Revisiting the Synergistic In Vitro Antimicrobial and Antibiofilm Potential of Chlorhexidine Gluconate and Cetrimide in Combination as an Antiseptic and Disinfectant Agent

Abstract

Chlorhexidine and cetrimide are often used as antiseptics and disinfectants. While their individual activities are well-documented, their synergism has rarely been evaluated. Here, we attempted to evaluate the antimicrobial and antibiofilm effects of the combination of these two antimicrobial agents against two environment isolates, viz., P. aeruginosa and S. aureus. The synergism was assayed by determining the fractional inhibitory concentrations, while the antibiofilm effects were determined using crystal violet staining and the resazurin assay. Further, the effects on the biofilms were visualized using brightfield and confocal laser scanning microscopy. Our results show that the combination of these antimicrobials resulted in synergistic inhibition of P. aeruginosa growth. When tested at concentrations below the individual MICs (one-quarter of the MICs), the combination was able to significantly reduce the adherence of S. aureus biofilms to a polystyrene surface, while no effect was observed for P. aeruginosa. The combination was also able to significantly reduce the viability of pre-formed biofilms of both bacteria, thereby showing its antibiofilm potential. Next, we evaluated the performance of this combination against a wide array of micro-organisms. This fixed-dose combination formulation exhibited a significant reduction in the viability of an array of clinically relevant micro-organisms, including ESKAPE pathogens, Mycobacterium sp., MRSA, Leptospira, Candida sp., norovirus and adenovirus. Overall, it can be inferred that the combination of chlorhexidine and cetrimide is a potential biocide that continues to be relevant for use in antisepsis and disinfection against infection-causing pathogens.

1. Introduction

Microbial contamination of biotic or abiotic surfaces often leads to intractable situations like biofilms, delayed wound healing, spread of infectious diseases, and overall negative effects on the environment and human health [1,2]. These contaminations are often caused by Gram-negative or Gram-positive bacteria, including P. aeruginosa and S. aureus [3]. Use of antiseptics and disinfectants is the primary method adopted for the prevention of microbial contamination [4]. Antiseptics and disinfectants are chemical or physical agents used on abiotic surfaces or on the skin and mucous membranes. Antimicrobial biocides are routinely used in commercially available antiseptics and disinfectants designed for healthcare and household settings. Chlorhexidine is one such prominently used antimicrobial agent because of its broad spectrum of activity. The efficacy of chlorhexidine depends on its ability to disrupt the bacterial cell membrane, leading to increased permeability and resulting in cell lysis. Low concentrations of chlorhexidine affect membrane integrity, whereas high concentrations lead to coagulation of cytoplasmic contents [5]. The use of chlorhexidine is generally considered safe for children and adults, with an excellent efficacy and persistent activity [6]. It has diverse applications, ranging from hand washing, preoperative skin preparation and treatment of gingivitis to household disinfection [6,7]. Cetrimide (CTR) is a cationic surfactant with proven antimicrobial efficacy. The combination of chlorhexidine gluconate (CHG) with CTR is widely used in antiseptic and disinfectant formulations for various applications like wound cleaning, medical bathing, etc. [8,9]. Many studies have been conducted in the past to establish the antimicrobial activity of these biocides individually [10,11,12], though limited data on the effect of these antimicrobials in combination are available. Here, we attempted to understand the synergistic efficacy of these biocides as antimicrobial and antibiofilm agents. The efficacy of the combination of CHG and CTR as an antimicrobial and antibiofilm agent against S. aureus, as a Gram-positive model organism, and P. aeruginosa, as a Gram-negative model organism, was determined. The synergy between CHG and CTR in combination was established by determining the fractional inhibitory concentration, followed by studying the effects on biofilm formation ability and pre-formed biofilms by the crystal violet staining method and the resazurin assay, respectively. Confocal laser scanning microscopy (CLSM) was used to understand the effect on the biomass of the biofilms. Based on the results obtained, we further evaluated the killing efficacy of a commercial formulation based on the combination of CTR and CHG in terms of log₁₀ reductions against a wide range of micro-organisms, including clinically relevant bacteria (e.g., ESKAPE pathogens, Mycobacteria, etc.), fungi (Candida sp.), Leptospira and viruses (DNA and RNA viruses like SARS-CoV-2, norovirus, rhinovirus, rotavirus, etc.).

2. Materials and Methods

2.1. Bacterial Strains, Growth Conditions, Media, Chemicals and Reagents

The bacterial strains used for the antimicrobial and antibiofilm studies included Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538P. Both are the reference representative strains for testing the antimicrobial activity of disinfectants under EN 1276 (the quantitative suspension test for the evaluation of the bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas). The bacterial, fungal and viral strains used to study the contact killing efficacy were reported accordingly. The bacterial cultures were grown on tryptic soy broth (TSB) medium (Himedia, Mumbai, India) at 37 °C overnight and maintained on Tryptic soy media with 1.5% agar. Mueller–Hinton cation-adjusted broth (Himedia, Mumbai, India) was used for determining minimum inhibitory concentrations. All the chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chlorhexidine gluconate IP 20% w/v solution and strong cetrimide 32.5% w/v solution were used to prepare the stocks for the experiments as required for the study. The concentrations of the antimicrobials are reported as mg/L and/or w/v percentages.

2.2. Determination of Minimum Inhibitory Concentrations of Planktonic Cells

Minimum inhibitory concentration is defined as the minimum concentration of an antimicrobial agent at which no visible growth of micro-organisms is observed. The minimum inhibitory concentrations (MICs) of CHG and CTR against micro-organisms were determined as per the CLSI, 2021 [13]. Briefly, the antimicrobial agents were serially diluted two-fold in a 96-well micro-titer plate to obtain the required concentration. Each well was then inoculated with ~10⁵ cells per well to make up the final volume of 200 µL and kept for incubation at 37 °C for 16–18 h. Further, MICs were estimated as the concentration of the well where no visible growth was observed.

2.3. Fractional Inhibitory Concentration Index Determination

Having determined the MIC values of CHG and CTR individually, the checkerboard assay was performed to assess the effect of the combination of CHG with CTR on antimicrobial activity [14]. The fractional inhibitory concentration index (FIC_I) at the concentrations where no visible growth was observed was calculated as follows:

FIC_I = FIC_CTR→CHG + FIC_CHG→CTR

where, FIC_CTR→CHG = MIC_{CHG combine}/MIC_{CHG alone}

FIC_CHG→CTR = MIC_{CTR combine}/MIC_{CTR alone}

And the results were interpreted as synergistic (FIC_I ≤ 0.5), additive (0.5 < FIC_I ≤ 1), no interaction (1 < FIC_I < 4) or antagonistic (FIC_I ≥ 4.0). Further, the extended evaluation of FIC data was performed for directionality and the nature of the interaction by plotting the individual FICs (FIC_CTR→CHG and FIC_CHG→CTR) on a two-axis plot [15].

