Is a bacterial biofilm in the lacrimal sac the cause of chronic refractory dacryocystitis? – A pilot study

ABSTRACT Purpose A pilot study to identify bacterial biofilm in the lacrimal sacs of patients with chronic dacryocystitis, and in patients with epiphora but without discharge, using scanning electron microscopy. Methods Five patients: two with nasolacrimal duct obstruction without dacryocystitis, and three with dacryocystitis refractory to antibiotics, underwent external dacryocystorhinostomy. One control patient without infection was included. Bacterial cultures were obtained from the lumen of the lacrimal sac to analyze possible bacterial growth, including antibiotic resistance. Biopsies were taken from all lacrimal sacs and prepared for light and scanning electron microscopy. Results Scanning electron microscopy of all the lacrimal sac samples revealed structures consistent with bacterial communities and adjacent extracellular material, indicating biofilm formation. This was most prominent in one of the patients with chronic dacryocystitis. Bacteria were found not only on the luminal surface of the sac, but also within the tissue of the sac. Bacterial growth was identified in samples from two patients with chronic dacryocystitis, whereas samples from the other three patients showed no bacterial growth. Conclusion Lack of patency of the lacrimal duct predisposes to bacterial growth, even in patients with no clinically confirmed infection of the lacrimal sac. The finding of a biofilm in patients with chronic dacryocystitis explains the lack of efficiency of antibiotic treatment at the concentrations used in clinical practice.


Introduction
Lacrimal problems are frequently encountered among ophthalmic patients.A large study carried out in England in the 1960's on the prevalence of lacrimal obstructions in adults showed that 22% of the population aged over 40 years had uni-or bilateral obstruction. 1 The overall annual incidence of acquired lacrimal duct obstruction has been reported to be 30.47/ 100 000; nasolacrimal duct obstruction (NLDO) being the most common form of stenosis, with an incidence of 20.2/100 000. 2 The symptoms of lacrimal stenosis vary from very mild epiphora to more severe problems with constant epiphora, mucus discharge and chronic dacryocystitis. 3Around one third of patients with an episode of dacryocystitis will have one or more episodes of dacryocystitis within three years, despite treatment with appropriate antibiotics. 4It is not known why so many patients with complete lacrimal duct obstruction have chronic or recurrent episodes of dacryocystitis, while others only have epiphora.
Patients with complete NLDO are usually treated with bypass surgery in the form of dacryocystorhinostomy (DCR).The surgical technique is the same for those with recurrent or chronic dacryocystitis and those with epiphora only.The aim of DCR is to construct an anastomosis between the lacrimal sac and the nasal mucosa.During surgery, the lacrimal sac is incised, making it possible to collect samples for bacterial culture directly from the inside of the lacrimal sac, and biopsies from the lacrimal sac can be examined using conventional light microscopy or electron microscopy.
Bacteria can live in a free-floating planktonic form, or in complex homogeneous or heterogeneous communities of biofilms attached to a surface. 5Bacterial biofilms are clusters of bacteria that are embedded in a self-CONTACT Karl Engelsberg karl.engelsberg@med.lu.seClinical Sciences, Department of Ophthalmology, Lund University and Skåne University Hospital, Ögonklinik A, Kioskgatan 1B, Lund SE-221 85, Sweden produced matrix consisting of substances such as proteins (e.g.fibrin), polysaccharides (e.g.alginate), as well as environmental DNA, and may be attached to inert or living surfaces. 6Under physiological conditions, once the biofilm has been established, bacteria predominantly grow in communities attached to a surface.Biofilms are often found on implants and prostheses, but can also be found on other surfaces such as biological tissue/ material. 7,8Common biofilm-forming bacteria are Staphylococcus, Streptococcus, Enterococcus, and Pseudomonas species.The most frequently isolated bacterial organisms from infected lacrimal sacs are Staphylococcus species. 9Other common pathogens causing dacryocystitis are gram negative bacteria such as Escherichia coli, Haemophilus influenzae or Pseudomonas species. 10he aim of this pilot study was to investigate whether biofilm-forming bacteria were present in the lacrimal sac, and whether biofilms could predispose to chronic or recurrent dacryocystitis refractory to antibiotics.

Ethics
The study was approved by the Swedish Ethical Review Authority (No. 2020-01983) and complied with the principles of the Declaration of Helsinki.Written consent to participate was obtained from all participants.

Subjects
The study included patients who were to undergo DCR at the Ophthalmology Clinic at Skåne University Hospital during the years 2022-2023.Patients considered eligible for the study were those who had complained preoperatively of epiphora or had several episodes of dacryocystitis, and in which lacrimal duct stenosis had been confirmed by lacrimal syringing or radiology (dacryocystography).Exclusion criteria were previous DCR on the same side, children (aged <18 years), and previous trauma to the orbit.They were offered treatment by external DCR and participation in the study.The characteristics of the patients are presented in Table 1.

