Lysozyme increases bactericidal activity of ceragenin CSA-13 against Bacillus subtilis

Introduction

Members of the genus Bacillus are Gram-positive, spore-forming, rod-shaped bacteria, widely distributed in natural environments, especially in soil, food products, air, and water. In addition, some species are members of human and animal intestinal microbiota [1]. Despite routine cleaning procedures Bacillus strains are also found in hospital settings in various reservoirs such as ventilation equipment, linen, medical devices, gloves, hands of medical staff, or alcohol-based hand disinfectant solutions, hence they are often regarded as environmental contaminants [2]. Although only Bacillus anthracis is considered a strict pathogen in this group, several other species, in particular B. cereus, B. subtilis, B. licheniformis, and B. thuringiensis, may cause various opportunistic infections. With the development of modern medicine, many immunocompromised individuals are able to live longer, and the clinical significance of these opportunistic bacilli is constantly growing [3, 4]. In general, eye infections, such as post-traumatic or post-surgery endophthalmitis, are among the most frequently described conditions. However, other conditions such as traumatic and surgical wound infections, bacteraemia, central venous catheter-linked infections, endocarditis, meningitis, brain abscesses, osteomyelitis, pulmonary infections, or neonatal infections are also commonly reported. The presence of intravascular devices, dialysis, alcoholism, trauma, intravenous drug use, haematological malignancies, and premature birth are important predisposing factors for such infections. Finally, B. cereus is a well-recognised enteropathogen, causing toxin-mediated food-borne acute gastroenteritis [5–12].
Lysozyme is a natural protein and a key player in innate immunity. It is found in tissues, body fluids, and cells exposed to the environment or involved in bacterial clearance (e.g. mucosal surfaces of respiratory, intestinal, urogenital tracts, tears, saliva, serum, breast milk, and urine as well as phagocytes, including macrophages, neutrophils, and dendritic cells). Generally, its two main tasks are (i) direct antibacterial activity and (ii) modulation of immune response in the site of inflammation [13, 14].
Ceragenins (CSAs) are cationic lipids synthesised using the structural base of cholic acid, and CSA-13 is the best-characterised molecule of its family [15, 16]. They mimic the amphiphilic character of natural antibacterial peptides such as cathelicidin LL-37, and are described by broad-spectrum antibacterial activity against aerobic and anaerobic Gram-positive and Gram-negative bacteria, as well as multidrug-resistant strains of fungi, parasites, and some viruses [17–20]. Furthermore, ceragenins are effective against microorganisms in both planktonic and biofilm form [21].

Aim of the research

In the present paper, our intention was to study the antibacterial activity of a combination of lysozyme and CSA-13 against a model bacterium B. subtilis.

Material and methods

Antimicrobial testing

Bactericidal activities of lysozyme from chicken egg white and ceragenin CSA-13 (synthesised as previously reported [22]), against the reference strain of B. subtilis ATCC 6051, were evaluated using a colony forming-unit (CFU) assay, as described previously [23]. Briefly, B. subtilis bacteria were grown overnight on Luria-Bertani (LB) agar plates (Difco, Sparks, MD) at 37°C to reach a mid-log phase of growth, resuspended in PBS, and brought to 108 CFU/ml (which corresponds to optical density of 0.5 at 600 nm). Next, the bacterial suspension was adjusted to 105 CFU/ml and treated with different concentrations of tested agents (0–50 U/ml of lysozyme, and/or 0–5 µM of CSA-13). After 1 h of incubation at 37°C the plates were transferred to ice and suspensions were diluted 10- to 1000-fold in PBS. Then, 10 μl aliquots were spotted on agar plates for overnight culture at 37°C, after which CFUs were determined. The CFUs (CFU/ml) of the individual samples were determined from the dilution factor and were used to calculate the percentage of bacterial outgrowth.

