Introduction

Infectious diseases caused by pathogenic microorganisms have now become one of the main causes of morbidity globally. One major reason is that pathogenic microorganisms can easily spread through daily behaviors and social interactions, including having dinner, unpacking mail, shaking hands, and hugging, as they can adhere to or colonize the surface of household items’ materials like glass, wood, plastic, and metal, as well as our body, such as skin and oral mucosa, and transmit by surface contact, posing serious challenges to personal hygiene and healthcare [1, 2]. The use of disinfectants has been considered an efficient approach to control the transmission of pathogenic microorganisms through the surface contact pathway [3]. Alcohol- and hypochlorite-based disinfectants are the most used ones in medical units and at home [4, 5]. However, such disinfectants have their shortcomings, including the strong irritant smell, rapid volatilization, the risk of contact dermatitis, and corroding, which restrict their application, especially in skin and oral disinfection [6, 7].

Due to the shortcomings of alcohol- and hypochlorite-based disinfectants, quaternary ammonium salts and biguanides reagents like cetylpyridinium chloride (CPC) and chlorhexidine acetate (CHA) are broadly applied for disinfection at present. Both these two drugs can damage the cell membrane, and induce leakage of cytoplasm and cell lysis [8, 9]. Compared with the alcohol- and hypochlorite-based agents, CPC and CHA exhibit bacteriostatic at low concentrations and non-toxic to mammalian cells at minimal biocidal concentration, making them more desirable for the preparation of hand-sanitizer-spray and oral rinse [10, 11]. Nevertheless, the high price and unpleasant taste restrict their broader application [12]. Moreover, the extensive use of CPC and CHA possesses the potential risk of antimicrobial resistance [13]. The combined use of existing reagents has become the main alternative method, since it has been proved to enhance the antimicrobial activity of the involved disinfectants and reduce the required dose, as the involved disinfectants normally work in different mechanisms, and the combined use of them may complement the shortcomings [14, 15], which is an effective approach to cut the cost, cut down unpleasant taste, and reduce the risk of antimicrobial resistance. Compared with the development of new pharmaceutical agents, another main alternative method, this approach is normally time-saving, costless, and low technique requirement [16]. The combined use of CPC or CHA with other disinfectants has been reported, e.g., nystatin [17], fluconazole [18], and chlorhexidine digluconate [19], which exhibited lower minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) against target bacteria or fungi than using either of them alone. Besides combining with other disinfectants, these two drugs have also been used in combination to eliminate living bacteria on in situ collected biofilms and virus [20, 21]. However, these reports only focused on the disinfection effect on a single type of pathogenic microorganisms. Since the public is normally suffering the risk of the transmission and infection of multiple types of pathogenic microorganisms, including gram-positive/gram-negative bacteria, fungi, and viruses, it is of great importance to develop a combination of existing drugs whose antimicrobial activity on multiple types of pathogenic microorganisms are fully investigated and confirmed [22].

Herein, the antimicrobial activity of the CPC/CHA combination was investigated. Despite these two drugs having been widely used for disinfection, no reports have comprehensively evaluated the antibacterial and antifungal activities of their combination. In this study, we tried to propose a universal concentration of CPC/CHA combination that would be applicable for the rapid killing of multiple types of pathogenic microorganisms by evaluating its antimicrobial activity against gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Bacillus cereus), gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium), and fungi (Candida lusitaniae and Aspergillus niger). In addition to antibacterial and antifungal activity, we also preliminarily investigated the antiviral activity of this combination. Moreover, the safety of CPC/CHA at this universal concentration was also evaluated by acute mucosal and skin irritation tests in vitro. Our work will open new perspectives for the development of board-spectrum disinfectants based on the combined use of present drugs, especially those to be used for skin and oral disinfection.

Materials and Methods

Strains and Reagents

S. aureus (ATCC 25923), B. subtilis (ATCC 11774), B. cereus (ATCC 14579), E. coli (ATCC 25922), P. aeruginosa (ATCC 15442), S. typhimurium (ATCC 14028), C. lusitaniae (ATCC 34449), and A. niger (ATCC 16888) strains were provided by the Affiliated Hospital of Qingdao University. CPC, fetal calf serum (FCS), Eagle’s minimum essential medium (EMEM), and agar powder were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). CHA and bovine serum protein (BSA) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). LB broth medium, Sabouraud dextrose broth medium, and D/E (Dey/Engley) neutralizing broth were purchased from Hope Bio-Technology Co., Ltd. (Qingdao, China). Glutaraldehyde solution (2.5%, v/v) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit was purchased from Thermo Fisher Scientific (China) Co., Ltd. (Shanghai, China). BCA Protein Colorimetric Assay Kit was purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). New Zealand white rabbits were purchased from Lukang Pharmaceutical Co., Ltd (Qingdao, China). All the other chemicals and reagents were of analytical grade.

