Introduction

Reasonable interest has been sparked by the necessity to treat illnesses caused by multidrug-resistant (MDR) microorganisms (Sharada et al. 2013). Disinfectants serve a key role in preventing infectious organisms and are a crucial component of the infection control strategy (Okore et al. 2014). Dettol is a basic chloroxylenol-based disinfectant that is frequently used in household and healthcare facilities for a variety of tasks, including the cleaning of environmental surfaces, items, and skin. Dettol-H and Alpilon were considered sufficient dilutions for the different organisms tested (Kapoor et al. 1998). Antimicrobial activity of Dettol adopting paper disc agar diffusion assay against pathogenic microorganisms reported (Mahmood et al. 2008) that strong micro biocidal activity is due to damage of the cell wall, cell membrane, and protein precipitation. The distinct antimicrobial properties of chloroxylenol, Dettol’s primary chemical component, and other chlorinated phenol are highly effective against a wide range of microorganisms known to kill bacterial vegetative cells, fungi, and most viruses. The nanobubble-based drug carrier system focuses on enhancing antimicrobial effectiveness properties against a wide range of pathogens known to cause severe health complications (Cavalli et al. 2016). The inactivation of the Ebola virus’s effect during outbreaks is possible by the effective use of the Dettol disinfection agent (Todd A. Cutts et al. 2019). A recent study on hygiene products such as Dettol and Lysol revealed that these products are efficient enough to break the chain of COVID-19 infection (M Khalid Ijaz et al. 2020). Established cell culture-based techniques can be used as a valuable tool for screening of toxicity of the various compounds (Li et al. 2022). For instance, P. aeruginosa and S. aureus could coexist, at least in the initial phases of infection, in acute wound infection models that include serum in the growth medium. Interspecific interactions therefore looked to be tightly controlled and reliant on the environment. Since P. aeruginosa excretes virulence agents, such as pyoverdine, pyocyanin, and elastases, intercellular communication impacts the synthesis of such component strains.

Previous study demonstrated the efficacy of the secondary metabolites (lariciresinol and berberine) of the North-Western Himalayan region's Zingiber officinale Roscoe against MDR Salmonella typhimurium by inhibiting the efflux pump (Parmanik et al. 2022). The results showed that lariciresinol decreased the MIC by two-to-fourfold, while berberine boosted antibacterial activity by two-to-fourfold. Lariciresinol was found to have the capacity to block the efflux pump and kill drug-resistant S. typhimurium. Saponins and bromo-polyphenols, which are plant-derived secondary metabolites, were discovered to be effective against MDR pathogens (E. coli and M. tuberculosis) isolated from Ganga river water, milk, and chicken flesh. Compared to the usual rifampicin antibiotics, there was a significantly greater inhibition of bacterial RNA polymerase. These phytoconstituents could be employed as therapeutic adjuncts in the future to help prevent and treat MDR bacterial infections; it was determined (Chakraborty et al. 2022).

There are numerous ways to block the systems that control MDR transporters (Akan et al. 2005). Cytotoxic medications cause the MDR protein gene to express in tumor cells, but a number of pharmacological inhibitors that interfere with signaling pathways prevent this gene expression (Feldmann et al. 2006). By directly interacting with and activating the nuclear steroid and xenobiotic receptor (SXR), taxol was shown to activate the expression of the MDR1 and cytochrome p450 3A4 (CYP3A4) genes, increasing drug resistance and accelerating drug clearance. Hence, to deal with the up-regulation of MDR1 and CYP3A4, antagonists of the nuclear steroid and xenobiotic receptor may be used in addition to anticancer medications.

Avoiding the total elimination of bone marrow stem cells is a critical dose-limiting toxicity factor for anticancer medications (Sarkadi et al. 2004). By transferring vectors containing the MDR1 cDNA into bone marrow cells, Gottesman et al. were able to create multidrug-resistant cells. With this process, they were able to administer a chemotherapy treatment at levels that would have previously been intolerable, defeating MDR (Asadi et al. 2020).

Current research suggests that a mutant ABCG2 protein is a prime option for gene therapy selectable markers and human stem cell protection (Chen et al. 2011). This protein’s cDNA is only approximately 2 kb in size, and the overexpressed cells spontaneously produce the active dimer. The R482G variation of ABCG2 offers a distinct benefit in gene therapy applications, because it has different substrate specificity from the wild-type protein. The use of an apoptosis-inducing monoclonal antibody targeted against the CD20 receptor is a unique method for evading MDR processes. Chemotherapy is more effective when drug-sensitive cells are forced to undergo apoptosis.