2.4. Effect on Biofilm Formation Ability of Micro-Organisms

The antibiofilm potential of the CTR and CHG combination was studied with respect to biofilm formation ability. The effect of the CTR and CHG combination on the biofilm formation ability of the test micro-organisms was assessed by staining the total biomass using the crystal violet staining method, as previously described, with modifications [16]. Briefly, different concentrations of CHG and CTR were introduced into a 96-well micro-titer plate containing the media with bacterial inoculums (~10⁵ cfu/mL) and incubated at 37 °C for 18–24 h. The OD_{600 nm} of planktonic cells was taken at the end of the incubation period. Further, the biofilms were washed thrice using 1× PBS for removal of planktonic cells, followed by staining using 0.2% crystal violet. The excess stain was washed off, and the stained dye was resolubilized using 30% acetic acid and quantified by taking the absorbance at 590 nm. The specific biofilm formation was calculated by using the following formula:

Specific biofilm formation (SBF) = [A_{590 nm} (Biofilms) − A_{590 nm} (Blank)]/A_{600 nm} (Planktonic)]

The alternation in the adhesion of biofilms in the presence of CHG and CTR were estimated based on the adhesion index as follows:

Adhesion index (AI) = [A]_T/[A]_C

where [A]_T is the A_{620 nm} of the solubilized dyes from the test wells and [A]_C is the A_{620 nm} of the solubilized dyes from the control wells (biofilm with no antimicrobials).

2.5. Effect on Pre-Formed Biofilms and Minimum Biofilm Eradication Concentration Determination

The effect on the pre-formed biofilms was determined by estimating the change in the metabolic activity of cells within the biofilms using the resazurin assay followed by the spot assay to confirm the minimum biofilm eradication concentrations (MBECs). The overnight-grown bacterial culture was diluted to ~10⁵ cfu/mL, and 150 µL of this culture was transferred into a 96-well micro-titer plate and incubated at 37 °C for 18–24 h. The planktonic cells were removed, and biofilms were washed thrice using 1× PBS. Further, various combinations of CHG (128–0.125 mg/mL) and CTR (2048–32 mg/L), along with the necessary controls, were added to the wells and incubated at 37 °C for 18–24 h. The next day, the wells were washed thrice with 1× PBS, and fresh medium was added. A quantity of 10 µL of resazurin (8 mg/mL) was then added to each well and incubated for 20 min, and the fluorescence was measured at 535 nm (excitation) and 590 nm (emission) using the Tecan Infinite M Plex multimode reader (Bioscreen, Tecan Trading AG, Seestrasse, Männedorf, Switzerland) to estimate the metabolic activity of the cells. The concentrations of the wells showing depletion of metabolic activity were considered as biofilm eradication concentrations.

The Calgary Biofilm Device (CBD) was used to grow biofilms for the determination of MBECs. This device comprises a 96-well micro-titer plate with a 96-peg lid. The device has been used to form biofilms of predictable and reproducible size on each peg of the CBD lid [17]. Experimentally, the overnight-grown culture was diluted to ~10⁵ cfu/mL in TSB, and 150 µL of this culture was transferred into the wells of the micro-titer plates, except for wells A12, B12 and C12, and the peg lid was carefully placed to immerse the pegs in the media. The plate was incubated for 18–24 h at 37 °C. Post-incubation, the biofilm formed on the pegs of the lid was washed thrice with 1× PBS to remove the planktonic cells. This was followed by transferring the peg lid to a 96-well micro-titer plate (the challenge plate) containing combinations of different concentrations of CHG and CTR as described for the resazurin assay. Medium was added to well A12 of the challenge plate, neutralizer to well B12 and 150 mM NaCl to well C12. This was followed by incubation at 37 °C for 24 h, and then the peg lid was transferred to a fresh micro-titer plate containing the neutralizing solution (Polysorbate 80, Lecithin, sodium thiosulfate, sodium dodecyl sulfate, Sodium chloride and Tryptone in 1000 mL distilled water). After 24 h, the biofilm was dispersed from the peg lid to the neutralizer solution by sonication. The dispersed cells from each well were spotted on a TSA plate, and the concentration at which no growth was observed is reported as the minimum biofilm eradication concentration.

2.6. Microscopic Analysis of the Biofilms

Brightfield microscopy was performed to assess the effect of the antimicrobials on the adhesion of S. aureus biofilms after crystal violet staining. For this purpose, the biofilms were grown on cover-slips in a 24-well plate along with different combinations of CHG and CTR. The formed biofilms were washed thrice with sterile water and stained with 0.2% crystal violet staining solution for 15 min. The stained biofilms were then washed thrice with sterile water and air-dried before undergoing brightfield microscopy (Evos-FL; Life Technologies, Thermofisher Scientific, Carlsbad, CA, USA).

Confocal laser scanning microscopy (CLSM) was performed to assess the effect of the antimicrobials on the viability of the cells within the biofilms and their three-dimensional structures by live/dead staining as per the manufacturer’s protocol (LIVE/DEAD™ BacLight™ Bacterial Viability Kit; Thermofisher Scientific, USA) using the Zeiss LSM 710 Confocal laser scanning microscope (Carl Zeiss, Thornwood, New York, NY, USA). For this purpose, the biofilms were formed on 35/10 mm confocal dishes (Greiner bio-one, Frickenhausen, Germany) and then treated with different concentrations of CHG and CTR and their combination for 24 h. After incubation, the treated biofilms were washed with sterile water and stained with 0.05 mL of 1:1000 diluted pre-mix solution of Syto9 and PI for 5 min, followed by visualization under the microscope. Images were acquired using a plan apochromat 40×/1.4 Oil DIC objective lens with 2× magnification and 512 × 512 dimensions. The relative fluorescence ratio of Syto9 to PI was calculated to estimate the cell viability within the biofilms, and the biofilm thickness was estimated using the comstat2 plugin embedded in ImageJ 1.54g [18,19,20].

2.7. Motility and Congo Red Binding

Swarming motility was assessed on Tryptic soy with 0.5% agar plates, while curli/slime formation was assessed on Congo red containing tryptic soy agar plates in the presence and absence of the CHG-CTR combinations. Different combinations of CHG and CTR were mixed with soy agar medium and poured into plates. About 2 μL of the overnight-grown culture was spotted on the center of each TSA plate. The TSA plates were then incubated for 48–72 h at 37 °C, followed by measurements of motility zones.

