Disruption of the Enterococcus faecalis–Induced Biofilm on the Intraocular Lens Using Bacteriophages

Abstract

Purpose: To compare the effects of bacteriophages (phages) and vancomycin on Enterococcus faecalis–induced biofilms on the intraocular lens.

Methods: E. faecalis strains EF24, GU02, GU03, and phiEF14H1 were used. The expression of the enterococcus surface protein (esp) gene was analyzed using polymerase chain reaction. Phages or vancomycin was added to the biofilms formed on culture plates or acrylic intraocular lenses. The biofilms were quantified after staining with crystal violet. The structure of the biofilms was analyzed using scanning electron microscopy.

Results: E. faecalis strains EF24, GU02, and GU03 formed biofilms on cell culture plates; however, the esp-negative GU03 strain had a significantly lower biofilm-forming ability than the esp-positive strains EF24 and GU02. The addition of phiEF14H1 resulted in a significant reduction in biofilm mass produced by both EF24 and GU02 compared with the untreated control. However, the addition of vancomycin did not degrade the biofilms. Phages significantly degraded biofilms and reduced the viable EF24 and GU02 bacteria on the intraocular lens.

Conclusions: Phages can degrade biofilms formed on the intraocular lens and destroy the bacteria within it. Thus, phage therapy may be a new treatment option for refractory and recurrent endophthalmitis caused by biofilm-forming bacteria.

Translational Relevance: Phage therapy, a novel treatment option for refractory and recurrent endophthalmitis caused by biofilm-forming bacteria, effectively lyses E. faecalis–induced biofilms.

Introduction

Postoperative endophthalmitis is a serious infectious ocular disease that can cause rapid and irreversible vision loss within hours to days of symptom onset. In particular, endophthalmitis caused by Enterococcus spp. has a poorer visual outcome than that caused by other bacteria, such as Staphylococcus aureus or coagulase-negative staphylococci.¹ In a 10-year multicenter study in Japan, all cases of endophthalmitis caused by Enterococcus faecalis had a final best-corrected visual acuity < 1.0 logarithm of the minimum angle of resolution (logMAR).² 

Biofilms are microbial accretions covered with a matrix of extracellular polymeric substances.³ Bacterial biofilm formation increases antimicrobial resistance 10- to 1000-fold to antimicrobial agents.^4,5 Enterococci are biofilm-forming bacteria and are one of the primary organisms causing postoperative endophthalmitis. Endophthalmitis caused by bacteria that form biofilms results in retinal structural and functional impairment and poor visual prognosis.⁶ In cases of biofilms formation on the intraocular lens, removal of the intraocular lens is often required to manage endophthalmitis.^7,8 Because biofilms are resistant to current antimicrobial treatment, a novel treatment is needed. 

Bacteriophages (phages) are viruses that specifically infect and lyse bacterial cells. Phages exist in natural environments where bacteria exist. We have previously reported the therapeutic and prophylactic effects of phage therapy on E. faecalis endophthalmitis. Phages also have the potential to lyse biofilms, and if they can dissolve biofilms formed on intraocular lenses then they may offer a novel therapeutic approach as an alternative to antimicrobial agents for refractory postoperative endophthalmitis.^9-11 Therefore, this study aimed to demonstrate the effect of phages on E. faecalis–induced biofilm formation on intraocular lenses. 

Methods

Bacteria and Phages

Vancomycin-sensitive E. faecalis strains EF24, GU02, and GU03 were isolated and prepared as previously described.^12,13 E. faecalis cells were cultured in tryptic soy broth (TSB; Beckton, Dickinson and Company, Franklin Lakes, NJ) supplemented with 0.75% glucose.¹⁴ The phage phiEF14H1 was isolated and amplified using E. faecalis strain EF14 as previously described.^13,15 To evaluate phage plaque formation, we used brain–heart infusion medium–based agar consisting of a double layer of 1.5% agar in the lower layer and 0.5% agar in the upper layer. 

