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. 2021 Apr 19;65(5):e02066-20. doi: 10.1128/AAC.02066-20

Inhibitory Effect of Host Ocular Microenvironmental Factors on Chlorhexidine Digluconate Activity

Chun-Hsien Chen a, Yu-Jen Wang a, Jian-Ming Huang b, Fu-Chin Huang c, Wei-Chen Lin a,b,d,✉
PMCID: PMC8092908  PMID: 33685899

Acanthamoeba spp. are free-living protozoans that cause a serious human eye disease called Acanthamoeba keratitis (AK). Several new and effective medical therapies for AK patients remain highly debated, and therefore, chlorhexidine digluconate (CHG) is still considered one of the first lines of treatment for AK patients.

KEYWORDS: ocular microenvironmental factors, Acanthamoeba keratitis, drug-resistance, chlorhexidine digluconate

ABSTRACT

Acanthamoeba spp. are free-living protozoans that cause a serious human eye disease called Acanthamoeba keratitis (AK). Several new and effective medical therapies for AK patients remain highly debated, and therefore, chlorhexidine digluconate (CHG) is still considered one of the first lines of treatment for AK patients. We hypothesized that ocular microenvironmental factors are responsible for Acanthamoeba drug resistance and clinical AK treatment failure. To investigate the influence of the ocular surface on CHG treatment, we tested the effect of several ocular elements on the antiamoeba activity of CHG. The suspected inhibitory elements, including mucin, albumin, human and amoeba cell lysates, live and heat-killed bacteria, and cornea, were added to the amoebicidal activity platform, where amoebae were incubated with CHG at various concentrations. Mucin showed a significant inhibitory effect on CHG activity against Acanthamoeba castellanii. In contrast, albumin did not affect CHG treatment. Furthermore, human and amoeba cell lysates and live and heat-killed bacterial suspensions also significantly inhibited CHG activity. Additionally, we found that pig corneas also reduced CHG activity. In contrast, dry-eye drops and their major component, propylene glycol, which is commonly used as eyewash material, did not have an impact on CHG activity. Our results demonstrate the effect of ocular microenvironmental factors on CHG activity and suggest that these factors may play a role in the development of amoeba resistance to CHG and treatment failure.

INTRODUCTION

Acanthamoeba spp. are opportunistic protozoan parasites that cause human disease, including blinding keratitis, which is a serious and sight-threatening corneal infection (1, 2). Like most free-living protozoans, Acanthamoeba has a life cycle with two distinct stages, the trophozoite stage (proliferative stage) and the cyst stage (dormant stage) (3). Due to their unique double cell wall, the cysts are highly resistant to chemicals, which leads to clinical treatment failure (4, 5).

However, after several considerable improvements, the current recommended regime uses chlorhexidine digluconate (CHG), which is one of the best antiamoeba agents (6, 7). CHG is an antiseptic that is used for skin disinfection during surgery and is widely used in dental practice for decreasing plaque formation and disinfecting the root canal (8). As a cationic antiseptic, binding to negatively charged cell membranes may be the major mechanism whereby CHG exerts its antiamoeba effect (9). Although CHG was found to destroy cysts and trophozoites in vivo, several clinical cases developed recurrent episodes despite prolonged treatment with CHG (10). A previous study showed that dentin interferes with the antibacterial activity of CHG. Previous studies demonstrated that mucins in the saliva decrease the bactericidal activity of CHG (11). Furthermore, the bactericidal activity of biguanide antiseptic agents is inhibited by human wound exudates (12). The ocular surface environment contains various elements, including layers of the tear film, normal flora, and ocular epidemic cells that maintain ocular health and protect it from environmental exposure (13–15). Consequently, due to the gap between the reported CHG effective amoebicidal activity in vitro and its weak effect in vivo, we hypothesized that complex ocular surfaces, such as components of the tear film, damaged cells, and opportunistic bacteria, are responsible for AK treatment failure by decreasing CHG activity. AK therapy may be difficult due to the lack of knowledge about the CHG mode of inhibition in the complex ocular environment. In this study, we propose to prove that some of the elements from the ocular surface may be involved in AK treatment failure by investigating the effect of these elements on CHG amoebicidal activity.