Procedures and microbial culturing
Patients undergoing treatment with external DCR were all operated on by the same surgeon (K.E.).Surgery was performed under general anesthesia according to standard procedure. 11Briefly, a skin incision was made and an ostium was created by removing the lacrimal bone and part of the frontal process of the maxilla.The nasal mucosa was incised, and the lacrimal sac opened.Bacterial sampling was performed from the inside of the lacrimal sac (eSwab ® , Copan Italia spa, Brescia, Italy) for bacterial culture and analysis using standard routine procedures at the Department of Clinical Microbiology, Skåne University Hospital.The isolates identified in the bacterial cultures were Haemophilus influenzae, Streptococcus intermedius, and Staphylococcus aureus.
A biopsy, 3 × 3 mm, was taken from the lacrimal sac.Flaps from the lacrimal sac were sutured to flaps from the nasal mucosa creating an anastomosis from the lacrimal sac into the nose.

Preparation of the biopsies for microscopy
For correlative light electron microscopy (CLEM), specimens were fixed with 2.5% glutaraldehyde in 150 mM sodium cacodylate, pH 7.2, in room temperature overnight, then washed with cacodylate buffer 4x.The tissues were rinsed with water and dehydrated in graded alcohol: 50%, 70%, 95%, and absolute alcohol (ethanol) After the final dehydration step, the tissues were cleared with Histo-Clear ® (xylene substitute), followed by incu- bation in hot (60°C) paraffin.The tissues were either embedded in paraffin in suitable embedding cassettes/ molds, or fixed tissues were rinsed with water followed by dehydration and embedding in paraffin.The tissues were then cut into 3-5 µm sections and mounted on glass slides.The sections were then heated, deparaffinized, and processed for target/antigen retrieval using Tris-EDTA buffer, pH 9.0.After target retrieval, the slides were rinsed with Tris-buffered saline with 0.1% Tween 20 (TBST) wash buffer, pH 7.4.The sections were then fixed again with 2.5% glutaraldehyde in 150 mM sodium cacodylate, pH 7.2, in room temperature overnight.Sections were washed with cacodylate buffer 4x, and then dehydrated using an ascending ethanol series from 50% (vol/vol) to absolute ethanol.The specimens were then subjected to critical-point drying with carbon dioxide, and absolute alcohol was used as an intermediate solvent.The tissue samples were mounted on aluminum holders, sputtered with 20 nm palladium/ gold, and examined in a correlative light and scanning electron microscope (DELPHI Phenom-World Delmic, the Netherlands).
For hematoxylin-eosin staining, thin sections (3-5 µm) were mounted on glass slides, heated and deparaffinized.After rehydration the sections were stained with hematoxylin-eosin followed by dehydration in graded alcohol, and cleared with Histo-Clear, as described above.The stained sections were coverslipped with Pertex ® mounting medium.

Results
Six patients fulfilled the inclusion criteria.One of the patients had canalicular stenosis of unknown etiology without any signs of previous or current infection, and was thus included in the study as a control as she had no previous disease associated with the lacrimal sac or lacrimal duct.She first underwent external DCR, followed by a second surgery to insert a Lester Jones bypass tube.The bacterial sample and biopsy were taken during the first surgery.
Three of the remaining five patients had signs of lacrimal sac infection (pus in the lacrimal sac).Cultures of the bacterial samples from the infected patients revealed the growth of Haemophilus influenzae, Staphylococcus aureus, and Streptococcus intermedius (Table 1).
Hematoxylin-eosin staining of the biopsies showed stratified columnar epithelium and surrounding connective tissue, when examined by light microscopy.Neutrophils were also found in several of the sections, however not in the lacrimal sac from the control patient.
Scanning electron microscopy of the lacrimal sac biopsies revealed structures consistent with bacterial colonies and extracellular material adjacent to the bacterial colonies, indicating biofilm formation (Figure 1, Table 1).This was most prominent in one of the patients with chronic dacryocystitis.Bacteria were found not only on the luminal surface of the sac, but also within the tissue of the lacrimal sac (Figure 2).The biopsy sample from the lacrimal sac of the control showed no bacterial colonies (Figure 3).