Strains Culture Conditions

Bacterial and fungal strains involved in this work were stored in 50% (v/v) glycerol solution at − 80 °C. Frozen stock cultures of the bacterial strains were incubated in LB broth mediums (1% NaCl, 1% tryptone, and 0.5% yeast extract, pH 7.0) and then cultured on LB agar plates at 37 ℃ overnight [23]. Frozen stock cultures of C. lusitaniae strain was incubated in Sabouraud dextrose broth mediums (1% peptone and 4% dextrose, pH 5.6) and then cultured on Sabouraud dextrose agar plates at 35 °C overnight [17]. Frozen stock cultures of A. niger strain were incubated in Sabouraud dextrose broth mediums and then cultured on Sabouraud dextrose agar plates at 28 °C for 7 days [24]. A single colony was transferred into 200 mL of relevant mediums and incubated at 37 °C (for bacteria), 35 °C (for C. lusitaniae), or 28 °C (for A. niger) with a reciprocal shaker (THZ-82A, Jinyi, Changzhou, China) for 18 h at 160 rpm.

MIC and MBC Determination

MIC was defined as the lowest concentration that inhibits the growth of microorganisms in a medium after 18–24 h of culture, which is commonly used to evaluate the antimicrobial activity of antimicrobics. CPC and CHA solutions were 2-folds diluted from 64 to 2 μM by sterile PBS, respectively, 100 μL of each solution was transferred into 96-well microtiter plates. Bacterial and fungal suspensions were diluted with sterile PBS to the final concentration of 5 × 105 CFU/mL and then inoculated into each well, respectively, to give a final volume of 200 μL. Unlike bacteria and yeasts, conidia are normally employed as a target for the research on the antimicrobial activity of antimicrobics against filamentous fungi. The conidia suspension of A. niger was prepared by gently scraping the culture surface using a sterile glass rod after adding 3–5 mL 0.1% aqueous solution of Tween 80, the density of which was determined and adjusted by hemacytometer after being filtered through gauze [25]. The plates were covered by sterile lids and incubated at 37 °C (for bacteria), 35 °C (for C. lusitaniae), or 28 °C (for A. niger) for 18–24 h with shaking at 160 rpm. Afterward, 50 μL of the mixtures were transferred into 450 μL D/E neutralizing broth and incubated at 37 ℃ for 10 min to terminate the disinfection. The turbidity of each well was measured by a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific Co., Ltd., MA, USA) at 600 nm before and after the incubation. The MIC was determined as the lowest concentration giving complete inhibition of growth (OD value equal to that of blank) [26]. Subsequently, the microtiter plates set up for the MIC determination were applied to determine MBC. Specifically, 100 μL of the mixtures whose OD value was equal to the blank after incubation were spread on LB agar plates and cultured at 37 °C overnight (for bacteria), or on Sabouraud dextrose agar plates and cultured at 35 °C (for C. lusitaniae), or 28 °C (for A. niger) overnight. The MBC was determined as the lowest concentration showing no growth after the culture [27].

Fractional Inhibition Concentration Determination

Synergistic activity between CPC and CHA was determined by measuring the fractional inhibition concentration (FIC) via a checkerboard synergy assay [28]. Briefly, CPC and CHA solutions with different concentrations from 1/32 to 4 folds of MIC were mixed and transferred into 96-well microtiter plates to give a final volume of 100 μL. Then 100 μL bacterial and fungal suspensions with the concentration of 5 × 105 CFU/mL were inoculated into each well. The plates were covered by sterile lids and incubated at 37 °C (for bacteria), 35 °C (for C. lusitaniae), or 28 °C (for A. niger) for 18–24 h with shaking at 160 rpm. Afterward, 50 μL of the mixtures were transferred into 450 μL D/E neutralizing broth and incubated at 37 °C for 10 min to terminate the disinfection. The turbidity of each well was measured at 600 nm before and after the incubation. FIC of CPC/CHA combination was calculated by the following formula:

$$FIC\,{\text{index}} = FIC_{{{\text{CPC}}}} + FIC_{{{\text{CHA}}}} = \frac{{MIC_{{{\text{CPC}}}}^{{{\text{Combination}}}} }}{{MIC_{{{\text{CPC}}}}^{{{\text{Alone}}}} }} + \frac{{MIC_{{{\text{CHA}}}}^{{{\text{Combination}}}} }}{{MIC_{{{\text{CHA}}}}^{{{\text{Alone}}}} }}$$