Biofilm Formation

Bacterial suspensions of E. faecalis strains EF24, GU02, and GU03 (10⁴ cells/mL) were added to 96-well flat-bottom polystyrene plates, and the plates were incubated to facilitate biofilm formation on the surface. Sterile hydrophobic acrylic intraocular lenses (SN60WF; Alcon, Geneva, Switzerland) were placed in 96-well plates and contaminated with 200- µL EF24 and GU02 bacterial suspension (10⁴ cells/mL) in TSB medium supplemented with 0.75% glucose. The 96-well plates were incubated at 37°C for 48 hours to allow for initial biofilm formation. Acrylic intraocular lenses were incubated at 37°C for 12, 24, 48, and 72 hours to form biofilms. After initial biofilm development, 96-well plates and lenses were rinsed with physiological saline to remove non-adherent cells. The contaminated 96-well plates and intraocular lenses were divided into three treatment groups: (1) control (no treatment), (2) phage only, and (3) vancomycin only. For the phage treatment groups, 96-well plates and lenses were exposed to phiEF14H1 (20–50 × 10¹⁰ plaque forming units). Vancomycin was added at 2.0 mg/mL, reflecting typical therapeutic levels; 200  µL of respective treatment solutions was added to each well and incubated for 6 hours at 37°C. 

Biofilm masses in 96-well plates and intraocular lenses were quantified using crystal violet staining. Briefly, 96-well plates and lenses were rinsed twice with physiological saline, stained with 0.2% crystal violet (Muto Pure Chemicals, Tokyo, Japan) for 15 minutes, and then rinsed. Bound crystal violet was solubilized in 200  µL ethanol–acetone (80:20, vol/vol). The absorbance of the solution was measured at 595 nm using a spectrophotometer (SpectraMax 190; Molecular Devices, Silicon Valley, CA) to quantify biofilm formation. 

Detection of the Enterococcus Surface Protein Gene in E. faecalis

The enterococcus surface protein (esp) gene was amplified using polymerase chain reaction (PCR) as previously described.¹⁶ PCR amplification was performed on a Takara Dice system (Takara Bio, Shiga, Japan) using ExTaq DNA polymerase (Takara Bio) and primer pairs for esp (Integrated DNA Technologies, Tokyo, Japan). 

Scanning Electron Microscopy

Scanning electron microscopy was used to visualize the structure and integrity of the biofilms after treatment. Intraocular lenses were fixed with 2% glutaraldehyde in 0.1-M phosphate buffer (pH 7.3) at 4°C overnight. After rinsing with phosphate-buffered saline (PBS), intraocular lenses were post-fixed with 1% OsO₄ in 0.1-M phosphate buffer (pH 7.3) at 4°C for 1 hour. After being rinsed again with PBS, the intraocular lenses were dehydrated using graded ethanol. After treatment with t-butyl alcohol, the intraocular lenses were frozen. The frozen intraocular lenses were then dried in a vacuum chamber using a rotary pump. The dried intraocular lenses were gold coated using an ion coater (Eiko IB-5; Eiko Engineering, Tokyo, Japan) and observed under a scanning electron microscope (JSM-6010LV; JEOL, Tokyo, Japan). 

Measurement of Viable Bacteria on Intraocular Lenses

The number of viable bacteria on intraocular lenses was measured as described previously,¹⁷ with slight modification. The intraocular lenses were then rinsed with sterile physiological saline. The biofilm on the surface of the intraocular lenses was disrupted three times with zirconia beads using a homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) at 6000 rpm for 30 seconds each time. The homogenates were diluted with saline, plated on tryptic soy agar (Becton, Dickinson and Company), and incubated at 37°C for 48 hours, using a track dilution method as described previously.¹⁸ 

Statistical Analysis

Statistical analysis was performed using Statcel 4 software (OMS Publishing, Saitama, Japan). Quantitative data are presented as mean ± standard error of mean and were analyzed using the Tukey–Kramer test or Student's unpaired t-test. Statistical significance was set at P < 0.05. 