Discussion
This pilot study revealed bacteria and the presence of a biofilm in lacrimal sacs from patients with a blockage  of the nasolacrimal duct.Lack of patency of the lacrimal duct predisposes to bacterial growth in the lacrimal sac, and in this study patients with no clinically proven infection of the lacrimal sac were also found to have bacteria in the lacrimal sacs upon examination with electron microscope.There was some discrepancy between the bacterial findings using cultures and SEM.One of the reasons for this is that cultures can only detect bacteria on the luminal surface of the lacrimal sac, while electron microscopy visualizes bacteria within the tissue of the lacrimal sac.Infiltration of bacteria into the tissue, and possibly intracellular evasive mechanisms, could cause the bacterial infection to be persistent and more resistant to the immune system and topically applied antibiotics. 12The presence of bacteria within the tissue of the lacrimal sac is also in line with the clinically well-known potential complication of dacryocystitis, namely anterior cellulitis.This complication is the result of bacteria migrating from the luminal side of the sac through the sac, and into the orbicularis oculi muscle and skin around the eye and, in the worst case, they can penetrate the septum and cause posterior orbital cellulitis.
The results of this study demonstrated the presence of a biofilm on the lumen of the lacrimal sac, even when bacterial culture revealed no bacteria.One explanation of this could be that traditional methods use to culture bacteria might not be sensitive enough to detect growth under certain circumstances. 13Other culture methods could have revealed bacteria in the samples showing no bacterial growth in this study.
Nasolacrimal duct obstruction is thought to develop due to recurrent infections and inflammation, which lead to nasolacrimal duct stenosis. 14The factors triggering the development of inflammation and fibrosis are not known, but the presence of a biofilm could play an important role.The presence of a biofilm in the lacrimal sac of patients with epiphora has been reported in one previous study. 15However, that study did not separate patients with only epiphora from patients with epiphora combined with dacryocystitis.Also, they did not take any cultures from the lacrimal sacs to correlate with the electron microscopic findings.In a study by Samini et al. 16 biofilm was found on lacrimal implants, but they did not investigate if there was biofilm in the lacrimal system of the patients.In the present study, we found more conspicuous biofilms in the patients with clinical signs of lacrimal sac infection.It is reasonable to assume that the formation of a bacterial biofilm increases the bacterial load, and thus the risk of a lacrimal sac infection with more pronounced clinical symptoms.
The finding of a bacterial biofilm in patients with chronic dacryocystitis explains the lack of efficacy of antibiotics at the concentrations normally used in clinical practice, as bacterial biofilms are 10-1000 times more resistant to antibiotic treatment than freefloating planktonic bacteria.This is in line with clinical studies showing that many patients with chronic dacryocystitis do not respond to antibiotics despite the fact that bacterial cultures show that the correct antibiotic has been administered.However, it would be difficult to administer a sufficiently high dose of antibiotics to eradicate a biofilm in the clinical setting.
An important finding of this study is that the lacrimal sac of the control patient did not exhibit any bacterial biofilm.Although we saw no bacterial colonization of the lacrimal sac in the control subject, bacterial biofilms may occur in the lacrimal sac of healthy individuals, and this must be investigated in future studies.However, the results presented here indicate that the combination of NLDO and a bacterial biofilm could be responsible for recurrent cases of dacryocystitis.As 90-98% of the patients who have undergone DCR do not have recurrent episodes of dacryocystitis [17][18][19] this raised the question of whether the biofilm vanishes due to the passage of tears through the open system.Whether or not there is still a biofilm on the lacrimal surface after such an intervention remains to be investigated.This pilot study was carried out on a small number of patients and that is a limitation to the study, and future studies should include more patients.It would also be of interest to examine postoperative samples from the lacrimal sac.However, despite the small number of patients in this study, we demonstrated the presence of a biofilm in the lacrimal sac of both patient groups.When the bacterial culture was negative, the presence of a bacterial biofilm was demonstrated by electron microscopy.
In conclusion, lack of patency of the lacrimal duct predisposes to bacterial growth, and we found signs of a bacterial biofilm even in patients with no clinically confirmed infection of the lacrimal sac.The finding of a biofilm in patients with chronic dacryocystitis explains the lack of efficacy of antibiotic treatment in clinical practice.

Figure 1 .
Figure 1.Scanning electron microscopy of lacrimal sac biopsies revealed structures consistent with extracellular material (red arrows) adjacent to the bacterial colonies, indicating biofilm formation.

Figure 2 .
Figure 2. Bacteria were located not only on the luminal surface of the sacs, but also within the tissue of the sac (red arrows).

Figure 3 .
Figure 3. Sample from the lacrimal sac of the control patient.Neither bacteria nor biofilm were found.Red blood cells resulting from the surgery can be seen on the luminal part of the sac.

Table 1 .
Characteristics of the patients and their lacrimal sacs.