2.8. Microbial Adhesion to Hydrocarbon (MATH) Assay

Bacterial cultures were grown in the presence and absence of different concentrations of combinations of CHG and CTR for 24 h at 37 °C. Cells were harvested by centrifugation at 8000 RPM for 10 min and washed thrice using 1× PBS. Finally, the cells were resuspended in 1× PBS, adjusting the OD_{600 nm} to ~0.9. This was followed by the addition of 1 mL hexane and vortexing for 1 min and maintenance in a static state for 10 min for the separation of the phases. When the aqueous and organic phases separated, the OD_{600 nm} was measured for the aqueous phase and the percent cell surface hydrophobicity (CSH) was calculated as follows:

CSH% = [1 − (OD_{600 nm} aqueous phase/OD_{600 nm} initial)] × 100

2.9. Preparation of the Chlorhexidine and Cetrimide-Based Formulation

An antiseptic and disinfectant solution (ASDL) was manufactured in the Savlon Swasth India Mission research laboratory, ITC Life Sciences and Technology Centre, Bangalore, India. The combination included CHG IP at 1.5% v/v and Strong Cetrimide solution BP equivalent to Cetrimide IP 3.0% w/v in an aqueous solution.

2.10. Standards and Guidelines Followed for Assessing Contact Killing

The efficacy testing of ASDL was performed following the standardized international guidelines:

Bactericidal activity (BS EN1276): Chemical disinfectants and antiseptics. Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas. Test method and requirements (phase 2, step 1) [21,22].

Bactericidal activity (IS 11479 (Adapted)): Antibacterial toilet soap—specification part 1: solid cake [23].

Anti-mycobacterial activity (EN 1656): Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area [24].

Mycobactericidal activity (EN 14348:2005): Quantitative suspension test for the evaluation of mycobactericidal activity of chemical disinfectants in the medical area, including instrument disinfectants (phase 2, step 1) [25].

Yeasticidal activity (BS EN 1650): Quantitative Suspension Test for the Evaluation of Fungicidal or Yeasticidal activity of Chemical Disinfectants and Antiseptics (phase 2, step 1) [26,27].

Virucidal activity (ASTM 1052): Standard Practice to Assess the Activity of Microbicides against Viruses in Suspension [28].

Virucidal activity (EN 14476:2013 + A2:2019): Quantitative suspension test for the evaluation of virucidal activity of disinfectants intended for use in the medical area (phase 2, step 1) [29,30].

Sporicidal activity 11. (BS: EN 13704: 2002) British Standards Institute, BS EN 13704, Chemical disinfectants–Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas—Test method and requirements (phase 2, step 1) [31].

2.11. Flow Cytometry-Based Assay for Anti-Leptospira Activity

The efficacy of ASDL against Leptospira interrogans, a pathogenic, spirochetal bacterium, was evaluated using flow cytometry [32]. Briefly, Syto9/PI-based live/dead staining was performed to evaluate the number of live and dead cells in a cell suspension using a flow cytometer.

3. Results

3.1. The CHG-CTR Combination Exhibited Synergistic Antimicrobial Activity Against P. aeruginosa

The individual antimicrobial activities of CHG and CTR were estimated by determining the minimum inhibitory concentrations against P. aeruginosa and S. aureus. Previous reports show that CHG exhibits antimicrobial activity against P. aeruginosa within the MIC range of 2–64 mg/L [33]. Our results showed that CHG was able to inhibit the growth of P. aeruginosa at 4 mg/L and that of S. aureus at 1 mg/L (

Table 1. Fractional inhibitory concentrations of different chlorhexidine gluconate and cetrimide combinations against P. aeruginosa.

P. aeruginosa
MICCHG	MICCTR	FICCTR-CHG	FICCHG-CTR	FICI	Interaction
4	256	0.5	0.5	1.0	Additive
4	128	0.5	0.25	0.75	Additive
4	64	0.5	0.125	0.625	Additive
4	32	0.5	0.063	0.563	Additive
4	16	0.5	0.031	0.531	Additive
4	8	0.5	0.016	0.516	Additive
4	4	0.5	0.008	0.508	Additive
4	2	0.5	0.004	0.504	Additive
2	256	0.25	0.5	0.75	Additive
2	128	0.25	0.25	0.5	Additive
2	64	0.25	0.125	0.375	Synergy
2	32	0.25	0.063	0.313	Synergy
2	16	0.25	0.031	0.281	Synergy
2	8	0.25	0.016	0.266	Synergy
1	256	0.125	0.5	0.625	Additive
1	128	0.125	0.25	0.375	Synergy
1	64	0.125	0.125	0.25	Synergy
1	32	0.125	0.063	0.188	Synergy
1	16	0.125	0.031	0.156	Synergy
0.5	256	0.063	0.5	0.563	Additive
0.5	128	0.063	0.25	0.313	Synergy
0.5	64	0.063	0.125	0.188	Synergy
0.5	32	0.063	0.063	0.125	Synergy
0.25	256	0.031	0.5	0.531	Additive
0.25	128	0.031	0.25	0.281	Synergy
0.25	64	0.031	0.125	0.156	Synergy
0.125	256	0.016	0.500	0.516	Additive
0.125	128	0.016	0.250	0.266	Synergy

). This is in line with previous reports suggesting that CHG is a better antimicrobial agent against Gram-positives than Gram-negatives, though a proven mechanism has yet to be discovered [34]. CTR exhibited growth inhibition against P. aeruginosa at 512 mg/L and against Gram-positive S. aureus at 8 mg/L (Table 1 and

Table 2. Fractional inhibitory concentrations of different chlorhexidine gluconate and cetrimide combinations against S. aureus.

S. aureus
MICCHG	MICCTR	FICCTR-CHG	FICCHG-CTR	FICI	Interaction
0.5	4	0.5	0.5	1	Additive
0.25	4	0.25	0.5	0.75	Additive
0.125	4	0.125	0.5	0.625	Additive
0.063	4	0.063	0.5	0.563	Additive
0.031	4	0.031	0.5	0.531	Additive
0.016	4	0.016	0.5	0.516	Additive
0.008	4	0.008	0.5	0.508	Additive
0.004	4	0.004	0.5	0.504	Additive
0.5	2	0.5	0.25	0.75	Additive

). Since we aimed to explore the effect of the combination of CTR with CHG, we further determined the FICs of the different combinations of these antimicrobial agents. Traditionally, an FIC_I below 0.5 is considered synergistic. The combination of CTR with CHG reduced the individual MICs of these antimicrobial agents against P. aeruginosa (>4-fold), leading to a fractional inhibitory concentration (FIC_I) of <0.5, thereby showing synergism. The combination did not show any synergistic effect against S. aureus, though an additive effect was observed at individual concentrations of ~0.25 mg/L of CHG and ~2 mg/L of CTR (Table 1 and Table 2). In such a case, the usual interpretation is that the tested antimicrobials contributed equally (FIC_individual ≤ 0.25) to the reduced concentration required for the antimicrobial activity.