RESULTS

Evaluation of optimal CHG experimental concentration.

Different strains of Acanthamoeba have distinct drug tolerance. Our previous test demonstrated that the ATCC 30010 strain has a higher susceptibility to polyhexamethylene biguanide (PHMB) and CHG than clinical strains (see Fig. S1 in the supplemental material). To investigate ocular surface interference, we first determined the optimal CHG working concentration by evaluating CHG amoebicidal activity on Acanthamoeba. As shown in Fig. 1, CHG decreased the number of amoebae in a concentration-dependent manner. Among the tested concentrations, the highest CHG concentration at which amoebae could survive was 0.0025%. Thus, 0.0025% CHG was chosen as a good concentration to evaluate ocular interference.

FIG 1.

FIG 1

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The activity of CHG against A. castellanii strain NCKU_SD. Amoeba cells were treated with 0.0003125, 0.000625, 0.00125, 0.0025, and 0.005% CHG for 30 min. The control group was treated with PAS. *, P < 0.05 compared with the control group.

Effect of mucin and albumin on CHG treatment.

The mucins of the ocular surface form a dense glycocalyx at the interface between the epithelium and tear film to protect epithelial cells (14). Therefore, we analyzed the effect of mucin on CHG activity. Our results showed that mucin significantly rescued the survival rate of Acanthamoeba in a dose-dependent manner (Fig. 2A). Previous studies suggested that albumin, which is also present in the mucosa, could suppress CHG bactericidal activity (16). Thus, we further evaluated the influence of albumin. Interestingly, we found no significant impact of albumin on the amoebicidal activity of CHG when used at the same concentrations as mucin (Fig. 2B). We observed that mucin inhibited CHG-dependent amoeba cell death (Fig. 2C). These results suggested that mucin, which is commonly present in the tear film, may reduce the amoebicidal activity of CHG during treatment.

FIG 2.

FIG 2

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Inhibitory effect of mucin and albumin on CHG activity. (A and B) Different concentrations of (A) mucin and (B) albumin were coincubated with amoeba cells and 0.0025% CHG for 30 min. The control group was treated with PAS. The CHG group was treated only with 0.0025% CHG. (C) Examination of the amoeba after treatment with albumin (0.5 mg/ml) or mucin (0.5 mg/ml) using a light microscope. Damaged amoeba cells are marked by arrowheads. *, P < 0.05 compared with the CHG-only group.

Effect of human and amoeba cell lysates and bacterial suspensions on CHG treatment.

CHG is applied to the eyes hourly after confirming the clinical diagnosis of AK. However, CHG therapy does not only kill Acanthamoeba but also causes ocular epithelial cell damage. Therefore, we used C6 and A549 cell lysates to investigate the effect of damaged human cells on CHG treatment. The results demonstrated that C6 and A549 cell lysates have a significant impact on CHG activity (Fig. 3A). Next, the effect of amoeba cell lysates on CHG activity was examined. As shown in Fig. 3B, raw amoeba cell lysates significantly reduced the amoebicidal activity of CHG in a dose-dependent manner (Fig. 3B). A control group in which raw amoeba cell lysates were coincubated with amoebae in the absence of CHG demonstrated that the lysates were not cytotoxic themselves. Next, we centrifuged the amoeba cell lysates to remove the cell debris, which was considered to contain a variety of negative charges. As shown in Fig. 3C, amoeba supernatant could also significantly inhibit the amoebicidal activity of CHG (Fig. 3C). The amoeba supernatant from the cell lysates included amoeba genomic DNA, which might take part in the inhibition effect. Thus, we further evaluated the effect of amoeba genomic DNA, which was shown to significantly affect CHG amoebicidal activity (Fig. 3D). Overall, these results demonstrated that damaged human cells and amoeba cells could inhibit the amoebicidal activity of CHG. Moreover, amoeba protein and genomic DNA were also involved in the drug-inhibition effect.