Results

Biofilm Formation by E. faecalis Strains

We first evaluated the ability of E. faecalis strains to form biofilms on cell culture plates. E. faecalis strain EF24 from vaginal discharge and strains GU02 and GU03 from patients with endophthalmitis were used in this study. GU03 had a significantly lower biofilm-forming ability than EF24 and GU02 (Fig. 1A). We detected the esp gene in EF24 and GU02 but not in GU03 (Fig. 1B). This correlated with the biofilm production results. Therefore, further studies were performed using the EF24 and GU02 strains. 

Figure 1.

 

Biofilm formation and detection of esp in E. faecalis. E. faecalis strains EF24, GU02, and GU03 were incubated on culture plates for 48 hours and then rinsed with saline to remove non-adherent bacteria. (A) The biofilms formed on the plates were then stained with crystal violet, and the extracts were quantified colorimetrically. Data are expressed as mean ± SEM. **P < 0.01 versus EF24, ††P < 0.01 versus GU02, ‡‡P < 0.01 versus GU03 (Tukey–Kramer test). (B) The presence of esp in E. faecalis strains was examined using PCR. Lane M, molecular marker; 1, EF24; 2, GU02; 3, GU03; 4, no bacteria.

Effect of Phage and Vancomycin on E. faecalis–Induced Biofilm

We examined the effects of phages and vancomycin on EF24- and GU02-induced biofilms. The treatment of phiEF14H1 resulted in a significant reduction in the biofilm mass produced by EF24 compared with that of the untreated control (P < 0.01). Vancomycin treatment did not reduce biofilm formation produced by EF24 (Fig. 2A). Similarly, phiEF14H1 significantly degraded biofilms produced by GU02, whereas vancomycin did not (Fig. 2B). 

Figure 2.

 

Effects of phage and vancomycin on E. faecalis–induced biofilm. (A, B) Biofilms formed by E. faecalis strains EF24 (A) or GU02 (B) on culture plates were incubated with a vehicle, phage, or vancomycin for 6 hours. The biofilms were then stained with crystal violet, and the extract was quantified colorimetrically. Three representative photographs of culture plates are shown for each group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 versus none (Tukey–Kramer test).

Effect of Phage and Vancomycin on E. faecalis–Induced Biofilm on Intraocular Lenses

We examined the effects of phages and vancomycin on E. faecalis–induced biofilm formation on intraocular lenses. Both EF24 and GU02 formed biofilms on intraocular lenses (Fig. 3A). Biofilms on the intraocular lens treated with phages displayed significant structural damage, with large areas of the intraocular lens surface appearing clear (Fig. 3B); however, no change in biofilm formation was observed in intraocular lenses treated with vancomycin (Fig. 3C). We then quantitatively evaluated the effects of phages and vancomycin on biofilms on intraocular lenses using crystal violet staining. We examined 12-, 24-, 48-, and 72-hour cultures of the clinical isolate GU02 derived from endophthalmitis. At 48 and 72 hours, the biofilms formed on the intraocular lens were detached even when gently washed. Therefore, because the 48- and 72-hour cultures were deemed unsuitable for analysis, we conducted the analyses at 12 and 24 hours. At 12 and 24 hours, phage treatment resulted in significant disruption of biofilm integrity and reduced biofilm formation compared with untreated controls. However, the addition of vancomycin did not degrade the biofilm on intraocular lenses (Fig. 4). Then, the number of viable bacteria on the intraocular lens was examined. The phage-treated group showed a significant decrease in viable bacteria compared with the control group. However, the vancomycin-treated group did not differ from the control group (Fig. 5). Finally, when EF24 was analyzed after 24 hours, similar to the results for GU02, phage treatment dissolved the biofilm, reduced biofilm formation compared with that in untreated controls, and decreased the number of viable bacteria. However, there was no difference between the vancomycin-treated group and the control group (Fig. 6). 

Figure 3.

 

Electron micrograph of the biofilm on the intraocular lens. (A–C) Intraocular lenses with biofilm formation by E. faecalis strain GU02 were incubated with a vehicle (A), phage (B), or vancomycin (C) for 6 hours. The intraocular lenses were then examined using scanning electron microscopy. Scale bar: 10  µm.