To obtain extended information from FICs, Fatsis-Kavalopoulos N et al. reported that plotting individual FICs (FIC_CTR→CHG vs. FIC_CHG→CTR) on a two-axis plot can describe the directionality of a synergism [15]. In a two-axis plot, directionality can be observed if the FIC_CTR→CHG ≤ 0.25, which indicates that CTR promotes the action of CHG. Alternatively, an FIC_CHG→CTR ≤ 0.25 indicates the reverse. Considering this, the trends in the graph depict that CHG and CTR contributed nearly equally to the synergistic antimicrobial activity against P. aeruginosa (Figure S1a), while the additive effect against S. aureus (Figure S1b) was most likely because of the addition of CTR to CHG (the quadrant at the left bottom of the red dotted lines represents the individual FIC ≤ 0.25).

3.2. Effect on Biofilm Formation

3.2.1. The Presence of CHG-CTR Reduced the Adhesion of S. aureus Biofilm on Surfaces

Owing to the aforementioned effect whereby the combination of CHG and CTR showed superior antibacterial activity synergistically against planktonic cells of P. aeruginosa and additively against S. aureus, we wished to further explore its effect on biofilm formation and disruption. In addition to its antiseptic nature, CTR’s being a surfactant can additionally affect the mechanical stability of biofilms [35]. The biofilm forming ability of S. aureus and P. aeruginosa in the presence of the CHG-CTR combination was estimated by determining the specific biofilm formation and the adhesion index using the crystal violet staining method. Although crystal violet staining does not reflect the viability of cells within biofilms, it can efficiently quantify biofilm formation with respect to a reduction in total adhered biomass. The presence of sub-inhibitory concentrations of CHG and CTR had no significant effect on the biofilm formation ability of P. aeruginosa (Figure S2). In contrast, there was a substantial reduction in the specific biofilm formation of S. aureus in the presence of sub-inhibitory concentrations (one-quarter of the individual MICs) of 0.25 mg/L and 2 mg/L of CHG and CTR, respectively (Figure 1a). The adhesion index also decreased with increasing sub-inhibitory concentrations of the CHG-CTR combinations (Figure 1b). Therefore, it can be concluded that the presence of CHG-CTR can help prevent the adherence of cells to a surface, thereby impeding biofilm formation. Further, a qualitative evaluation of the effect of sub-inhibitory concentrations on the biofilm formation of S. aureus was performed.

3.2.2. Microscopic Analysis of the S. aureus Biofilms Corroborated the In Vitro Quantitative Analysis

The effect of these antimicrobial agents on the biofilm formation of S. aureus was visualized using brightfield microscopy of the crystal violet-stained biofilms (Figure 1c). Crystal violet staining showed a reduction in biofilm formation in the presence of the combination of antimicrobials, as depicted by the reduced density of crystal violet stain. The effect of these antimicrobials was also visualized with Syto9-stained biofilms, imaged using CLSM (Figure 2a–d). Syto9 dye (green channel) can stain total cells, live or dead. We quantified the relative fluorescence units (RFUs) of this channel in the presence of the antimicrobials (Figure 2e). Overall RFUs were drastically decreased in the presence of the individual antiseptics and their combination, which implies reduced biomass in the presence of the antimicrobials. This reduced signal intensity observed in the Syto9-stained biofilms correlates with the effect on the adhesion index quantified using crystal violet staining.

We also quantified the biovolume of the S. aureus biofilms in the presence of CHG and CTR in the three-dimensional stacked images obtained via CLSM. The presence of a sub-inhibitory concentration of CHG substantially reduced the biovolume of S. aureus biofilm, whereas CTR and its combination with CHG drastically reduced the overall biomass, as can be seen in the microscopy images. In conclusion, the addition of CTR along with CHG synergistically inhibited the growth of P. aeruginosa, though no effect on biofilm formation was observed. In the case of S. aureus, an additive inhibitory effect was observed, along with significant inhibition of adhesion to the surface, leading to reduced biofilm formation. Such effects can be attributed to different cell surface architectures as well as peptidoglycan structures in both bacteria. Further, we ascertained the effect of these antimicrobials on the pre-formed biofilms of S. aureus and P. aeruginosa.

3.3. Effect on the Pre-Formed Biofilms

3.3.1. Treatment with CHG-CTR Led to Reduced Metabolic Activity of Cells Within the Biofilms

To understand the effect on the eradication of the pre-formed biofilms (24 h), metabolic activity assessment using the resazurin assay was carried out. The MBEC of CHG was observed to be 128 mg/L against pre-formed P. aeruginosa biofilms, which is 32-fold higher than the MIC against planktonic cells (Figure 3a). The MBEC of CTR alone was 1024 mg/L, which is twice the MIC of CTR against planktonic cells. This observation suggests that CTR is relatively more efficient than CHG in inhibiting the metabolic activity of P. aeruginosa biofilms. Interestingly, the combination of CTR with CHG reduced the MBEC of CHG by >4-fold as compared to its individual MBEC (Figure 3a). In the case of S. aureus, the presence of CHG alone at a concentration of 32 mg/L inhibited the metabolic activity, while CTR inhibited the activity at 64 mg/L. The combination of CTR (32 mg/L) with CHG exhibited a substantial reduction in the metabolic activity (Figure 3b). Further, the MBECs were confirmed by the spot assay. For this purpose, the Calgary Biofilm Device containing a 96-peg lid plate was used. The biofilms were grown on these peg lids and challenged by the antimicrobials. The cells dispersed from the biofilms post-sonication were spotted on TSA plates. The concentrations at which no growth was observed were considered as biofilm eradication concentrations. For S. aureus, the individual MBECs were observed to be ≥32 mg/L for CHG and ≥64 mg/L for CTR, while for P. aeruginosa the observed MBEC values were ≥128 mg/L and ≥1024 mg/L for CHG and CTR, respectively, corroborating the resazurin assay results.

3.3.2. CLSM Assessment of the CHG-CTR-Treated Pre-Formed Biofilms of P. aeruginosa and S. aureus

The effect on pre-formed biofilms was also evaluated using CLSM to determine the Syto9 (green)-to-PI (red) ratio (Figure 4). As discussed previously, Syto9 dye binds to both live and dead cells, as it can penetrate live cells. In contrast, PI cannot penetrate live cells and binds to dead cells only. Therefore, the ratio of Syto9 to PI can depict the viability of cells. A decrease in cell viability in terms of the Syto9/PI ratio was observed in the presence of the antimicrobials. The combination caused the highest reduction in cell viability as compared to the individual compounds. Therefore, CHG and CTR in combination possess significant potential as antimicrobial and antibiofilm agents against P. aeruginosa and S. aureus.