Though, glutathione (GSH) is likely to play a role in the development of the resistance, the precise mechanism by which MRP1 is implicated in multidrug resistance is yet unclear. A pro-glutathione medication is called N-acetylcysteine (NAC). BSO, or dl-Buthionine (S,R)-sulfoximine, prevents the production of GSH. We recently looked at how NAC and BSO affected the MRP1-mediated resistance to vincristine and doxorubicin in human embryonic kidney (HEK293) and its MRP1 transfected 293MRP cells. A plasmid encoding the entire MRP1 gene was transfected into human embryonic kidney (HEK293) cells.

Materials and methods

Reagents, chemicals, and culture media

Dettol (Reckitt Benckiser Private Ltd, Mysore, Karnataka, India) as a commercially formulated product was obtained from the Medical store, Sholinganallur, Chennai. Active ingredients of this formulation are as follows: Chloroxylenol 4.8% (v/v), Tertineol − 9.0%, Alcohol (denaturized)—13.1% Reagents, and chemicals used for further studies were obtained as analytical grade used without any purification. The culture media used for the antibacterial study were purchased from Hi-Media (Mumbai), India. Media and reagents for the cell culture studies were obtained from Sigma-Aldrich. Dimethyl Sulphoxide (DMSO) was obtained (Merck). All solutions were made with Millipore water.

Preparation of nanobubble formulated Dettol (NB-D)

The Nanobubble formulation generation was carried out by minor modification of our recent study using a household hand mixer as per the procedure stated in our earlier publication (G. Senthilkumar et al.), as shown in Fig. 1.

Fig. 1
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3D image of nanobubble generator (household mechanical mixer or stirrer)

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The size of NBs was in the order of 80–100 nm, as revealed by AFM. The dynamic light scattering (DLS) was used to examine the particle-size distribution. The primary characterization of NB-D was carried out by atomic force microscopy (AFM). Sample preparation is an essential part of AFM analysis, which involves placing 100 µL of the sample in a clean glass slide and allowed to dry at room temperature to get a thin film of the sample. The AFM analysis was performed using AFM Ntegra Prima-NTMDT, Ireland, and the zeta potential and particle size were performed using the Zetasizer (Malvern, UK) at 25 °C. Averages of three replicates are taken for results and discussion.

Antibacterial activity

The determination of antibacterial activity was carried out by the modified method of agar diffusion assay (qualitative) and microdilution broth turbidometric assay against human pathogenic strains Pseudomonas aeruginosa (ATCC 10,145) and Staphylococcus aureus (ATCC 12,034). The effect of NB-D on the survival rate and death rate was also studied. Further, the biofilm inhibition adopting spectrophotometric crystal violet assay was performed to confirm the antibacterial activity.

Bacterial strains

American Type culture collection strains Pseudomonas aeruginosa (ATCC 10,145) and Staphylococcus aureus (ATCC 12,034) were selected in the study. The respective strain on trypticase soy agar slant was maintained with pure culture. Subculturing was done every 10 day interval using trypticase soy broth.

Inoculum preparation

Trypticase soy broth was employed for inoculum preparation. Tryptic soy broth was inoculated with a loopful slant culture of bacterial strain and incubated at 37 ℃ for 12 h under vibrating condition.

Evaluation of stability

Stability of Nanoformulation was checked by exposing the sample at ambient temperature (30 °C) for 30 days. At different time periods, the sample was withdrawn aseptically, and the stability studies were carried out. Stability study for antibacterial activity of NB-D samples collected at different time intervals using well-diffusion assay, microdilution broth turbidometric assay, and determination of survival rates against the tested bacterial strains was conducted.

Results

Characterization of nanobubble formulation

Figure 1 shows three-dimensional images of the mechanical stirrer used for Nanobubble generation. AFM and particle-size analysis did the primary characterization of Nanobubble formulation. A glass sheet was taken in which 100 µL of Nanoformulation was kept till it was dried and subjected to AFM. Figure 3 depicts the surface morphology of generated nanobubbles, with surface roughness of 1.546 nm. Figures 2 and 3 show a 2D, 3D morphology in horizontal and vertical axis (1 micrometer = 100 nanometers). The respective graphs of each bubble are shown in the right side of Fig. 4.

Fig. 2
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AFM micrograph of 2D image of NB-D

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Fig. 3
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AFM micrograph of 3D image of NB-D

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Fig. 4
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Measurement of two different bubbles BY AFM

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Evaluation of enhanced antibacterial activity

The zone of inhibition of Dettol against P. aeruginosa and S. aureus was found to be 12 0.0 and 14.4 mm, and an increase in zone of inhibition against both the tested strains was increased in Nanobubble treatment, which recorded 21.0 and 24.0 mm as revealed from Fig. 5. Table 2 shows that the Nanobubble exhibited inhibition of both the tested strains at a lower concentration.