Evaluation of the antibacterial activity of CSA-13/lysozyme combinations

To evaluate possible synergy between ceragenin CSA-13 and lysozyme, microbial outgrowth reduction and synergistic effect values were calculated as presented previously [24]. A mutual antimicrobial effect of the analysed compounds was calculated as follows: synergistic effect value = A – (B + C), where A is the reduction of colonies from combined CSA-13/lysozyme treatment, B is the reduction from CSA-13 treatment alone, and C is the reduction from lysozyme treatment alone. A synergistic effect value > 0 indicates beneficial and synergistic interaction between tested agents, and values < 0 suggest decreased benefit of combining treatment. A synergistic effect value of 0 indicates that there is no synergistic effect of combining the individual treatments.

Atomic force microscopy (AFM) analysis

To visualise the effects of CSA-13 and lysozyme combinations on bacteria, topography images of treated bacterial cells were recorded using atomic force microscopy, as a qualitative assessment of antibiotic-mediated cell destruction. In all AFM-based experiments B. subtilis cells were resuspended in distilled water (OD600 ~ 0.1), and incubated with 0.5–1 µM of CSA-13, 5 U/ml of lysozyme or CSA-13 and lysozyme in combinations (the concentrations were estimated based on the maximum numerical synergistic value, see results), at 37°C for 60 min. Then, 200 µl of bacterial samples were transferred to a mica surface precoated with 5% (3-aminopropyl)triethoxysilane (APTES) in water. Bacteria attachment to functionalised mica was achieved during 20-min incubation. Images were collected using a Nano Wizard 4 BioScience AFM (JPK Instruments, Germany) working in Contact Mode and Quantitative Imaging (QI) Mode. App Nano NITRA-TALL-V-G triangular pyramid shaped tips with a nominal spring constant equal to 0.24 N/m were employed. Initially, the tip was brought into contact with the surface of a bacterial cell until a given deflection of the cantilever was reached. The scanning was then started with a constant velocity of 3 µm/s. Three signals were recorded simultaneously while scanning the sample surface: topography, vertical deflection of the cantilever, and error signal, with a resolution of 128 pixels per line. Topography images serve as a qualitative assessment, while vertical deflection and error signal uncover surface features with better clarity. Due to the high adhesion between the AFM tip and damaged cells after incubation and lateral forces during contact mode scanning, a force curve-based imaging mode was used (QI mode) with the resolution of 128 pixels per line, using the same cantilever.

Results

Lysozyme and ceragenin CSA-13 exert antibacterial activity against B. subtilis ATCC 6051

As shown in Figure 1 A and B, a decline in the survival of B. subtilis cells after incubation with various concentrations of lysozyme and CSA-13 was observed, indicating that both agents display activity against B. subtilis. Complete inhibition of bacterial growth occurred when 25 U/ml of lysozyme and 5 µM of CSA-13 were applied.

Lysozyme exerts synergistic effects with ceragenin CSA-13 against B. subtilis

The decrease of bacterial survival and colony forming capability upon combined treatment with ceragenin CSA-13 and lysozyme is presented in Figure 2 and Table 1. As shown in Figure 2, the combination of natural and synthetic-based agents significantly reduced the number of bacterial colonies upon 1-h incubation. The reduction of B. subtilis colonies was 0.17 ±0.22 to 4.48 ±0.06 log CFU/ml, depending on the concentrations of tested agents. It was shown that an increase in CSA-13 concentration resulted in a decrease in bacterial viability, and this effect was further strengthened by relatively low doses of lysozyme, which suggest synergistic interactions between these compounds. The maximum numerical synergistic value was 1.38 log CFU/ml after combined treatment with CSA-13 and lysozyme in doses of 1 µM and 5 U/ml, respectively. Accordingly, a significant decrease in B. subtilis viability by 4.48 ±0.06 log CFU/ml was observed (Figure 2 D, Table 1). In contrast, treatment of B. subtilis with CSA-13 and lysozyme alone led to a decrease of bacterial outgrowth by 1.82 ±0.02 and 1.28 ±0.246 log CFU/ml, respectively. Such lysozyme-mediated improved bactericidal activity of ceragenin was also observed when lysozyme was co-applied with lower doses of CSA-13, i.e. 0.25 µM and 0.5 µM (Figures 2 B and C), and the numerical difference was 0.63 to 0.88 log CFU/ml. These results strongly suggest that combined treatments with CSA-13 and lysozyme result in a greater microbial reduction than the treatments applied individually, and this effect is particularly prominent with higher doses of both agents.