Synergism, addictive effect, indifference effect, and antagonism by the checkerboard assay were defined at an FIC index of ≤ 0.5, > 0.5 and ≤ 1, > 1 and ≤ 2, and > 2, respectively.

Growth Curve Determination

Growth curves of a gram-positive bacterium (S. aureus), a gram-negative bacterium (E. coli), and a fungus (C. lusitaniae) contacting with CPC, CHA, and CPC/CHA combination were determined to evaluate their dynamic combined effects on different types of microorganisms. Specifically, suspensions of these three microorganisms were first diluted to the concentration of 5 × 105–6 × 105 CFU/mL by the LB broth medium (for S. aureus and E. coli) and the Sabouraud dextrose broth medium (for C. lusitaniae). Then the suspensions were mixed with CPC, CHA, and CPC/CHA combination to a final volume of 5 mL, respectively, followed by the turbidity measured at 600 nm. The concentrations of CPC and CHA used in this assay were equal to the MIC in their combination. Suspensions of tested microorganisms and blank mediums mixed with sterile PBS were employed as the positive and negative control, respectively. Subsequently, the suspensions were incubated at 37 ℃ (for S. aureus and E. coli) or 35 ℃ (for C. lusitaniae) for 18 h with shaking at 160 rpm. The turbidity of the suspensions was monitored by measuring the OD value every 2 h intervals [19].

Rapid Biocidal Activity Assay

Minimal biocidal concentrations in 5 min (MBC5min) of CPC, CHA, and CPC/CHA combination were also determined to evaluate the rapid biocidal activity. Briefly, 100 μL of bacterial and fungal suspensions with the concentration of 1.0 × 108 CFU/mL were mixed with 100 μL BSA solution (3%, w/v) and 800 μL solution of CPC, CHA, or CPC/CHA combination, respectively. The final concentrations of CPC and CHA were 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 μM, respectively. After incubating at 37 °C for 5 min, 50 μL of the mixtures were transferred into 450 μL D/E neutralizing broth and incubated at 37 °C for 10 min to terminate the disinfection. Then 100 μL of the neutralized mixtures were spread on LB agar plates and incubated at 37 °C overnight (for bacteria), or on Sabouraud dextrose agar plates and incubated at 35 °C (for C. lusitaniae) or 28 °C (for A. niger) overnight. The MBC5min was determined as the lowest concentration showing no growth after the 5 min treatment, which was the concentration of CPC and CHA used for the following assays. Subsequently, 500 μL of the neutralized mixtures were coated on a relevant medium plate and cultured at 37 °C (for bacteria), 35 °C (for C. lusitaniae), or 28 °C (for A. niger) overnight [29]. The rest neutralized mixtures containing S. aureus, E. coli, or C. lusitaniae cells were centrifuged at 4000 rpm for 5 min to collect the cells, which were washed with sterile PBS twice, followed by staining with a Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit according to the manufacturer’s instruction. Specifically, all the cells were stained green by SYTO 9, while the dead cells were stained red by propidium iodide (PI). Finally, the stained cells were observed by an Eclipse Ni-U Upright Microscope (Nikon, Tokyo, Japan).

Scanning Electron Microscopy Observation

One milliliter of S. aureus, E. coli, and C. lusitaniae suspensions were centrifuged at 4000 rpm for 5 min to collect cells, which were washed with sterile PBS. Then the cells were resuspended with 100 μL BSA (3%, w/v) and treated with CPC, CHA, and CPC/CHA combination at room temperature for 30 min, followed by neutralization by D/E neutralizing broth. The concentrations of CPC and CHA were equal to the MBC5min of them in the combination. Subsequently, the suspensions were centrifuged (8000 rpm) at 4 °C for 10 min, the cells were collected and washed twice with sterile PBS, which were fixed at 4 °C with 25 μL glutaraldehyde (2.5%, v/v) overnight [30]. The samples were fixed on a steel sheet and sprayed with a layer of platinum in a vacuum sputter coater, and then observed by a Regulus8100 Field Emission SEM (HITACHI, Tokyo, Japan).