Figure 4.

 

Effects of phage and vancomycin on E. faecalis GU02–induced biofilm on intraocular lenses. Intraocular lenses with biofilms formed by E. faecalis strain GU02 for 12, 24, 48, and 72 hours were incubated with a vehicle, phage, or vancomycin for 6 hours. The intraocular lens was then stained with crystal violet, and the extract was quantified colorimetrically. Three representative photographs of intraocular lenses are shown for each time point. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 versus none (Tukey–Kramer test).

Figure 5.

 

Effects of phage and vancomycin on viable bacterial load on intraocular lenses. (A, B) Intraocular lenses with biofilms formed by E. faecalis strain GU02 for 12 hours (A) and 24 hours (B) were incubated with a vehicle, phage, or vancomycin for 6 hours. Viable bacterial loads were measured in the intraocular lens after incubation with a vehicle, phage, or vancomycin for 6 hours. Data are presented as mean ± SEM. **P < 0.01 versus none (Tukey–Kramer test).

Figure 6.

 

Effects of phage and vancomycin on E. faecalis EF24–induced biofilm and viable bacterial load on intraocular lenses. (B) Intraocular lenses with biofilms formed by E. faecalis strain EF24 for 24 hours were incubated with a vehicle, phage, or vancomycin for 6 hours. The intraocular lens was then stained with crystal violet, and the extract was quantified colorimetrically. (A) Three representative photographs of intraocular lenses are shown for each time point. (C) Viable bacterial load was measured in the intraocular lens following incubation with a vehicle, phage, or vancomycin for 6 hours. Data are presented as mean ± SEM. **P < 0.01 versus none (Tukey–Kramer test).

Discussion

In this study, we demonstrated that phages lyse E. faecalis–induced biofilms. Despite sensitivity to vancomycin, its administration did not dissolve the biofilm or reduce the viable bacterial count of E. faecalis. However, the administration of phages led to both dissolution of the biofilm and a decrease in the viable bacterial count of E. faecalis. These results suggest that phage therapy is a novel treatment option for refractory and recurrent endophthalmitis caused by biofilm-forming bacteria. 

Cases of postoperative endophthalmitis after cataract surgery that recurred despite appropriate antibiotic treatment and were resolved by intraocular lens removal have been reported.^7,8 In these cases, biofilms formed on the removed intraocular lens. These reports suggest that biofilms on intraocular lenses can cause recurrent endophthalmitis, and intraocular lens exchange may be considered in cases of postoperative endophthalmitis caused by biofilm-forming bacteria to reduce the risk of recurrent infection. Lodha et al.⁶ also prospectively reported on the treatment and visual outcomes of endophthalmitis cases with and without biofilm formation. This study revealed that endophthalmitis caused by biofilm-forming bacteria required more surgical intervention and had worse anatomical and functional outcomes than non–biofilm-forming bacterial endophthalmitis, despite similar antimicrobial resistance patterns between the two groups. The results of this study, in which phages disrupted the biofilm on the intraocular lens and reduced the number of bacteria, suggest that phages can treat cases of endophthalmitis caused by biofilm-forming bacteria without removing the intraocular lens. 

Several hypotheses have been proposed regarding the mechanisms through which phages dissolve biofilms. It is widely recognized that numerous phage genomes encode enzymes that can degrade polymeric substances, including lipopolysaccharides (LPSs), capsular polysaccharides (CPSs), and exopolysaccharides (EPSs).¹⁹ These phage-derived enzymes, known as depolymerases, are predominantly components of tail fibers or tail spike proteins of phages. They specifically bind to the LPSs, CPSs, or EPSs of host bacteria, thereby facilitating phage adsorption. Following successful adsorption, phages overcome the final barrier and cell wall and infect the cells.²⁰ Phage depolymerases are also believed to play a pivotal role in phage–host interactions within biofilms by promoting matrix disruption. This disruption enhances the diffusion of phages through the biofilm structure, allowing them to target bacterial cells.²¹ Furthermore, phages can penetrate inner layers of biofilms, unlike antibiotics, which primarily destroy surface bacteria. They can dissolve the biofilm matrix by producing enzymes or inducing enzyme production in the bacterial host. Phages infect bacteria within biofilms and destroy the extracellular matrix using phage-derived enzymes. The proliferating phages lyse the bacteria deeper within the biofilm.^9–11 In our study, antibiotics had no effect on the biofilm, whereas phages were able to dissolve it. Given the reduction in viable bacterial counts, it is plausible that, similar to previous reports, phages lysed the bacteria within the biofilm on intraocular lenses and continued to proliferate, thereby further dissolving the surrounding biofilm. 