FIG 3.

FIG 3

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Inhibitory effect of C6, A549, and amoeba cell lysates and amoeba protein and genomic DNA on CHG activity. (A) 0.1 and 0.5 mg/ml raw C6 and A549 cell lysates coincubated with amoeba cells and 0.0025% CHG. (B) Raw amoeba lysates and (C) amoeba protein coincubated with amoeba cells and 0.0025% CHG. The 0.5 mg/ml-only group consisted of a coculture of raw amoeba lysates and amoeba. (D) Amoeba genomic DNA coincubated with amoeba cells and 0.0025% CHG. All incubations lasted 30 min. *, P < 0.05 compared with the CHG group.

Additionally, the conjunctiva and eyelids harbor a variety of microorganisms from the environment that constitute the normal flora. Staphylococcus epidermidis is one of the bacteria commonly present on the ocular surface. We isolated S. epidermidis from a sample of an AK patient and evaluated the impact of bacteria on the amoebicidal activity of CHG. The results showed that both live and heat-killed S. epidermidis suspensions significantly reduced the amoebicidal activity of CHG (Fig. 4A). We speculated that the binding between positively charged CHG and negatively charged ocular factors could be responsible for these inhibitory effects. Therefore, we further investigated whether preincubation of CHG with these inhibitory factors could reduce the effect of CHG. We preincubated CHG with S. epidermidis and removed the remaining bacteria. Then, the prepared medium was applied to Acanthamoeba to measure the amoebicidal activity of CHG. The results showed that preincubation of CHG with S. epidermis reduced the amoebicidal activity of CHG (Fig. 4B). Together, these results demonstrated that elements from the ocular surface, such as tear film, damaged cells, and regular bacterial flora, can significantly inhibit CHG activity.

FIG 4.

FIG 4

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Inhibitory effect of live and heat-killed bacterial suspensions and pig cornea on CHG activity. (A) Live and heat-killed bacterial suspensions at OD600 of 0.3 and 0.8 were added to the CHG test platform. (B) Preincubation (PI) of CHG with bacterial suspensions at an OD600 of 0.3 reduced the effective concentration of CHG. (C) After the protein quantification using the protein assay, raw lysates (0.1 mg/ml) from pigs, rats, and mice were added to the CHG test platform. (D) Incubation with the combination of mucin with the other tested factors (Acanthamoeba, bacteria, cells, and pig cornea) reduced the antiamoeba activity of CHG more prominently than the incubation with individual factors (M, 0.025 mg/ml mucin; B, OD600 0.3 bacteria; A, 0.1 mg/ml amoeba lysates; C, 0.1 mg/ml cell lysates; P, 0.1 mg/ml pig cornea). All incubations lasted 30 min. *, P < 0.05 compared with the CHG group.

Influence of animal cornea on CHG amoebicidal activity.

Based on the above-described data, we found that ocular surface elements may affect CHG treatment. However, Acanthamoeba cells not only adhere to the ocular epithelium but also invade the stroma by activating the degradation components of the extracellular matrix. Thus, the influence of stroma on CHG should be considered. Therefore, we investigated the effect of the animal cornea. Our results showed that the homogeneous pig, mouse, and rat cornea solution significantly inhibited CHG activity (Fig. 4C). Collectively, our findings demonstrated that not only the ocular surface elements but also the cornea may disturb the antiamoeba activity of CHG. Furthermore, Fig. 4D showed that the combination of mucin and the other inhibitory components enhanced the effect on the inhibition of CHG activity.

Effect of eye drops on the CHG antiamoeba activity.

Corneal debridement and topical drug therapy enhance the production of ocular elements such as damaged cells and opportunistic bacteria. Eye drops could rinse the ocular surface and wash away irritants, including cell debris and bacteria. We found that eye drops for dry eye syndrome did not interfere with the amoebicidal activity of CHG (Fig. 5A). Furthermore, propylene glycol, which is the dominant compound in the eye drops, showed similar results to the full eye drop product (Fig. 5B). Overall, our findings suggested that dry-eye drop utilization does not affect CHG treatment.