Soluble Protein Content Determination

The suspensions of S. aureus, E. coli, and C. lusitaniae were pre-treated by following the protocol described in the section above to the step of centrifuging at 8000 rpm for 10 min. Then the supernatants were collected, the soluble protein content of which was determined by a BCA Protein Colorimetric Assay Kit according to the manufacturer’s instruction.

In Vitro Acute Eye and Skin Irritation Test

In vitro acute eye and skin irritation test was carried out according to the previous report with some modifications. Briefly, for in vitro acute eye irritation test, 0.1 mL of CPC/CHA combination at the concentration equivalent to the MBC5min against A. niger conidia cells was dropped into the conjunctival sac of the left eyes of three New Zealand white rabbits, making this eye close for 4 s, which was then rinsed with saline 30 s later, followed by observing whether conjunctival congestion and edema as well as iris and corneal damage occurred after 24 h, 48 h, and 72 h, respectively, and obtaining the eye irritation response score at each time point accordingly. The irritation index of CPC/CHA combination to eyes in vitro was assessed based the average score of each test rabbit [31]. An equal volume of saline was dropped into the right eyes of the same test rabbits to serve as the control. For in vitro acute skin irritation test, the back fur on both sides of the spine of the test rabbits was shaved to make the skin with an area of 3 × 3 cm exposed. Then 0.5 mL of CPC/CHA combination was applied to the left exposed skin, which was rinsed with warm water 4 h later. An equal volume of saline was applied to the right exposed skin of the same test rabbits to serve as the control. This test was carried out on consecutive days for 14 days, and the skin damage (e.g., erythema and edema) was daily observed to obtain skin irritation scores [32]. The in vitro acute skin irritation index was assessed based on the average score per day of each test animal.

Statistical Analysis

The assays were performed at least in triplicate on separation occasions. The data collected in this study were statistically analyzed by unpaired two-tailed t-test of variance with GraphPad Prism, version 6 (GraphPad Software Inc., CA, USA). The differences were statistically significant (*), very significant (**), and sharply significant (***) when the p values were less than 0.05, 0.01, and 0.001, respectively. The sign n.s. represented not significant.

Results

MIC and MBC

MIC and MBC values of CPC and CHA are presented in Table 1. Both of these reagents showed an inhibitory effect on the growth of the tested microorganisms, as well as a biocidal effect on them. Figure S1 displays that the turbidity was the same as the blank control at the concentration of MIC, while no colonies appeared on the plates at the concentration of MBC. The MIC and MBC against the tested microorganisms are consistent with the previous literatures [13, 33]. Moreover, the MIC and MBC against B.subtilis, B. cereus, C. lusitaniae, and A.niger of CHA were lower than or equal to the CPC, while the MIC and MBC against S. aureus and tested gram-negative bacteria of CPC were not higher than CHA. CPC and CHA possessed the same MIC and MBC against E. coli.

Table 1 Inhibitory and biocidal activity of CPC and CHA
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The Synergy Between CPC and CHA

The synergistic activity of CPC and CHA is detailed in Table 2 and Fig. S2. The results of the checkboard assay show that CPC and CHA had a synergic effect against S. aureus, B. subtilis, B. cereus, and P. aeruginosa, giving FIC indexes of ≤ 0.5. This combination failed to reach the FIC and needed to be considered synergistic against other tested microorganisms, the index of which were 0.75 or 1, respectively, illustrating CPC and CHA had an addictive effect on these microorganisms.

Table 2 Antimicrobial activity of CPC and CHA in combination
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Dynamic Combined Effects of CPC and CHA

Growth curve assays with CPC, CHA, and CPC/CHA combination against S. aureus, E. coli, and C. lusitaniae are shown in Fig. 1. Exponential phase of the bacterial and fungal cells in control groups started at approximately 2 h and 4 h, respectively, as the OD value significantly increased compared with that at the initial time point (p < 0.001, unpaired student t-test). Individual treatment by CPC or CHA showed an inhibitory effect on the tested microorganism, which delayed the active exponential phase of the planktonic cells by 2–6 h. Compared with the individual treatment by CPC or CHA, CPC/CHA combination almost completely inhibited the growth of the planktonic cells, exhibiting as the turbidity of the culture mediums hardly changed compared with that at the initial time point (p > 0.5) within 16 h. After 16 h of incubation, the OD value of the microbial cells in the mediums containing CPC, CHA or CPC/CHA combination was significantly lower than that of the microbial cells in control group (p < 0.01).