3.3.3. Effect on Cell Surface Hydrophobicity, Motility and Congo Red Binding

Cell surface hydrophobicity (CSH) is a biophysical measurement of the preference of bacterial cells towards hydrophobic or hydrophilic environments. Cells prefer a hydrophobic environment if CSH is higher, and vice versa. This parameter affects cell–cell and cell–surface interactions and is therefore crucial to understanding the virulence and the biofilm formation of microbes. Here, we calculated the cell surface hydrophobicity of S. aureus and P. aeruginosa using the Microbial Adhesion to Hydrocarbon test (MATH) in the presence of CHG and CTR. However, no significant difference was observed in bacterial preference for an organic solvent in the presence of these antimicrobials at sub-inhibitory concentrations (Figure S3). Bacterial motility helps bacteria to move towards favorable conditions. In biofilms, bacteria usually exhibit non-motile behavior. The inhibitory effect of antimicrobials on biofilms is usually correlated with decreased motility, though no visible difference in motility was observed. One of the major factors associated with the adherence of biofilms is the production of an amyloid fiber known as curli as a part of the biofilm’s extracellular matrix. There was no difference in the curli formation of P. aeruginosa in the presence of CHG-CTR as assessed by the Congo red agar assay. Though curli formation does not occur in S. aureus, phenol-soluble modulins are involved in forming curli-like structures, thereby enhancing adherence to the surface. However, extracellular polymeric substances bind to Congo red dye, showing the presence of biofilm. No difference was observed in the morphological features of S. aureus in the presence of CHG-CTR on Congo red agar plates as compared to the control.

3.3.4. Antimicrobial Efficacy of the Formulation

The combination of CHG and CTR as an antiseptic and disinfectant (ASDL) has been on the market for decades. Given the synergistic/additive effects of CHG and CTR on planktonic cells and biofilms, we looked at the antimicrobial efficiency of a formulation containing CHG and CTR against a wide range of micro-organisms, including those of clinical relevance in the present day. This formulation contains 1.5% v/v of chlorhexidine gluconate IP and 3.0% w/v of CTR strong solution IP. A comprehensive illustration of the spectrum of activity of this formulation against clinically relevant and evolved bacteria, fungi and viruses is still lacking. The antibacterial efficacy against bacteria was estimated by the contact-time killing method, as per the guidelines prescribed by International standards (

Table 3. Antibacterial efficacy of antiseptic and disinfectant liquid.