FIG 5.

FIG 5

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Inhibitory effect of dry-eye drops and propylene glycol on CHG activity. (A) Different concentrations of dry-eye drops added into the CHG test platform. The 0.5 mg/ml-only group consisted of a coculture of dry-eye drops and amoeba. (B) Amoeba incubated with different concentrations of propylene glycol in combination with 0.00125 or 0.0025% CHG.

DISCUSSION

In the past decades, many researchers have studied the development of new therapeutic drugs against Acanthamoeba due to the development of drug resistance during first-line treatment for AK. However, CHG remains the first choice for AK treatment (17). Apart from amoeba resistance to CHG, the reason why CHG may not successfully inhibit amoeba infection is still unknown. Thus, in this study, we investigated the effect of different elements from the ocular surface on CHG amoebicidal activity and proved that some of these elements may be involved in AK treatment failure. The tear film is the front-line barrier, whereas CHG is applied to the eye surface. Several glands and tissues secrete the multiple complex layers of the tear film (18). The inner layer is the mucous layer, which consists of various secreted mucins produced by the lacrimal gland, apical cells, and goblet cells (13–15). Our results showed that mucin significantly inhibits the amoebicidal activity of CHG (Fig. 2A). This was consistent with the findings from previous studies (19–21). We found that the same concentration of albumin did not impact CHG activity. These results contradict previous research suggesting that albumin could reduce the bactericidal activity of CHG (16). We speculate that the discrepancy may be due to the different treatment processes and concentrations of albumin used. A previous study preincubated a high concentration of albumin with CHG for an hour, and then the bactericidal activity of CHG was evaluated. Here, we added a low concentration of albumin and coincubated it for 30 min, resembling more closely the conditions during clinical AK treatment. Thus, the shorter interaction time and the lower concentration of albumin in this study may have led to different results from those of previous studies. Overall, in this study, we demonstrated the significant inhibitory action of mucin on CHG.

Cornea curettage and aggressive utilization of CHG in the AK therapy process leads to corneal epithelial damage (22, 23). Our results indicated that raw cell lysates from C6 and A549 cell lines significantly reduced CHG activity (Fig. 3A). Human wound exudates consist of many organic components, such as platelets, and cell lysates from these exudates showed a similar inhibitory effect on CHG (12). We also demonstrated the inhibitory effect of raw amoeba cell lysates (Fig. 3B). Next, we removed the cell debris from the amoeba cell lysates and explored the influence of amoeba protein and genomic DNA (Fig. 3C and D). The interaction between amoeba nucleic acids and the cationic antiamoeba agent PHMB has been reported (24, 25). Here, we demonstrated that the interaction between amoeba DNA and CHG rescued amoeba survival. As a broad-spectrum antimicrobial agent, CHG use in skin disinfection and dental practice is effective at preventing and controlling bacterial invasion (26–28). A previous study demonstrated that the bactericidal activity of CHG is inhibited by preincubation with heat-killed bacteria (11). To mimic clinical conditions, we isolated S. epidermidis from corneal samples of patients with AK. Our results showed that live and heat-killed bacterial suspensions affected CHG amoebicidal activity (Fig. 4A). Therefore, we suggest that ocular bacteria disturb CHG activity during AK therapy. Furthermore, we showed that the preincubation of CHG with all the tested inhibitory elements reduced the amoebicidal activity of CHG (Fig. 4B, Fig. S2). Previous research shows that preincubation with microorganisms inactivates the bactericidal activity of CHG, which agrees with our results (11). We suggest that the preincubation of these elements provides a longer interaction time and reduces the effect of CHG, resulting in a stronger inhibitory effect on CHG than that during coincubation.