Fig. 1
figure 1

Effects of CPC/CHA alone, or combination on the growth of different microorganisms. Growth curves of (A) S. aureus cells incubated with the culture medium containing 0.5 μM CPC or/and 1 μM CHA, (B) E. coli cells incubated with PBS, 4 μM CPC and/or 2 μM CHA, as well as (C) C. lusitaniae cells incubated with PBS, 8 μM CPC and/or 2 μM CHA in 16 h. All assays were performed in triplicate on separation occasions. Significant differences compared to the control for each group (by unpaired two-tailed t-test) are indicated as follow: **P < 0.01 and ***P < 0.001

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Rapid Biocidal Activity Assay

Table 3 shows the minimal concentration of CPC or CHA alone and in combination that achieved the biocidal effect in 5 min. MBC5min of CPC and CHA was 40 to 80 folds as MBC that reflects biocidal activity in 18–24 h. Moreover, the combined use of CPC and CHA significantly decreased the MBC5min of them, which was 1/8 to 1/4 as that against the tested microorganisms alone, respectively. Fig. S3 shows that no colonies appeared on the plates coated with the suspensions treated with CPC/CHA combination. Furthermore, the rapid biocidal activity of CPC, CHA, and CPC/CHA combination against S. aureus, E. coli, and C. lusitaniae is also displayed in fluorescence microscopy images (Fig. 2), revealing that the treatment of the CPC/CHA combination triggered the cell death of all these three microorganisms, as almost only dead cells (stain red) could be observed after the treatment.

Table 3 Rapid biocidal activity of CPC and CHA
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Fig. 2
figure 2

Fluorescence microscopy images of (A) S. aureus cells treated with PBS, 80 μM CPC or/and 160 μM CHA, (B) E. coli cells treated with PBS, 40 μM CPC and/or 80 μM CHA, and (C) C. lusitaniae cells treated with PBS, 160 μM CPC and/or 160 μM CHA for 5 min at 37 °C. Bacterial or fungal cells were stained for 15 min with SYTO 9 and PI

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SEM Observation

The ultrastructural changes in surface morphology and membrane integrity of S. aureus, E. coli, and C. lusitaniae cells were observed by SEM (Fig. 3). The SEM images of control groups show that the cells had their original globular shape (S. aureus), rod shape (E. coli), an oval shape (C. lusitaniae), as well as an intact and smooth surface. After the treatment of CPC or CHA alone, the original morphology of most of the cells was not changed, only a few of them exhibited visible partial pore formation and blurry cell envelopes, while the combined use of CPC and CHA further destroyed the cells and caused more remarkable structural changes, showing as more pores on the membrane, as well as ruptured and aggregated cells.

Fig. 3
figure 3

SEM images of (A) S. aureus cells treated with PBS, 80 μM CPC or/and 160 μM CHA, (B) E. coli cells treated with PBS, 40 μM CPC and/or 80 μM CHA, and (C) C. lusitaniae cells treated with PBS, 160 μM CPC and/or 160 μM CHA for 30 min at 37 °C

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Soluble Protein Content

Soluble protein content in the supernatant after the treatment of CPC, CHA, and CPC/CHA combination is shown in Fig. 4. Protein leaked from all three kinds of cells into the solution was significantly increased with the prolonging of the processing time. Compared with the cells treated with CPC or CHA alone, markedly more protein was released from those treated with the combination, indicating the combined use of the two drugs enhanced destructive effects on the cells (p < 0.01). Besides, the results also showed that the effect of CHA on protein leakage was stronger than that of CPC in general, though the data presented no statistical significance between some groups.