Micro-Organism	Dilution	Contact Time (min)	Log Reduction
Bactericidal activity shown as per BS EN 1276
Escherichia coli ATCC 10536 *	1:15	1	>5
Pseudomonas aeruginosa ATCC 15442 *	1:15	1	>5
Enterococcus hirae ATCC 10541 #	1:15	1	>5
Staphylococcus aureus ATCC 6538 #	1:15	1	>5
Bactericidal activity shown as per IS 11479
Escherichia coli ATCC 10536 *	1:15	1	>5
Salmonella typhi AB 1468 *	1:15	1	>6
Salmonella typhi AB 1649 *	1:15	1	>6
Klebsiella pneumonia NCIM 5215 *	1:15	1	>6.78
Klebsiella pneumonia ATCC 700603 *	1:15	1	>5.93
Klebsiella pneumonia BAA 2146 *	1:15	1	>6.14
Vibrio cholerae MTCC 3906 *	1:15	1	>5.77
Escherichia coli ATCC 32518 *	1:15	1	>6.39
Escherichia coli ATCC 25922 *	1:15	1	>6.46
Escherichia coli MTCC 448 *	1:15	1	>6.75
Escherichia coli ATCC 8739 *	1:15	1	>6.31
Escherichia coli ATCC 10536 *	1:15	1	>6.57
Salmonella typhimurium ATCC 14028 *	1:15	1	>6.52
Enterobacter aerogenes ATCC 13048 *	1:15	1	>6.82
Salmonella enterica MTCC 1166 *	1:15	1	>6.64
Salmonella enterica MTCC 9844 *	1:15	1	>6.79
Salmonella enterica MTCC 3219 *	1:15	1	>6.42
Salmonella enterica MTCC 3223 *	1:15	1	>6.34
Salmonella enterica MTCC 3232 *	1:15	1	>6.38
Salmonella enterica ATCC 13076 *	1:15	1	>6.75
Salmonella typhi AB 1468 *	1:15	1	>6.31
Salmonella typhi AB 1649 *	1:15	1	>6.43
Shigella flexneri ATCC 12022 *	1:15	1	>6.65
Salmonella typhimurium ATCC 14028 *	1:15	1	>6.52
Salmonella enterica MTCC 1166 *	1:15	1	>6.64
Salmonella enterica MTCC 9844 *	1:15	1	>6.79
Salmonella enterica MTCC 3219 *	1:15	1	>6.42
Salmonella enterica MTCC 3223 *	1:15	1	>6.34
Salmonella enterica MTCC 3232 *	1:15	1	>6.38
Salmonella enterica ATCC 13076 *	1:15	1	>6.75
Shigella flexneri ATCC 12022 *	1:15	1	>6.65
Enterobacter aerogenes ATCC 13048 *	1:15	1	>6.82
Enterobacter durans MTCC 3031 *	1:15	1	>6.75
Haemophilus influenza ATCC 49247 *	1:15	1	>6.61
Haemophilus paraphrophilus ATCC 49917 *	1:15	1	>6.94
Haemophilus parainfluenzae ATCC 7901 *	1:15	1	>6.77
Acinetobacter baumannii ATCC 19606 *	1:15	1	>5.74
Acinetobacter baumannii BAA 1605 *	1:15	1	>6.01
Camphylobacter jejuni ATCC 49301 *	1:15	1	>6.11
Helicobacter pylori ATCC 700392 *	1:15	1	>5.84
Helicobacter pylori NCTC 11637 *	1:15	1	>6.04
Neisseria meningitides ATCC 13090 *	1:15	1	>5.93
Prevotella bivia ATCC 29303 *	1:15	1	>6.49
Yersinia pseudotuberculosis ATCC 13979 *	1:15	1	>5.74
Serratia marcescens ATCC 14041 *	1:15	1	>6.92
Neisseria meningitides ATCC 13090 *	1:15	1	>5.93
Haemophilus influenzae ATCC 49247 *	1:15	1	>6.61
Moraxella catarrhalis ATCC 25238 *	1:15	1	>6.56
Bacillus cereus NCIM 2155 #	1:15	1	>5.9
Streptococcus pneumonia ATCC 12344 #	1:15	1	>6.81
Streptococcus pneumoniae ATCC 700674 #	1:15	1	>6.61
Streptococcus pneumoniae ATCC 35088 #	1:15	1	>6.16
Streptococcus pneumoniae ATCC 49150 #	1:15	1	>6.1
Streptococcus pneumoniae ATCC 49616 #	1:15	1	>6.03
Corynebacterium diphtheria ATCC 13812 #	1:15	1	>6.61
Staphylococcus aureus ATCC 6538 #	1:15	1	>6.04
Staphylococcus aureus ATCC 33591 #	1:15	1	>6.87
Staphylococcus aureus ATCC 43300 #	1:15	1	>6.47
Staphylococcus aureus MTCC 96 #	1:15	1	>6.54
Staphylococcus aureus ATCC 700698 #	1:15	1	>6.49
Staphylococcus aureus ATCC 29213 #	1:15	1	>6.72
Staphylococcus aureus ATCC 6538P #	1:15	1	>6.54
Staphylococcus aureus MTCC 3103 #	1:15	1	>6.64
Staphylococcus aureus MTCC 1144 #	1:15	1	>6.75
Corynebacterium striatum MTCC 8963 #	1:15	1	>6.2
Enterococcus faecium ATCC 700221 #	1:15	1	>6.54
Enterococcus faecium ATCC 49624 #	1:15	1	>6.37
Enterococcus faecium ATCC 51558 #	1:15	1	>6.23
Staphylococcus epidermidis ATCC 700570 #	1:15	1	>6.48
Staphylococcus epidermidis ATCC 3086 #	1:15	1	>6.22
Staphylococcus epidermidis ATCC 26936 #	1:15	1	>6.23
Staphylococcus epidermidis MTCC 3086 #	1:15	1	>6.11
Actinomyces viscosus MTCC 7435 #	1:15	1	>6.48
Lactococcus latics ATCC 11454 #	1:15	1	>6.58
Streptococcus pyogenes ATCC 432202 #	1:15	1	>6.58
Streptococcus pyogenes BAA 946 #	1:15	1	>6.75
Streptococcus pyogenes MTCC 1928 #	1:15	1	>6.25
Streptococcus pyogenes MTCC 1924 #	1:15	1	>6.64
Streptococcus pyogenes MTCC 1926 #	1:15	1	>6.37
Streptococcus pyogenes MTCC 1927 #	1:15	1	>6.33
Clostridium perfringens ATCC 13124 #	1:15	1	>5.85
Clostridium perfringens ATCC 3624 #	1:15	1	>6.62
Clostridium sporogenes ATCC 11437 #	1:15	1	>6.6
Leuconostoc mesenteroides MTCC 867 #	1:15	1	>6.69
Finegoldia magna ATCC 29328 #	1:15	1	>6.62
Arthobacter sp MTCC 1726 #	1:15	1	>6.69
Lactobacillus acidophilus MTCC 10307 #	1:15	1	>6.86
Bactericidal activity shown against antibiotic-resistant pathogens as per IS 11479
Streptococcus pneumoninae ATCC 700674 # (intermediate resistance to penicillin)	1:15	1	>6.61
Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 #	1:15	1	>6.87
Pseudomonas aeruginosa (broad spectrum of resistance to various commercial antibiotics) *	1:15	1	>6.60
Enterococcus fecalis (VRE) # (resistant to Gentamicin and Vancomycin)	1:15	1	>7.38
Enterococcus fecium (VRE) ATCC 700221 # (resistant to Vancomycin)	1:15	1	>6.54
Anaerobic microbes shown as per IS 11479
Clostridium perfringens ATCC 13124 #	1:15	1	>5.85
Prevotella bivia ATCC 29303 *	1:15	1	>6.49
Clostridium difficile BAA 1382 #*	1:15	1	>6.03
Camphylobacter jejuni ATCC 49301 *	1:15	1	>6.11
Clostridium perfringens ATCC 3624 *	1:15	1	>6.62
Clostridium sporogenes ATCC 11437 #*	1:15	1	>6.6
Propionibacterium acnes MTCC 1951 #	1:15	1	>6.36
Propionibacterium acnes ATCC 11827 #	1:15	1	>6.91
Propionibacterium acnes MTCC 3297 #	1:15	1	>6.74
Finegoldia magna ATCC 29328 #	1:15	1	>6.62
Mycobacterium activity shown as per EN 1656 (1999) 1, EN14348 2and IS11479 3
Mycobacterium tuberculosis H37Rv 1	1:15	1	>4.81
Mycobacterium pheli NCIM 2240 1	1:15	1	>4.81
Mycobacterium terrae 2	-	60	>4.51
Mycobacterium smegmatis ATCC 607 3	1:15	1	>5.95
Mycobacterium smegmatis mc 2 155 ATCC 700084 3	1:15	1	>6.03
Bactericidal reduction and inactivation of Leptospira interrogans
Leptospira interrogans *	1:15	5	>3.0
Sporicidal activity shown as per EN 13704
Bacillus subtilis # ATCC 6633	1:1	60	>3

^# Gram-positive; * Gram-negative; ¹ EN1656(1999), ² EN14348, ³ IS11479

). The formulation exhibited significant inhibitory activities against clinical (nosocomial pathogens, opportunistic pathogens, drug resistant bacteria) and environment relevant bacteria. Moreover, this formulation was able to kill the anerobic microbes. Similarly, the formulation showed >4 log reduction against the Mycobacterial sp. We enumerated the live/dead cells after 5 min of treatment with ASDL using flow cytometry and observed a >99.9% reduction in Leptospira interrogans viability. To our knowledge, this is the only antiseptic and disinfectant liquid with proven efficacy against Leptospira interrogans.

Moreover, CHG-CTR combination showed a rapid killing efficacy (>5 log reduction within 10 s) against a set of microbes which are relevant to our day-to-day life (

Table 4. Rapid killing efficacy (within 10 s) of antiseptic and disinfectant liquid against a set of bacteria and fungi.

Micro-Organism	Dilution	Log Reduction
Pseudomonas aeruginosa ATCC 15442	1:15	>6.89
Escherichia coli ATCC 10536	1:15	>6.57
Staphylococcus aureus ATCC 6538	1:15	>6.95
Enterococcus hirae ATCC 10541	1:15	>6.40
Corynebacterium striatum ATCC 6940	1:15	>6.18
Propionibacterium acnes MTCC 1951	1:15	>7.04
Staphylococcus aureus (MRSA) ATCC 3359	1:15	>6.56
Streptococcus pneumoniae ATCC 700674 (penicillin-resistant)	1:15	>5.56
Candida albicans ATCC 10231	1:15	>5.28

).

