fluorescent molecule 2,7-dichlorofluorescein (DCF) by the cellular ROS (Handali et al. 2019). Finally, the amount of DCF produced by the microorganism could be recorded in a fluorescence spectrophotometer at excitation and emission wavelength of 488 nm and 535 nm, respectively. To examine the effect of 1,4-naphthoquinone on the accumulation of ROS in the biofilm cells of S. aureus, 1 mL biofilm cell suspension was mixed with 50 μL DCFDA (0.1 mM) solution. After that, the tube was shaken for 30 min and incubated at 37 °C in dark. Thereafter, the DCFDA exposed biofilm cells were washed twice with sterile TSB to remove the unreacted dye. Then, a nearly equal number of DCFDA exposed cells were distributed into three experimental sets. In the first set, the mentioned biofilm cells were treated with 1,4-naphthoquinone. In the second set, the biofilm cells were exposed to both 1,4-naphthoquinone and ascorbic acid (30 µg/mL). However, in the third set, the biofilm cells were treated with neither 1,4-naphthoquinone nor ascorbic acid. All the discussed experimental sets were incubated at 37 °C for 6 h. After the desired period of incubation, the cellular accumulation of ROS was determined in the biofilm cells by following DCFDA method (Paul et al. 2021).

eDNA extraction and quantification

The preformed biofilm cells of S. aureus were incubated with 1,4-naphthoquinone for different period (0 h, 2 h, 4 h and 6 h) of time. At different time intervals, biofilm cells were collected from each set by following the protocol described by Chakraborty et al (2021). The collected biofilm cells were then vortexed and centrifuged at 8000 rpm for 10 min. The eDNA extracted from the supernatant was subsequently quantified by following the protocol mentioned by Sahu et al. (2012).

Statistical analysis

Each experiment was repeated three times to achieve statistical confidence. One-way analysis of variance (ANOVA) was used to perform the significance test. The degree of significance was incorporated as P value < 0.05 (*), P value < 0.01 (**), and P value < 0.001 (***) in contrast to control. P values more than 0.05 indicated that there was no significant difference and hence presented as N.S. (no statistical difference). A contour plot was constructed to establish a relationship among three variables using the software NCSS.