Fig. 5
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Inhibition zone (mm) of NB-D alongside tested strains

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Stability

The stability of NB-D was checked by exposing the formulation at ambient temperature (30 °C) for 10, 20, and 30 days. After the respective period of incubation, the average particle size and roughness were measured using atomic force microscopy (AFM).

Discussions

Ultra-small gas bubbles in liquid with diameters of micron and submicron-order, the so-called micro-Nanobubbles, play a significant role in various fields of science and technology, and their application in diverse fields is massive due to specific characteristic physical and chemical properties like buoyancy behavior (slow), unique surface charges (negative), free radical formation, and increased water molecule mobility. In this study, Nanobubble generated chloroxyphenol based antiseptic agent Dettol was prepared by the method of our recent study using mechanical stirring (Senthilkumar et al. 2018).There are various mechanisms by which the primary confirmation study with a good diffusion assay indicates an effective inhibition of both the tested bacterial strains. It can be seen that both the tested strains were susceptible to the respective samples (Table 1).

Table 1 Inhibition zone (mm) of nanobubble against pathogenic bacteria
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However, a significant difference was recorded in Nanobubble samples (P = 0.005), which reveals enhanced activity against both the tested strains (Table 1). Figure 5 shows the antibacterial activity of samples, which indicates an increase in the inhibition zone in Nanobubble concentration.

A similar effect was observed in the MBC study, indicating the high antibacterial potential of Nanobubble samples. The biofilm inhibitory effect against tested bacterial strains was characterized with spectrophotometric assay. Figure 6 indicates an influence on the formation of biofilm NB-D formulation.

Fig. 6
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Influence of NB-D on the biofilm inhibition (%) of tested bacterial strains

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MIC of Dettol against P. aeruginosa and S. aureus were 0.25 and 0.30 ml, and the Nanobubble samples show 0.12 and 0.15 ml, respectively (Table 2).

Table 2 Effect of nanobubble on MIC and MBC
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Adherent bacterial communities on solid surfaces covered with a protective matrix of extracellular polymeric substance (EPS) termed as biofilm now recognized as the major virulence factors of most of the pathogenic microorganisms by aiding in attachment or adherence of the bacterial cells to the host tissue, prevent the entry of antibiotics or other chemotherapeutic molecules and serve to promote bacteria persistence by resisting antibiotic treatment and host immune responses (Baker and Banfield 2003, Baillie and Douglas 2000). In this study, the biofilm inhibitory effect of NB- D was determined by determining the optical density or absorbance of ethanol solubilized mixture of adherent biofilm in a microtitre plate. The results were represented as an inhibitory percentage. However, free Dettol brought about biofilm inhibition. The inhibitory effect was observed as a dose-dependent manner, which reveals that Dettol's maximum dosage recorded high inhibition with the tested samples, unlike the high inhibitory effect of NB-D with all the dosages. The vulnerability of the cell aggregates caused damages in both free and Nanoformulation. Both of the examined bacterial strains' biofilm showed total biofilm destruction due to fragmentation or disintegration into free-floating cells after receiving NB-D treatment, which was followed by complete dehydration (Figs. 7, 8).

Fig. 7
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SEM image of biofilm of S. aureus: (a) Control (b) Dettol (c) NB-D

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Fig. 8
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SEM image of biofilm of P. aeruginosa: (a) Control (b) Dettol (c) NB-D

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Figure 9 illustrates data derived from variations in the total viable count of respective bacterial strains treated with NB-D. These data show that entire viability was lost in both free and NB-D treatments, and the rate of decrease was found to be higher at longer time intervals.

Fig. 9
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Influence of normal and NB-D on the viability (%) of tested bacterial strains

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DRT was substantially higher than free Dettol for both of the tested strains, as shown in Fig. 10. Free Dettol (FD) reported 5.01 min for P. aeruginosa, compared to 1.21 min for NB-D. S. aureus showed a similar result.

Fig. 10
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Influence of normal and NB-D on the decimal reduction time (DCR)

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The enhanced anti-biofilm effect of the Nanoformulation may be the result of the biochemical components of the biofilm matrix degrading, which in turn affects the biofilm's structural integrity. Additionally, the Nanobubble created in the antiseptic solution facilitated the rapid diffusion of chloroxylenol, an active biocidal compound of Dettol, through the channels present in the matrix, followed by the sustained release, which results in effective antimicrobial activity. To further investigate the antibacterial activity of NB-D, total viability (%), decimal reduction time, lag L, and lag E were determined (extrapolation number). study of the death curves and lag Nt/No of the tested bacterial strains. According to Fig. 11, the NB-D therapy showed a substantial difference in lag L and Lag E. Compared to free Dettol, NB-D therapy resulted in a lower lag L and a greater lag E. (FD). It is evident from the results that NB-D had a longer initial growth time (Lag phase) and a smaller lag E (active phase), which may be related to the controlled or sustained release of chloroxylenol, an active ingredient in Dettol that promotes interaction with viable cells of the tested pathogenic strains.