Acanthamoeba not only adheres to the ocular surface but also invades the cornea, which leads to serious ring infiltration (29). The pig cornea is considered a more feasible graft than rodent or rabbit cornea for xenocorneal transplantation (30). To reveal the possible effect of the cornea during treatment, we performed coincubation experiments with pig, rat, and mouse corneal lysates and found that they significantly disturbed CHG amoebicidal activity (Fig. 4C). Collagen fibrils are important elements of the cornea, and fibers in the peripheral cornea appear to be more loosely stacked than in the central cornea (31). Our findings are in line with previous studies showing that collagen inactivates the antibacterial activity of CHG (11). Furthermore, we showed that the combined incubation of mucin and the other inhibitory substances resulted in higher inhibition (Fig. 4D). Based on these results, we suggest that mucin enhances a stronger inhibitory effect on CHG in the ocular environment. However, it was difficult to determine whether the inhibition of CHG was antagonistic, synergistic, or additive since the combination index of these ocular factors was distinct and unique in the individual cases. Overall, we found that most of the tested ocular elements had the potential to reduce the amoebicidal activity of CHG and may therefore play a role in the failure of AK treatment.

In previous studies, dry-eye drops were reported to wash irritants away and promote corneal reepithelialization in a rabbit model (32, 33). Here, we evaluated the effect of eye drops on CHG activity and showed that they did not have an effect (Fig. 5). Therefore, rinsing the ocular surface with dry-eye drops may help to maintain the action of CHG, improve tear stability, and promote corneal reepithelialization in AK patients after corneal curettage and aggressive drug treatment.

In conclusion, our results indicate that complex ocular elements may play an important role in the observed gap between in vitro drug analysis and clinical treatment. Since drug resistance may be caused, at least in part, by the action of elements from the ocular surface, we suggest that washing with dry-eye drops should be done hourly during topical AK therapy to eliminate the inhibitory action of the ocular elements and enhance CHG effectiveness.

MATERIALS AND METHODS

Free-living amoeba cultivation.

The Acanthamoeba castellanii NCKU_SD strain was isolated from the cornea of patients diagnosed with AK at the National Cheng Kung University Hospital. Acanthamoeba was axenically cultured in a protease peptone-yeast extract glucose medium at 28°C in cell culture flasks and routinely cultured as monolayers at 28°C in proteose peptone-yeast extract glucose (PYG) medium.

Cell line cultivation.

Rat glial C6 cells (ATCC CCL-185) and human lung carcinoma A549 cells (ATCC CCL-107) were cultured in 6 ml Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (50 μg/ml streptomycin and 100 U/ml penicillin) at 37°C. After 24 h of incubation, cells were washed and resuspended in Page’s amoeba saline (PAS) buffer for homogenization.

Determination of CHG antiamoeba activity.

To determine antiamoeba activity, chlorhexidine digluconate (CHG) was diluted in Page’s amoeba saline (PAS) to different working concentrations (0.005%, 0.0025%, 0.00125%, 0.000625%, and 0.0003125% wt/vol) and cultured with 8 × 105 NCKU_SD cells. The amoeba cells were incubated for 30 min at 28°C, after which the survival rate was evaluated using a trypan blue viability cell counting assay.

Mucin and albumin treatment.

To investigate the effect of mucin (Sigma-Aldrich, Germany) and albumin (Leadgene, Taiwan) on CHG activity, 8 × 105 NCKU_SD cells were coincubated with 0.0025% CHG and different concentrations (0.025, 0.05, 0.10, 0.15, and 0.5 mg/ml) of mucin and bovine serum albumin diluted in PAS. After 30 min at 28°C, the amoeba survival rate was measured using a trypan blue viability cell counting assay and a CellR light microscope (Olympus CellR, Japan). PAS was applied to the medium as a control group.

Treatment with human and amoeba cell lysates, bacterial suspensions, and animal cornea.

Raw cell lysates of NCKU_SD, C6, and A549 cells were obtained with ultrasonic treatment. Additionally, raw amoeba lysates were centrifuged at 16,000 × g for 15 min at 4°C, and the pellets were removed to collect the amoeba cell protein fraction. We used a protein assay (Bio-Rad, USA) to measure the protein concentration in the raw cell lysates and amoeba protein fractions. Raw cell lysates and amoeba protein were diluted to 0.10, 0.15, and 0.5 mg/ml with PAS.