Results and discussion

1,4-Naphthoquinone exhibited disintegration of pre-existing biofilm of S. aureus

Staphylococcus aureus can cause several persistent and recurrent infections by involving biofilm (Azmi et al. 2019). Biofilm formation appears to promote microbial pathogenesis by exhibiting tolerance against various drugs and increasing the release of virulence factors (Lewis 2005). The existing literature revealed that most biofilm management strategies are aimed at preventing the initial phases of biofilm formation (Chakraborty et al. 2018a, 2018b; Paul et al. 2021). However, a sustainable strategy for the efficient management of the pre-existing biofilm is yet to be explored. 1,4-Naphthoquinone being a natural phytochemical has been reported to exhibit considerable antimicrobial properties against S. aureus wherein the minimum bactericidal concentration (MBC) of the compound was found to be 100 µg/mL (Paul et al. 2021). However, in the current study, efforts have been aimed to investigate the disintegration of the pre-existing biofilm under the influence of the same compound. Hence, cells were first allowed to develop biofilm in different tubes by growing in sterile TSB media for 24 h at 37 °C. At different time points, the degree of biofilm formation by S. aureus was tested by following CV assay. The result indicated that the biofilm formation by S. aureus was found to increase with the progression of time (Fig. 1A). To validate the observation of CV assay, a microscopic study was conducted in which an equal number of cells were inoculated in several tubes carrying autoclaved TSB media. Sterile coverslips were added to each tube to allow the organism to develop biofilm over it. Coverslips were diligently recovered from the growth media at regular time intervals. The colonized cells on the coverslips were stained with acridine orange and observed under a fluorescence microscope. The result showed that the organism could develop biofilm efficiently on the coverslips with the advancement of time (Fig. 1B). Thus, based on the observations of CV assay and fluorescence microscope, it appeared that the organism could develop substantial biofilm after 24 h of incubation. Thereafter, efforts were directed to investigate the biofilm disintegrating properties of 1,4-naphthoquinone by challenging the pre-existing biofilm with the compound. To this end, cells were separately grown in sterile TSB media for 24 h at 37 °C for the substantial development of microbial biofilm within the mentioned time. After the incubation, the planktonic cells were removed and the developed biofilm in tubes was subsequently exposed to 1,4-naphthoquinone. All the tubes were then incubated at 37 °C for different time points as mentioned before. At regular time intervals, tubes were recovered and the extent of biofilm disintegration under the influence of 1,4-naphthoquinone was measured by CV assay. The result showed that with the progression of incubation, the intensity of the CV stain got reduced when the developed biofilm was challenged with 1,4-naphthoquinone (Fig. 2A). The result indicated that 1,4-naphthoquinone could show an efficient biofilm disintegration property against S. aureus. To quantitatively estimate the biofilm disintegration of S. aureus under the influence of 1,4-naphthoquinone, the OD of CV suspension was measured at 630 nm using a spectrophotometer. The result showed that the highest biofilm was observed in a tube which was not incubated with 1,4-naphthoquinone (Fig. 2B). However, the degree of biofilm got reduced significantly when incubated with 1,4-naphthoquinone for an extended period (Fig. 2B). The result revealed that the maximum biofilm disintegration occurred when the pre-existing biofilm was incubated with 1,4-naphthoquinone for a period of 6 h (Fig. 2B). To validate the observation of the CV assay, we had determined the total biofilm protein as in literature it has been found to be a very useful method of biofilm quantification (Mukherjee et al. 2013; Sharma et al. 2015). The result showed that the highest level of protein was recovered from the biofilm which was not incubated with 1,4-naphthoquinone (Fig. 2C). However, protein recovery from the biofilm got reduced significantly when it was challenged with 1,4-naphthoquinone for an extended period of incubation (Fig. 2C). The result of the Lowry method also revealed that the highest biofilm disintegration took place when the biofilm was incubated with 1,4-naphthoquinone for 6 h (Fig. 2C). Furthermore, a microscopic study was conducted to confirm the disintegration of pre-existing biofilm under the exposure of 1,4-naphthoquinone. To do the experiment, the cells were first allowed to form biofilm over coverslips by growing cells in sterile TSB growth media for a period of 24 h at 37 °C. After that, the pre-existing biofilm over the coverslips was exposed to 1,4-naphthoquinone and incubated for another 6 h as the previous results indicated that the highest biofilm disintegration occurred in 6 h of incubation. However, a control set was also made in which the developed biofilm was not exposed to the compound (1,4-naphthoquinone) but incubated under similar conditions (6 h at 37 °C). Post-incubation, all the coverslips were carefully recovered and observed under a fluorescence microscope by staining with acridine orange. The result revealed that no considerable disintegration of the pre-existing biofilm occurred when the developed biofilm was incubated for 6 h in the absence of 1,4-naphthoquinone (Fig. 3A). The result also suggested that a noticeable disintegration of pre-existing biofilm occurred when the biofilm was incubated with 1,4-naphthoquinone for 6 h (Fig. 3A). Hence, the results demonstrated that 1,4-naphthoquinone was found to considerably disintegrate the pre-existing biofilm of S. aureus. Besides, non-biofilm cells of a culture media was also compared to address the biofilm disintegration properties of 1,4-naphthoquinone against S. aureus. In this regard, cells were allowed to form biofilm first by growing in TSB media as discussed already. The developed biofilm was then incubated with 1,4-naphthoquinone for different length of time. However, a control set was prepared in the same fashion in which the developed biofilm was incubated without 1,4-naphthoquinone. At different time intervals, the culture broth (100 µL) was separately collected from both 1,4-naphthoquinone treated and untreated growth media followed by measuring the non-biofilm cells through colony counting. The result showed that with the increase of incubation time, a sharp increase of non-biofilm cells was noticed when the pre-existing biofilm cells were exposed to 1,4-naphthoquinone (Fig. 3B). The result also indicated that the highest count of non-biofilm cells was observed when the pre-existing biofilm was exposed to 1,4-naphthoquinone for a period of 6 h (Fig. 3B). In the control set, with the advancement of incubation, we did not find any significant change in the count of non-biofilm cells (data not shown). Non-biofilm cells of a culture media could be explained by the planktonic as well as the cells which were disintegrated from the pre-existing biofilm (Fig. 3C). The disintegration of biofilm could lead to the dislocation of biofilm cells leading to the accumulation of non-biofilm cells (Fig. 3C). Hence, the enhanced accumulation of non-biofilm cells could be attributed to the disintegration of pre-existing biofilm by 1,4-naphthoquinone.