Apart from bacteria, fungal species are also the major cause of infections and adverse outcomes in hospitals as well as personal settings. This formulation was able to reduce the viability of the fungal species like Candida, Malassezia, Crptococcus etc. (

Table 5. Antifungal efficacy of antiseptic and disinfectant liquid.

Micro-Organism	Dilution	Contact Time	Log Reduction
Yeasticidal/Fungicidal Activity Shown as per IS 11479
Cryptococcus albidus ATCC 26902	1:15	1	>6.65
Candida krusei ATCC 6258	1:15	1	>4.93
Filobasidiella neoformans var. bacillispora (Cryptococcus neoformans var. gattii) MTCC 1347/ATCC 32269	1:15	1	>5.08
Cryptococcus laurentii MTCC 2898	1:15	1	>4.4
Candida albicans ATCC 10231	1:15	1	>6.06
Malassezia furfur ATCC 14521	1:15	1	3.02
Malassezia globosa NBRC 101597	1:15	1	>4.92
Malassezia restricta ATCC MYA 4611	1:15	1	>4.94
Candia albicans NCIM 3102	1:15	1	>5.01
Malassezia pachydermatis MTCC 1369	1:15	1	>4.61
Yeasticidal Activity Shown as per BS EN 1650
Candida albicans ATCC 10231	1:15	15	>4

).

The use of chemical disinfectants is widely advocated to avoid contamination with viral agents like HIV-1 (human immunodeficiency virus type 1) and SARS-CoV-2 [36]. Therefore, we investigated the effectiveness of the test product against prominent viral infection agents (

Table 6. Antiviral efficacy of antiseptic and disinfectant liquid.

Virus Name	Dilution	Contact Time	Log Reduction
Virucidal Activity Shown as per ASTM E1052 1 and EN 14476 2
Human immunodeficiency virus type 1 ^,1	1:1	1	>4.10
Duck hepatitis B $ virus 1	1:1	2	2
H1N1 influenza A ^ virus 2	1:15	2	1.75
H9N2 ^ (avian flu) 1	1:15	5	>4.67
hRSV ^,1	1:15	5	2
Adenovirus $,2	1:15	5	2.17
SARS-CoV-2 ^ (COVID-19 virus) 1	1:15	2	≥ 3.10
Zika ^ virus 1	1:1	5	2
Monkeypox virus $,1	1:15	5	>4.42
Murine coronavirus ^,2	1:15	2	>4
Murine norovirus ^,2	1:15	5	2.67
Simian rotavirus ^,2	1:15	1	>4.4

^{^} RNA virus; ^$ DNA virus; Tested using method ¹ ASTM E1052, ² EN14476

).

4. Discussion

The post-COVID-19 era has seen a substantial increase in health and hygiene measures in personal and institutional settings. The presence of microbial contamination in surroundings can cause infections, and the use of antiseptics and disinfectants is one of the primary methods to prevent such microbial contamination of living and non-living surfaces.

The combination of CHG and CTR is one of the most preferred antiseptics and disinfectants to be used in institutional settings. Approved over-the-counter topical antiseptic formulations containing the combination of chlorhexidine gluconate and cetrimide are available in different countries and have been used in first-aid and other topical applications for many decades [37,38]. This information is documented in monographs in the pharmacopoeias and formulary publications of different countries (the National Formulary of India, Section 12.1, Antiseptics, and the British Pharmaceutical Codex).

Although the combination of chlorhexidine gluconate and cetrimide has been used as a key antiseptic and disinfectant for several decades, a thorough re-investigation is required to assess its antimicrobial potential against rapidly evolving microbes. A recent meta-analysis indicates that microbes are still susceptible to CHG, and very little resistance has been documented [39]. This gives it an edge over other well-known antiseptics like povidone iodide. However, further studies are required to understand the efficacy of the combination of CHG and CTR.

Therefore, we investigated the synergistic antimicrobial and antibiofilm potential of CHG and CTR against two key microbial species, namely, P. aeruginosa and S. aureus. We also evaluated the antimicrobial efficacy of the combination against several important pathogens—bacteria, fungi and viruses relevant to the present-day scenario, including MDR strains, ESKAPE pathogens and SARS-CoV-2 virus.

P. aeruginosa is a Gram-negative bacterium widely present as a surface contaminant and forms difficult-to-treat biofilms. In the present study, we observed that CHG could inhibit the growth of P. aeruginosa at 4 mg/L. This is in line with previous studies which report that the MIC of CHG against P. aeruginosa ATCC 15442 ranges from 4 to 64 mg/L, subject to experimental conditions [10,40,41]. Interestingly, the addition of CTR significantly reduced the MIC against P. aeruginosa to a level demonstrative of synergy. CHG and CTR are both cationic molecules which disturb cell membrane integrity. The fractional inhibitory concentration curve showed the nearly equal distribution of the plot along the x- and y-axes, which indicates the equal contribution of both antimicrobials in achieving the synergistic antibacterial activity. It is noteworthy that CTR alone exhibited growth inhibition at a comparatively higher concentration (512 mg/L) than CHG alone, and that is the primary reason for its use in selection media for P. aeruginosa [42]. These results emphasize that although CTR alone may not have high antibacterial activity against P. aeruginosa, its combination with CHG can inhibit the growth of this bacterium at a much lower concentration. Similarly, in the case of S. aureus, previous reports suggest that CHG alone can inhibit growth in the MIC range of 0.125–8 mg/L [10,43]. Our results showed that S. aureus growth was inhibited at a minimum concentration of 1 mg/L of CHG and 8 mg/L of CTR. Though CHG-CTR did not show synergistic activity against S. aureus, an additive effect was observed. Therefore, it can be inferred that the CHG-CTR combination possesses more potential as an antibacterial agent against P. aeruginosa and S. aureus as compared to CHG and CTR alone. Also, considering the limitations of CHG alone as an efficacious antiseptic against certain microbes [10], its potential can be re-evaluated in combination with CTR.