Morphology of B. subtilis cells subjected to lysozyme/CSA-13 treatment

As indicated in Figure 3, B. subtilis cells exposed to lysozyme and CSA-13 revealed changes in surface morphology that indicate bacterial membrane damage. The extent of membrane blebbing and the presence of cell fragments and aggregations increased when combined treatment was applied. Overall, these changes were very similar to the membrane damage observed previously for Pseudomonas aeruginosa PA01 and LESB58 strains [25].

Discussion

The growing level of resistance to antimicrobials among pathogens involved in human infections requires the development of novel therapeutic options, as well as searching for new antibiotics. Combining antimicrobials is a promising approach, not only for improving antimicrobial therapy but also for controlling resistance developing through natural evolution. When drugs are combined, the synergistic, antagonistic, or additive effects on bacterial cells may be observed. In general, the mode of action of the drugs determines the interaction that occurs between them [26].
One possible approach for use of antimicrobial combinations for medical benefit is the analysis of the role of natural peptides present on the skin, mucosal surfaces, and in body fluids [27], in combination with exogenous antimicrobials [28]. In our study, we examined a combination of ceragenin CSA-13 and lysozyme against B. subtilis ATCC 6051, a model organism for other species of bacilli. Apart from the ability to produce virulence factors, there are no significant differences in susceptibility to antimicrobials among various species of Bacillus, so our results can be related to other members of genus Bacillus. Notably, many Gram-positive bacilli, other than B. anthracis (the causative agent of the deadly disease anthrax), are well-recognised opportunistic pathogens responsible for systemic diseases in immunocompromised patients as well as local, post-surgical, and post-traumatic infections [29]. For instance, B. cereus, due to secretion of toxins detrimental to ocular tissue, rapid intraocular migration, and the ability to stimulate a strong inflammatory response, is described as an aetiological agent of dangerous, sometimes fulminant infective endophthalmitis, which demands early and aggressive therapy [30]. In addition, similarly to other pathogens, multidrug or even extensive drug-resistant strains of bacilli, especially among B. cereus isolates, have been reported [31, 32]. As reported previously, ceragenins have well-documented antibacterial properties. They act against a broad spectrum of Gram-positive and Gram-negative bacteria, including multidrug-resistant organisms [33]. The activity of CSA-13 against B. subtilis, both vegetative cells and spores, has also been described [23]. Our data confirm this observation with Bacillus vegetative forms and demonstrate that the bacterial killing activity of CSA-13 is enhanced by lysozyme, as supported by AFM imagining.
In general, synergy can be observed when two antimicrobials associate with various targets in bacterial cells. The primary mode of ceragenin action against bacteria is concentration-dependent cell membrane depolarisation causing cellular dysfunction. Electrostatic interactions between cationic ceragenin and negatively-charged bacterial membrane molecules such as phosphatidylglycerol and LPS effectively kills bacteria, and this mechanism of action is unlikely to induce resistance [34]. Lysozyme can act through two different mechanisms. The primary mechanism leads to cell wall instability and subsequently to cell death as the result of hydrolysis of the -(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan (PG). The secondary mode, less important in Gram-positive bacteria, is independent of PG hydrolysis. Lysozyme, being highly cationic, can insert into negatively charged bacterial membranes and form pores [14]. So, the explanation for the synergistic interaction between CSA-13 and lysozyme against Gram-positive bacteria lies in the different modes of action of CSA-13 that targets the cell membrane and lysozyme that targets the cell wall peptidoglycan. In this combination one antimicrobial increases the permeability of the cell’s external barrier to another. Earlier in vitro studies also demonstrated that the activity of CSA-13 against Pseudomonas aeruginosa and Helicobacter pylori was augmented by host-secreted antimicrobial agents (including lysozyme), although it is known that lysozyme activity against Gram-negative bacteria is weak. In this situation, lysozyme acts by the membrane-dependent mechanism, so an additive but not synergistic effect was observed [35]. This study confirms that enhanced antibacterial activity of CSA-13 against bacteria might be expected at sites where lysozyme is present. Despite the reports on different biological features of hen egg white lysozyme (used in this study) and human