Fig. 1
figure 1

Efficient biofilm formation by S. aureus. An overnight grown culture of S. aureus was separately grown in sterile TSB media in the presence of glass coverslips. After the desired period of incubation, the extent of biofilm formation was measured by performing CV assay and microscopic studies. A Microbial biofilm profile. CV assay was performed to stain and quantify the degree of biofilm formation by S. aureus. The error bars indicated the mean ± standard deviation. Statistical significance among the results was determined using ANOVA. P values < 0.05 were defined as (*), P values < 0.01 were indicated as (**), and P values < 0.001 were introduced as (***). B Microscopic analysis. After the desired period of incubation, coverslips were recovered, stained with acridine orange and viewed under fluorescence microscope (Color figure online)

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Fig. 2
figure 2

1,4-Naphthoquinone showed promising disintegration of the pre-existing biofilm of S. aureus. The test organism was grown in freshly prepared TSB media for 24 h to allow the development of biofilm. This developed biofilm was then exposed to 1,4-naphthoquinone for different time points. Post-incubation, CV assay and the estimation of total biofilm protein were carried out to examine the extent of pre-existing biofilm disintegration. A CV staining profile. At different time points, the degree of biofilm cells colonized over the tubes were first stained with CV and then dissolved into glacial acetic acid. B CV assay. The variation in the color intensities among the samples were estimated by recording the OD at 630 nm. C Total biofilm protein estimation. After the incubation, the extent of biofilm cells adhered to the tubes was determined by estimating the total biofilm protein. The error bars indicated the mean ± standard deviation. ANOVA was used to test the statistical significance among the results. P values < 0.05 were defined as (*), P values < 0.01 were indicated as (**), and P values < 0.001 were introduced as (***) (Color figure online)

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Fig. 3
figure 3

1,4-Naphthoquinone exhibited considerable accumulation of non-biofilm cells. A Fluorescence microscopic image analysis. The pre-existing biofilm cells were either exposed to 1,4-naphthoquinone or left untreated. Then, the change in biofilm profile under the influence of 1,4-naphthoquinone was confirmed by fluorescence microscopy. B Estimation of the non-biofilm cells. The pre-existing biofilm cells were incubated with 1,4-naphthoquinone for a period of 6 h. At different time intervals, the non-biofilm cells were counted by following the CFU assay. The error bars indicated the mean ± standard deviation. Significance test among the results was performed using ANOVA. P values < 0.05 were defined as (*), P values < 0.01 were indicated as (**), and P values < 0.001 were introduced as (***). C Schematic representation of the accumulation of non-biofilm cells. Biofilm disintegration resulted in the release of biofilm-associated cells to the media. Planktonic (blue), as well as biofilm disintegrated cells (red) could be clubbed together to define non-biofilm cells (Color figure online)

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1,4-Naphthoquinone exhibited a considerable reduction in the metabolic activity of pre-existing biofilm

Biofilm often promotes the secretion of various virulence factors including proteases towards spreading microbial pathogenicity inside the host (Granato et al. 2018). Microbial proteases happen to be a key factor in breaking the protein layer enabling the organism to enter the host tissue comfortably (Chakraborty et al. 2018a). Since the results so far revealed that 1,4-naphthoquinone displayed efficient disintegration of the pre-existing biofilm, efforts are also being made to examine whether the same compound could show any modulation in the metabolic activity of the pre-existing biofilm. To test the hypothesis, cells were allowed to form biofilm followed by exposing the same to 1,4-naphthoquinone as mentioned previously. A control set was also prepared wherein the pre-existing biofilm was not challenged with 1,4-naphthoquinone. All the experimental sets including the control were incubated under similar conditions. At regular time intervals, non-biofilm cells were removed from each tube and the metabolic activity of the pre-existing biofilm was measured by following FDA assay. The result revealed that with the progression of incubation, the metabolic activity of the biofilm got reduced significantly when the pre-existing biofilm was incubated with 1,4-naphthoquinone (Supplementary Fig. 1A, B). However, at different time points of incubation, we did not observe any significant variation of the metabolic activity of the biofilm cells when incubated in the absence of 1,4-naphthoquinone (data not shown). Thus, the result suggested that 1,4-naphthoquinone could attenuate the metabolic activity of the biofilm considerably. The present report indicated that the compound may reduce the microbial pathogenicity by inhibiting the secretion of various hydrolytic enzymes including protease.