Biofilms are aggregates of bacteria adhered to a surface. They form a protective environment and allow bacteria to exhibit increased resistance against antimicrobials. Biofilm formation in healthcare settings and in wounds can render typical antiseptics and antibiotics less effective. As per previous reports, the associated use of CTR and CHG exhibited better efficacy in biofilm eradication against Gram-positive E. faecalis biofilms than the individual antimicrobials [44]. Also, it has been shown that the presence of CHG reduces adherence of E. faecalis to dental surfaces [45]. In the present study, the presence of these antimicrobials in sub-inhibitory concentrations did not affect the biofilm formation of P. aeruginosa. However, considering the synergistic antimicrobial activity of CHG and CTR against this organism, the combination of these antimicrobials is likely to negatively affect biofilm formation by reducing the overall growth of bacteria. In the case of S. aureus, the combination of CHG and CTR drastically reduced adherence to the surface. Interestingly, CTR alone affected biofilm formation to an extent similar to the combination. The substantial antibiofilm formation effect of the combination is likely a result of the presence of CTR. Therefore, it can be asserted that the combination of CHG and CTR can work as an effective antimicrobial and antibiofilm agent. We explored the mechanistic basis for this reduced adherence of S. aureus cells. Interestingly, the concentrations of CHG-CTR that reduced biofilm formation of S. aureus did not alter the cell surface hydrophobicity, curli formation or other slime formations. We speculate that the combination of these two cationic molecules may affect the overall surface phenomenon of biofilm attachment.

Disinfectants are invaluable in hospital settings in the eradication of biofilms in catheters, central lines, etc. The combination of CHG and CTR was able to effectively eradicate the pre-formed biofilms of both tested micro-organisms. The MBEC for CHG and CTR in combination against S. aureus was observed to be two-fold lesser than the individual biofilm eradication concentrations, while a substantial reduction in metabolic activity was observed at 32 mg/L CTR with different CHG concentrations. In the case of P. aeruginosa, the combination of CTR with CHG reduced the MBECs of CHG by >4-fold. CTR is a cationic surfactant, which can also affect the mechanical stability of biofilms. Our data show that the combination of CTR and CHG can effectively eradicate pre-formed biofilms as compared to these antimicrobials alone. Based on the above observations, it can be established that although CHG and CTR are used individually as antiseptic agents, the combination of these antimicrobials can compensate for the limitations of the individual antimicrobials and can significantly improve their overall efficacy as antimicrobial and antibiofilm agents.

Further, evaluation of a formulation comprising CHG-CTR was performed. The tested formulation exhibited >99.9% inhibition after 1 min of treatment against the tested bacteria at a final concentration of 6.25% (1:15). These bacteria included various Staphylococcus aureus strains, which are causative agents for multiple infectious diseases, including food poisoning, endocarditis, impetigo, Kawasaki disease, folliculitis, etc. [46]. The combination was also effective against the bacteria responsible for central nervous system, abdominal, lung and multi-organ-related infections, among many other bacteria [47]. The rising threat of antimicrobial resistance has limited the use of antibiotics. The use of proper hygiene measures is an effective method to prevent the dissemination of AMR-causing factors [48]. ASDL also shows >99.9% efficacy in the eradication of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli (ESKAPE pathogens), which are the leading cause of hospital-acquired infections (HAIs) [49]. This formulation was also effective against antimicrobial-resistant pathogens like MRSA and VRE and nosocomial fungal strains like Candida sp. and Cryptococcus sp. Moreover, the tested formulation exhibited rapid killing (in less than 10 s) against clinically relevant micro-organisms.

Further, we tested the efficacy of ASDL against a range of anaerobic bacteria. These anaerobic bacteria colonize the internal parts of organs with limited access to oxygen, causing brain, lung and abdominal abscesses (Clostridium sp.); dental infections; aspiration pneumonia (Prevotella bivia); bite infections; necrotizing infections of soft tissues; and acne (Propionibacterium sp.) [50]. The test product exhibited a >99.99% kill percentage against all the tested anaerobes.

Mycobacterium tuberculosis is still one of the leading causes of infection in India. Also, non-tuberculous mycobacterium (NTM) species, which colonize and cause infections, are present in the environment [51]. These infections are mostly caused by nosocomial exposures and highlight the need for proper disinfection in healthcare settings. It has been reported that CHG alone has limited antimicrobial efficacy against clinically relevant micro-organisms like Mycobacterium sp., except for M. smegmatis [52]. The formulation comprising the combination of CHG and CTR exhibited a >4-log reduction in the viability of Mycobacterium tuberculosis after 5 min of contact time. ASDL showed remarkable effectiveness (>4-log reduction) against the other tested mycobacterium strains, viz., M. smegmatis and M. pheli.

ASDL was also evaluated against Leptospira interrogans, a highly pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis [53]. Leptospira are excreted in the urine of animals, and they affect humans, directly or indirectly, when exposed through contact with an environment contaminated by the urine of infected animals, such as soil and surface water [54]. A promising application of this formulation arises from its ability to inhibit Leptospira interrogans.

Chemical disinfectants can eradicate the environment contamination of viral agents like HIV-1 (human immunodeficiency virus type 1) and SARS-CoV-2 [31]. The transmission routes of pathogens to hosts are complicated and difficult to investigate; however, enough evidence supports the transfer of viruses through surface contamination and aerosols. Viruses can be enveloped or non-enveloped, with DNA/RNA as their genetic material. Non-enveloped viruses like norovirus and adenovirus can be more virulent and resistant to harsh treatments than enveloped viruses like H1N1, hRSV, etc. Moreover, the absence of any approved vaccination against such viruses (e.g., HIV) leaves disinfection as the primary and crucial mode of prevention of infection. The test product showed >99.9% killing of HIV-1 and SARS-CoV-2 at a 6.25% concentration. The efficacy was also confirmed against a range of viruses, as shown in Table 4. The formulation exhibited antimicrobial activity, i.e., a 2-log reduction in the viability of non-enveloped viruses like hepatitis B, adenovirus, norovirus and rotavirus.

Chlorhexidine also imparts antifungal activity and antiviral activity. The mechanism of action for fungi is very similar to that of bacteria. A fungus uptakes CHG in a short amount of time and it impairs the integrity of the cell walls and the plasma membranes, entering the cytoplasm, resulting in the leakage of cell contents and cell death. Candida spp. are well-known infectious agents that affect the mucous membrane and moist areas of the skin. The severity of infection can vary from mild to more serious systemic infections, especially in immunocompromised individuals [55]. Immunocompromised individuals are also prone to Cryptococcus sp. infections, though limited reports have shown that these fungi can affect healthy individuals [56]. The use of antiseptics and disinfectants has therefore become a necessity to avoid critical consequences. The antifungal efficacy of the ASDL formulation was also evaluated (Table 5). ASDL exhibited a ≥4-log₁₀ reduction for both the Candida sp. and Cryptococcus sp. tested. Moreover, ASDL was also effective against the hair folliculitis-causing Malassezia sp. [57].

Therefore, it can be concluded that the age-old combination of CHG and CTR still possesses significant potential as an antiseptic and disinfectant and that it can be used for multiple applications in personal and institutional or healthcare settings.