Fig. 11
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Influence of normal and NB-D on the lag L and extrapolation number lag E

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Additionally, the free lag Nt/No and the NB-D reveal a negative slope (Fig. 12). These results show that NB-D had stronger antibacterial activity against both of the tested bacterial strains, which was supported by the successful viability reduction.

Fig. 12
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Influence of normal and NB-D on log No/Nt against the tested bacterial strains

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Results indicate that particle size and measured roughness were not changed in all the tested time periods (Fig. 13). The particle size and roughness pattern remain the same in the respective time period (Fig. 14).

Fig. 13
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Influence of storage on the diameter (nm) of NB-D

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Fig. 14
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Influence of storage on the average roughness of NB-D

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No significant difference (P = 0.5%) in both tested parameters of the NB-D during the incubation period indicates the high state of stability. The Nanobubble stability was influenced by various parameters like time of mechanical stirring, the addition of various adjuvants. A study by Ohmori et al. reveals the addition of ethanol and NaCl to Nanobubble water caused specific changes in the particle size. Further, stability was determined by the evaluation of enhanced antibacterial activity. Further, stability was examined by measuring antibacterial activity changes, adopting well-diffusion assay, turbidometric microdilution assay, and determination of survival rates. The significant difference in the zone of inhibition, minimum inhibitory concentration (MIC), viability, decimal reduction time, lag L and Lag E, and log No/Nt parameters was not noticed in the samples exposed to different time periods. In well-diffusion assay, the zone of inhibition recorded at respective storage time of NB-D treatment did not exhibit any changes (Table 3).

Table 3 Effect of storage of NB-D on the inhibition zone (mm) of nanobubble
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A similar result was also observed in MIC and MBC studies, showing no remarkable effect (Table 4). Further confirmation of stability was also inferred from the total count data, revealing no count reduction in the NB-D kept at the respective periods. No significant variation on the viability (%), decimal reduction time, lag L and extrapolation number, and log No/Nt in the NB-D were exposed to different time periods (Figs. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).

Table 4 Effect of storage (days) of NB-D on MIC and MBC against tested bacterial strains
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Fig. 15
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Influence of 30 day storage of NB-D on the total viability (%) against the tested bacterial strains

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Fig. 16
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Influence of 30 day storage of NB-D on the decimal reduction time (days) against the tested bacterial strains

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Fig. 17
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Influence of 30 day storage of NB-D on the lag L and extrapolation number lag E

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Fig. 18
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Influence of 30 day storage on log No/Nt of P. aeruginosa

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Fig. 19
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Influence of 30 day storage on log No/Nt of S. aureus

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Fig. 20
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Influence of 60 day storage of NB-D on the viability (%)

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Fig. 21
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Influence of 60 day storage of NB-D on the decimal reduction time

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Fig. 22
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Influence of 60 day storage of NB-D on the lag L and extrapolation number lag E

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Fig. 23
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Influence of 60 day storage of NB-D on log No/Nt of S. aureus

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Fig. 24
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Influence of 60 day storage of NB-D on log No/Nt of P. aeruginosa

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Conclusion

In recent years, Nanobubble-guided delivery of chemotherapeutics has been the focus of increasing attention to enhance antimicrobial activity. In the present study, the Nanobubble formulation of commonly used chloroxylenol-based antiseptic agent Dettol has been evaluated for the enhanced antibacterial activity strains and its non-target effect. The mechanical stirring process was employed for NB-D preparation, and this process brought about high stable Nanobubble with a size range of 260–290 nm. Prepared NB-D exhibited enhanced antibacterial activity by adopting well-diffusion assay, microtitre plate spectrophotometric crystal violet assay, turbidometric broth dilution assay, and total viability (%) decimal reduction time, lag L & lag E (extrapolation number), log Nt/No studies, respectively, than free Dettol. Such NB-D motifs have a high safety profile, according to in vivo safety assessments. These tiny compounds have the potential to be researched further and developed into fresh antibacterial treatments for MDR infections. The solvent exchange method, hydrodynamic cavitation method, electrolysis, gas–liquid circulation method, and temperature change method can be adopted for NB-D preparation to test the efficacy, as future study.