Staphylococcus epidermidis, isolated from corneal samples of AK patients at the National Cheng Kung University Hospital, was grown overnight in lysogeny broth (LB) on a rotary shaker (170 rpm). Then, bacterial cells were harvested in LB to optical densities at 600 nm (OD600) of 0.3 and 0.8. Then, LB was replaced with PAS. Additionally, bacteria were heat-killed by incubation at 95°C for 10 min, and the turbidity of the suspension was adjusted to OD600 of 0.3 and 0.8. To investigate the effect of live and heat-killed bacteria on the effect of CHG, we preincubated different concentrations of either live or heat-killed bacteria with CHG for 30 min and then removed the remaining bacteria by filtration. Then, Acanthamoeba was added to the medium for 30 min to examine whether the preincubation step reduced the effect of CHG.

Pig eyes were obtained from a local swine slaughterhouse and stored on ice. Rat and mouse corneas were obtained from sacrificed Sprague-Dawley (SD) rats and C57BL/6 mice without ocular disorder. The rat and mouse animal experimental procedures were conducted as reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Center at National Cheng Kung University (NCKU). The rat and mouse animal studies were performed following a protocol approved by the IACUC of NCKU (approvals NCKU-IACUC-106-013 and NCKU-IACUC-106-308). The corneas were then collected in the laboratory. The pig, rat, and mouse corneas were minced with scissors just before homogenization. The homogeneous corneas were prepared in PAS as the cornea solution using a sonic reactor. Then, we used a protein assay (Bio-Rad, USA) to measure the protein concentration in all raw animal cornea lysates.

As mentioned below, different concentrations of the test substances were applied to NCKU_SD cells in combination with 0.0025% CHG. After 30 min at 28°C, the amoeba survival rate was determined using the trypan blue counting assay.

We combined individual factors with the mucin at their lowest working concentrations and incubated them with Acanthamoeba to examine the interaction between the mucin and these components (cell lysates, Acanthamoeba lysates, bacteria, and pig cornea lysates).

Treatment with dry-eye drops.

Systane Ultra lubricant eye drops (Alcon Laboratories, Texas) were prepared at different concentrations (0.10, 0.15, and 0.5 mg/ml). One of the major components of the eye drop, polyethylene glycol 400, was quantified as a marker. Propylene glycol was diluted to 0.5 mg/ml in PAS. A prepared eye drop was applied to NCKU_SD cells in combination with 0.0025% CHG. Diluted propylene glycol was added to cells in combination with 0.0025% and 0.00125% CHG. After 30 min of treatment, amoeba cells in all groups were counted.

Genomic DNA extraction and treatment.

To measure the influence of amoeba DNA, protozoan cells were grown in PYG medium and cultured subsequently. The cell pellets were suspended in PAS for genome extraction. Genomic DNA was purified using a LabPrep DNA minikit (Taigen Bioscience Corporation, Taiwan). Genomic DNA was quantified using an ND-1000 instrument (Thermo Fisher, USA). As mentioned above, different concentrations of prepared amoeba genomic DNA (400, 800, 1,200, 4,000, 8,000, and 10,000 ng/ml) were incubated with CHG and Acanthamoeba for 30 min, and then the amoeba survival rate was measured.

Statistical analysis.

All experiments were performed as three independent experiments. Statistical analysis was calculated using Student’s t test. P < 0.05 (*) was considered significantly different from the control or CHG group.

Supplementary Material

Supplemental file 1
AAC.02066-20-s0001.pdf (93.5KB, pdf)

ACKNOWLEDGMENTS

We acknowledge Jyh-Wei Shin for providing reagents/materials/analysis tools. We thank Editage for English language editing.

This research was supported by the Ministry of Science and Technology (MOST) to W.-C.L. (grant MOST 109-2628-B-006-022).

Footnotes

Supplemental material is available online only.

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