lysozyme, we suggest that the same phenomenon might occur in human hosts [36]. It is established that in the site of infection exogenous antibiotics act in the presence of various natural host antibacterial products, and their combined action can influence the final antimicrobial effect. Such complex interactions of antimicrobial peptides are found in airway surface liquid, which include lysozyme, lactoferrin, secretory leukocyte protease inhibitor (SLPI), human b-defensins, and LL-37. Singh et al. [37] observed synergistic or additive effects of tobramycin with these natural defences against Escherichia coli. Notably, a triple combination of lysozyme, lactoferrin, and SLPI displayed even greater synergy with tobramycin than did pairs of antibacterial factors. Moreover, the study revealed that antimicrobial potency in body sites, where multiple antimicrobial compounds are present, can be increased by their interactions. However, some inhibitory factors, such as increased salt concentrations, may diminish these synergistic effects and inhibit local mucosal defences [37]. The enhanced activity of exogenous antibiotics by endogenous defences, other than lysozyme, present on skin and mucosal surfaces, has also been demonstrated in related studies. In one of these, the synergy between cathelicidin LL-37 and conventional, bactericidal antibiotics (amoxicillin with clavulanic acid and amikacin) was described [38]. On the other hand, the combination of LL-37 and bacteriostatic tetracycline or erythromycin resulted in antagonism [38]. It is worth underlining that the mucosal concentration of LL-37 increases in the course of infection [27]. Interestingly, in patients with atopic dermatitis decreased expression of antimicrobial peptides is observed [28]. As far as the effectiveness of antimicrobial treatment is concerned, the interactions between various exogenous as well as endogenous antibacterial compounds at the site of infection seem to play a key role in overall antimicrobial activity, especially in local infections. Therefore, the combination of lysozyme and ceragenins examined in our study might be a promising form of supplementary therapy in ocular infections caused by Bacillus. The combination could also be considered in the development of products for wound healing for topical applications. Ceragenins, with their: (i) strong antimicrobial activity against a variety of microorganisms, even Bacillus spores, (ii) ability to inhibit biofilm formation, (iii) biological activity in body fluids, (iv) good safety profile, and (v) anti-inflammatory properties, could be excellent candidates for potential use also in such infections as endophthalmitis [39]. Endophthalmitis following ocular trauma can be caused by a wide range of bacteria and fungi, including multidrug-resistant organisms. Because endophthalmitis is a serious and devastating condition, an early broad-spectrum antibiotic therapy should be introduced before results from susceptibility testing are available [40]. Antibiotics can be administered by topical, systemic, or intravitreal route [41]. CSA-13, thanks to its wide spectrum of activity and synergy with lysozyme that is naturally present in tears but also can be administrated as an exogenous drug, seems to be a reasonable option for ocular infections. Another important value is the synergistic effects of CSA-13 in combination with conventional antibiotics against selected clinical resistant strains, as demonstrated in previous studies [42, 43]. In our study we used lysozyme isolated from chicken egg white. Even though chicken and human lysozyme display similarities concerning the primary sequence and molecular weight, they differ in terms of enzymatic activity. Because human lysozyme is approximately threefold more active than chicken lysozyme we might assume that its lower concentration will be sufficient to observe synergy when combined with CSA-13 [36, 44].

Conclusions

Our data demonstrate and highlight the synergy of lysozyme and CSA-13 and confirm the concept that activity of exogenous antimicrobials is enhanced by host factors. A better description of synergy between natural substances found in inflammatory sites and locally administrated antimicrobials may be a potential direction of research for novel strategies for the treatment of topical infections.

Acknowledgments

This work was supported by a program of the Minister of Science and Higher Education under the name “Regional Initiative of Excellence” in 2019-2022, project number: 024/RID/2018/19, financing amount: 11,999,000.00 PLN. Part of the study was conducted with the use of equipment purchased by the Medical University of Białystok as part of the RPOWP 2007-2013 funding, Priority I, Axis 1.1, contract No. UDA- RPPD.01.01.00-20-001/15-00 dated 26.06.2015.

Conflict of interest

The authors declare no conflict of interest.

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