Fig. 4
figure 4

The leakage of soluble protein from (A) S. aureus cells treated with PBS, 80 μM CPC or/and 160 μM CHA, (B) E. coli cells treated with PBS, 40 μM CPC and/or 80 μM CHA, and (C) C. lusitaniae cells treated with PBS, 160 μM CPC and/or 160 μM CHA for 5 min, 15 min and 30 min at 37 °C. All assays were performed in triplicate on separation occasions. Significant differences between each group (by unpaired two-tailed t-test) are indicated as follow: n.s., not significant, *P < 0.05, **P < 0.01, and ***P < 0.001

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In Vitro Acute Eye and Skin Irritation Test

The results of in vitro acute eye irritation test show that no conjunctival congestion and edema as well as iris and corneal damage were observed in the rabbit eyes direct contact with CPC/CHA combination or saline after 24 h, 48 h, and 72 h (Fig. 5A). In addition to the eyes, the direct contact with CPC/CHA combination would not cause any acute irritation to the skin either. As shown in Fig. 5B, no erythema or edema appeared on the skin after 14 days treatment with CPC/CHA combination, whose appearance was the same as that treated with saline. Accordingly, both the in vitro acute eye and skin irritation indexes were zero, indicating the CPC/CHA combination had no irritation to mammal mucosal and skin.

Fig. 5
figure 5

Results of in vitro acute eye and skin irritation test. A The rabbit eyes before the treatment and after 24, 48, and 72 h of CPC/CHA combination treatment. B The exposed rabbit skin before the treatment and after 7 days and 14 days of CPC/CHA combination treatment

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Discussion

Combined use of the present antimicrobial drugs has been proved to be an ideal approach for novel antimicrobial agents’ development in addition to synthesizing new chemical drugs. CPC is a quaternary ammonium cationic surfactant able to replace the positive ions like Mg2+ and Ca2+ required to stabilize the cell membrane, which can reduce the fluidity of the plasma membrane. Additionally, the hexadecane chain further disorganizes the lipid membrane and triggers the complete rupture of the cell membrane [34]. The biguanide cationic antimicrobial drug CHA can be adsorbed on phosphate-containing protein in the cell wall, penetrates, and disrupts the cytoplasmic membrane, which leads to the leakage of cytoplasmic. Moreover, CHA can also damage the cells by forming irreversible precipitates with intracellular ATP and nucleic acids [35]. This study first evaluated the antimicrobial activity of the CPC/CHA combination against gram-positive/gram-negative bacteria and fungi. As expected, the combined use of CPC and CHA enhance their antimicrobial activity, exhibiting as the MIC of them in combination was 1/8–1/2 as those used alone. The variability in antimicrobial activities against the tested microorganisms can be attributed to the simple composition of the cell wall of gram-positive bacteria, leading to both CPC and CHA having an increased affinity to the cell wall of gram-positive organisms [33, 36]. In contrast, both CPC and CHA showed low affinity to the cell wall of the conidia, exhibiting several folds higher MIC and MBC against A. niger than other tested microorganisms. FIC assay demonstrated CPC and CHA were synergistic with all the three tested gram-positive strains and P. aeruginosa, suggesting CPC/CHA combination may possess an excellent biocidal effect on gram-positive bacteria. We speculate the membrane destabilization and permeability produced by CPC likely allow better access for CHA, which can coagulate with ATP and nucleic acid, and promote cell death. Despite CPC/CHA combination failing to reach the cut-off for being considered synergistic against the E. coli, S. typhimurium, C. lusitaniae, and A. niger, they would not interfere with each other and showed an additive effect. Growth curves of S. aureus, E. coli, and C. lusitaniae contacting with CPC, CHA, and CPC/CHA combination illustrate the dynamic combined effects against these tested microorganisms. Although the MIC of CHA was twice and the same as CPC when they were used in combination against S. aureus and E. coli, respectively, the exponential phase of both S. aureus and E. coli cells contacting with CPC started 4 h later than those contacting with CHA. Additionally, besides the delayed exponential phase, the destiny of the cells contacting with the drugs was also significantly lower than those in control groups when they reach the plateau phase, exhibiting lower turbidity, demonstrating CPC possessed a greater bactericidal activity against S. aureus and E. coli [37]. In contrast, CHA exhibited stronger inhibitory effects against C. lusitaniae, since C. lusitaniae cells contacting with CHA exhibited a later exponential phase, as well as lower turbidity in the plateau phase than those contacting with CPC, though MIC of CPC was 4 folds as that of CHA in combination. Compared with either of these two reagents alone, CPC/CHA combination performed superior inhibitory activity against all the tested microorganisms, which almost completely inhibited the growth of these cells, demonstrating this combination has dynamic combined effects against gram-positive/gram-negative bacteria and fungi. All these results are consistence with that of the MIC assay.