1,4-Naphthoquinone exhibited considerable accumulation of ROS that disintegrated the pre-existing biofilm

ROS happens to be the collection of various free radicals (superoxide, hydrogen peroxide, hydroxyl radical, etc.) which could inhibit diverse biological functions including the formation of microbial biofilm (Acker et al. 2016; Paul et al. 2021; Chakraborty et al. 2021). However, to date, the effect of ROS accumulation on biofilm disintegration has not been explained comprehensively. Hence, efforts are being put together to establish whether ROS has any effect on the disintegration of pre-existing biofilm. To validate this hypothesis, different experimental sets were prepared wherein in one set, the developed biofilm cells were only treated with 1,4-naphthoquinone. However, in the second set, the developed biofilm cells were treated with both 1,4-naphthoquinone and ascorbic acid. In the control set, the developed biofilm was treated with neither 1,4-naphthoquinone nor ascorbic acid. All the experimental sets were incubated under similar conditions. Post-incubation, ROS accumulation, and the biofilm disintegration were measured in each experimental condition. The result showed that 1,4-naphthoquinone was found to increase the accumulation of ROS in the biofilm cells with the advancement of incubation (Fig. 4A). We observed the highest accumulation of ROS in the biofilm cells that were exposed to 1,4-naphthoquinone for 6 h (Fig. 4A). Besides ROS measurement, we had also determined the extent of biofilm disintegration in all the experimental sets. The result showed that the extent of biofilm disintegration got increased when the biofilm cells were exposed to 1,4-naphthoquinone for an extended period of incubation (Fig. 4B). The result showed that the ROS accumulation and biofilm disintegration followed the same pattern in the current study (see Fig. 4A, B). The result indicated that cellular ROS accumulation could play an important role in the 1,4-naphthoquinone induced disintegration of pre-existing biofilm. In this context, we noticed that ascorbic acid was found to reduce the accumulation of ROS considerably (Fig. 4C). As expected, the biofilm disintegration got retarded efficiently under the influence of ascorbic acid (Fig. 4D). Moreover, a strong positive correlation (r = 0.98) was spotted between ROS accumulation and biofilm disintegration suggesting that ROS accumulation could promote the disintegration of pre-existing biofilm. To gain more confidence, a contour plot was constructed to define relationship (if any) among the exposure time of 1,4-naphthoquinone, accumulation of ROS, and the biofilm disintegration. The result showed that with the progression of exposure time, the cellular accumulation of ROS got increased that resulted in the enhancement of biofilm disintegration (Supplementary Fig. 2). Taken together, the results showed that ROS accumulation was found to cause the disintegration of pre-existing biofilm.

Fig. 4
figure 4

1,4-Naphthoquinone induced ROS accumulation exhibited considerable disintegration of pre-existing biofilm. A ROS profile. The pre-existing biofilm cells were challenged with 1,4-naphthoquinone for different length of time. At regular time span, cellular ROS generation was measured in the biofilm cells by following the DCFDA method. B Biofilm profile. CV assay was followed to determine the extent of biofilm disintegration under the presence and absence of 1,4-naphthoquinone. C Ascorbic acid inhibited the generation of ROS. The pre-existing biofilm cells were challenged with either 1,4-naphthoquinone alone or in a combination with ascorbic acid for 6 h. Post-incubation, the generation of ROS was measured by DCFDA method. D Ascorbic acid inhibited biofilm disintegration. CV assay was performed to examine the degree of biofilm disintegration of S. aureus under the influence of 1,4-naphthoquinone and ascorbic acid. The error bars indicated the mean ± standard deviation. Statistical significance among the results was confirmed by ANOVA. P values < 0.05 were mentioned as (*), P values < 0.01 were defined as (**), and P values < 0.001 were incorporated as (***) (Color figure online)

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1,4-Naphthoquinone did not show any considerable antimicrobial activity against the pre-existing biofilm

Though 1,4-naphthoquinone exhibited efficient biofilm disintegration property, the antimicrobial properties of the same against the pre-existing biofilm cells are yet to be tested. Hence, efforts have been put together to examine the antimicrobial activities of the compound against the pre-existing biofilm cells. To understand the same, cells were first allowed to form biofilm in several tubes by growing in TSB media for 24 h. After that, the developed biofilm cells were treated with 1,4-naphthoquinone for 6 h. In the control set, the developed biofilm was incubated for 6 h under similar conditions without being exposed to 1,4-naphthoquinone. Post-incubation, biofilm cells of both 1,4-naphthoquinone treated and untreated cells were stained with acridine orange and ethidium bromide. Acridine orange, being a small molecule was found to stain all the cells as it can enter both live and dead cells. Then, the acridine orange-stained cells were stained with ethidium bromide. Ethidium bromide happens to be a large molecule that can only enter the dead cells. After that, both acridine orange-stained (green-colored spot) and ethidium bromide-stained (red-colored spot) images were merged. The result of dual staining showed no yellow spots (indicator of dead cells) in the merged frame (Supplementary Fig. 3) suggesting that the compound did not show any antimicrobial properties against the pre-existing biofilm cells. Hence, the present report revealed that 1,4-naphthoquinone showed efficient disintegration of pre-existing biofilm without exhibiting any antimicrobial properties.