Biofilms are microbial communities attached to surfaces and encased in an extracellular matrix. They exhibit increased resistance to antibiotics compared to planktonic cells. The minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) of vancomycin against biofilms formed by enterococci are reportedly higher than the minimum inhibitory concentration (MIC) against planktonic enterococci. The E. faecalis strains we used were sensitive to vancomycin, and their growth was inhibited by vancomycin in planktonic cultures. However, we found that, despite this sensitivity, the biofilms formed by the enterococci, due to their antibiotic resistance mechanisms, were not dissolved by vancomycin.^22,23 The mechanisms by which biofilms confer resistance to antibiotics include interactions between antibiotics and matrix components, delayed antibiotic penetration through the biofilm matrix, reduced growth rates of organisms within biofilms, and various actions of specific genes that determine antibiotic resistance.^24,25 

Verma et al.²⁶ compared the in vitro therapeutic efficacy of phages alone and in combination with ciprofloxacin against Klebsiella pneumoniae–induced biofilms. Although phages alone were able to eradicate biofilms and there was no significant difference in their ability to do so in combination with antibiotics, combination therapy significantly prevented the emergence of resistant mutants. Therefore, the combination of phages and antibiotics may be an effective strategy to combat the emergence of treatment-associated resistant bacteria.²⁶ 

Even within the same bacterial species, different genes can result in differences in biofilm-forming abilities among strains; esp is not essential for biofilm production, but its presence reportedly promotes biofilm formation.²⁷ In the present study, the biofilm-forming ability differed depending on the presence or absence of esp. E. faecalis strains with esp formed more biofilms. Therefore, we focused our investigation on the strains EF24 and GU02 that formed more robust biofilms. Intraocular lenses used in cataract surgery are mainly comprised of three types of materials: acrylic, silicone, and polymethylmethacrylate. Acrylic intraocular lenses are currently the most widely used.²⁸ Kobayakawa et al.¹⁴ compared the characteristics of biofilm formation among these intraocular lens materials and revealed that enterococci formed the greatest amount of biofilm on acrylic intraocular lenses. Due to their higher propensity for biofilm formation than other materials, we selected acrylic intraocular lenses in this study. 

Baillif et al.²⁹ developed an in vitro model to study Staphylococcus epidermidis–induced biofilm formation on intraocular lenses and found that biofilm development was modulated by environmental factors. In our in vitro study, in which the conditions were uniformly controlled, it is likely that a more homogeneous and robust biofilm was formed on intraocular lenses than those formed in vivo. Our findings indicate that phages were effective against uniformly and robustly formed biofilms, suggesting their effectiveness against biofilms formed on intraocular lenses in vivo. 

This study has some limitations. First, the results obtained in this study were based on in vitro experiments and might not directly reflect in vivo reactions. In vivo, various immune cells are present, and the response to biofilms may differ from the results obtained here; therefore, further studies using in vivo models are necessary. Second, there was no investigation into the optimal dosage of phages. It is necessary to determine the most effective phage dosage in future studies. 

Acknowledgments

The authors thank the Division of Biological Research, Science Research Center, Kochi University, for the use of research instruments. 

Supported by a Kochi Medical School Hospital President's discretionary grant and by JSPS KAKENHI (grant number JP24K19791). 

Disclosure: T. Kishimoto, None; K. Fukuda, None; W. Ishida, None; A. Kuwana, None; D. Todokoro, None; J. Uchiyama, None; S. Matsuzaki, None; K. Yamashiro, None