The evaluation of antimicrobial activity based on MIC or MBC may not be sufficient because disinfectants normally will not remain on the surface and in contact with the microorganisms attached or colonized on it for a long time, thus they are expected to kill microorganisms rapidly, particularly for those applied for skin and oral disinfection [38]. We performed a killing assay using a 5 min exposure in planktonic cells to evaluate the antimicrobial activity of the CPC/CHA combination and concluded a concentration that allowed this combination to achieve rapid killing of microorganisms in 5 min. Moreover, considering the proteins in the skin and mucosa surface may affect antimicrobial activity, BSA was employed to simulate the disinfection environment [39]. The results show that CPC and CHA have stronger synergy in the rapid killing of gram-negative bacteria and fungi, as the MBC5min against E. coli, S. typhimurium, P. aeruginosa, C. lusitaniae, and A. niger in combination was only 1/8–1/4 as they were used alone, while this ratio in MIC was 1/4–1/2, demonstrating CPC/CHA combination is desirable for rapid disinfection. Photos of the medium plate and fluorescence microscopy images also display the excellent antimicrobial activity of the CPC/CHA combination. Notably, consistent with the result of the FIC assay, the microscopy images also demonstrated this combination is most suitable for killing gram-positive bacteria, as almost all the S. aureus were dead after 5 min treatment. Besides, these images also show CPC have stronger antibacterial activity than CHA, since the ratio of dead cells of S. aureus and E. coli treated with CPC was higher than those treated with CHA, despite the concentration of CHA was double as CPC, while in contrast, CHA performed better in antifungal activity. In addition to the distribution of the dead cells, their ultrastructural was also observed by SEM. SEM images display pores appearing on the cell surface, as well as some cells, were shrunken after the treatment with CPC or CHA, demonstrating the penetration of the cell wall and the leakage of the internal cellular constituents, which is consistence with the reports that CPC and CHA kill the microorganisms mainly by increasing the permeability of the cell membrane [40]. The treatment of the CPC/CHA combination causes more significant damage to cells, showing as ruptured and aggregated cells [41], especially for S. aureus. The amount of leaked protein also demonstrates CPC/CHA combination causing significantly more serious protein leakage than single used after only 5-min of contact, suggesting that CPC/CHA combination accelerates the process of cell membrane disintegration [23].

Besides bacteria and fungi, the viruses attached on the surface are also common causes of diseases, since the virions have been proven to maintain infective activity for hours to days in vitro. Antiviral activity of the CPC/CHA combination was also preliminarily evaluated since these two drugs were reported to be applied for virus inactivation [42, 43]. In addition to strong antimicrobial activity against bacteria and fungi, CPC/CHA combination also exhibited robust virus inactivation activity that could decrease the viral titer of Human Coronavirus 229E (HCoV-229E, ATCC VR-740) from 106.66 to 102.05 TCID50/0.1 mL within 5 min at the concentration equivalent to the MBC5min against A. niger conidia cells, which means 99.998% of the tested virions were rapidly inactivated (Fig. S4). The excellent antiviral activity against the tested coronavirus suggests this CPC/CHA combination possesses the potential for the prevention and control of COVID-19 at the MBC5min against A. niger conidia cells. More importantly, the acute eye and skin irritation test illustrate this broad-spectrum antimicrobial agent would not cause any mucosal or skin damage at this concentration, validating the safety of CPC/CHA combination for routine use in hands and oral disinfection besides household items and medical articles [44].

Conclusion

Herein, CPC/CHA combination were employed for the preparation of disinfectant, whose antimicrobial activity was evaluated. These two drugs exhibited a synergic effect against the tested gram-positive bacteria and an addictive effect against the tested fungi and most of the tested gram-negative bacteria, as the combined use of them significantly decreased the MIC and MBC, which is essential to cut down the unpleasant taste and reduce the risk of antimicrobial resistance. Moreover, CPC/CHA combination showed dynamic combined effects against the tested microorganisms at the concentration equal to MIC. Besides, this combination could also rapidly kill the tested microorganism within 5 min when the concentration of CPC and CHA raised to 320 μM and 640 μM, respectively, even in the presence of organic interfering substances. More importantly, this combination showed no irritation to mucosa or skin at the concentration that could rapidly kill the tested microorganism. In all, the combination use of CPC and CHA performed superior antimicrobial effects against these tested microorganisms, which provides a novel option of preparing effective disinfectant for preventing infections at home and in hospital.