The disintegration of pre-existing biofilm could be attributed to the degradation of extracellular DNA (eDNA)

Staphylococcal biofilm gets matured when the biofilm cells release an extracellular matrix (ECM) comprising of polysaccharides, proteins, and eDNA (Gupta et al. 2016). It was reported that eDNA has wide-ranging influences on the structural integrity of biofilm (Das et al. 2013). Hence, in the current study, we have made an attempt to examine whether eDNA has any role in biofilm disintegration. To do the test, cells were allowed to form biofilm in several tubes as mentioned earlier. Post-incubation, the developed biofilm was treated with DNaseI while in the control set, the preformed biofilm cells were left untreated. However, both the sets were incubated under similar conditions for 6 h. After the incubation, the extent of biofilm disintegration was examined in each tube by performing the CV assay. The result showed that the disintegration of pre-existing biofilm got increased when the biofilm was exposed to DNaseI (Fig. 5A). The result indicated that eDNA could hold the complex structure of biofilm efficiently as the degradation of the same was found to promote the disintegration of pre-existing biofilm. To investigate the mechanism of 1,4-naphthoquinone induced disintegration of pre-existing biofilm, we hypothesized that the accumulation of ROS by 1,4-naphthoquinone might degrade the eDNA resulting in the exhibition of biofilm disintegration. To validate this hypothesis, in one tube, the pre-existing biofilm was exposed to 1,4-naphthoquinone. However, in the second tube, the biofilm was exposed to both 1,4-naphthoquinone and ascorbic acid. In the control set, neither 1,4-naphthoquinone nor ascorbic acid was added to the pre-existing biofilm. All the experimental sets were incubated under similar conditions for 6 h. After the desired incubation, the extent of eDNA produced by the biofilm, the accumulation of cellular ROS, and the disintegration of biofilm were examined accordingly. The result showed that the amount of eDNA recovered from the microbial biofilm got reduced by 57% when the pre-existing biofilm was exposed to 1,4-naphthoquinone for a period of 6 h (Fig. 5B). The result also indicated that ROS accumulation got increased significantly with the progression of incubation (Fig. 5C). Therefore, the result revealed that eDNA recovery and ROS generation followed an inversely proportional relationship suggesting that whenever ROS generation got increased, the recovery of eDNA got decreased concomitantly (see Fig. 5B, C). The result further suggested that the generation of ROS might degrade the eDNA that resulted in the lesser recovery of eDNA from the biofilm. Taken together, the results suggested that the exposure of 1,4-naphthoquinone was found to increase the generation of ROS which could affect the eDNA thereby causing the disintegration of pre-existing biofilm.

Fig. 5
figure 5

1,4-Naphthoquinone-induced ROS accumulation exhibited efficient disintegration of the pre-existing biofilm. A Biofilm disintegration under the influence of DNaseI. The pre-existing biofilm cells were incubated in the presence and absence of DNaseI and subjected to the estimation of biofilm disintegration by following the CV assay. B eDNA estimation. The pre-existing biofilm cells were incubated with different combinations of 1,4-naphthoquinone and ascorbic acid for 6 h. After that, eDNA was quantified in each set by measuring the absorption at 260 nm. C ROS profile. The pre-existing biofilm cells were challenged with different doses of 1,4-naphthoquinone and ascorbic acid. Post-incubation, the accumulation of ROS in each experimental set was measured by following the DCFDA method. The error bars indicated the mean ± standard deviation. Statistical significance among the results was assessed by performing ANOVA. P values < 0.05 were incorporated as (*), P values < 0.01 were defined as (**), and P values < 0.001 were mentioned as (***) (Color figure online)

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Conclusion

Microbial biofilm tends to portray a serious threat to public healthcare as the microorganisms in biofilm often exhibit high degree of drug resistance. Thus, effective strategies are continuously being explored globally to combat such challenges. In the present study, 1,4-naphthoquinone has been proposed as an efficient antibiofilm molecule as it exhibits promising disintegration property against the pre-existing biofilm of S. aureus by increasing the generation of ROS. Thus, 1,4-naphthoquinone could be used alone or in combination with other reported antibiofilm agents to manage the threats of microbial biofilm in a sustainable and meaningful fashion.