Analysis of Intraocular Lens Biofilms and Fluids After Long-Term Uncomplicated Cataract Surgery


Postoperative endophthalmitis is a potentially sight-threatening complication of cataract surgery. However, the pathophysiological mechanisms are not completely understood. We sought to study and evaluate the intraocular environment (aqueous and vitreous humors), the capsular tissue, and the intraocular lens (IOL) surfaces of normal eyes after long-term uncomplicated cataract surgery.


Experimental laboratory investigation.


We studied 69 eyes donated for transplantation that had previously undergone cataract surgery with posterior chamber IOL implantation and that had no recorded clinical history of postoperative inflammation. We assessed the intraocular environment (DNA traces and biofilm formation) by microbiological evaluation of intraocular fluids using conventional microbiology and molecular techniques, including assessment for the presence of microbes (biofilm formation) on the IOL surface by scanning electron microscopy and ultrastructural capsular remnants by transmission electron microscopy.


Isolated or aggregated cocci were probable in 18.8% of IOL optic surfaces (n = 13) studied by scanning electron microscopy, suggesting the presence of bacterial biofilm. In 3 intraocular fluid samples for IOLs with biofilm, we identified 16S rDNA by polymerase chain reaction and sequencing. No microbial contamination was found in intraocular fluids by conventional microbiological methods.


Our data suggest the possibility of bacterial biofilm formation on the optic surface of IOLs in normal eyes after long-term uncomplicated cataract surgery even in the absence of clinical or subclinical symptoms.

Cataract removal and lens replacement with an artificial intraocular lens (IOL) is the most frequent surgery performed by ophthalmologists. The most fearsome complication of cataract surgery is postoperative endophthalmitis, which is defined as a severe inflammation of both the anterior and posterior segments of the eye secondary to an infectious agent. With incidence rates of 0.05%–0.34%, postoperative endophthalmitis is clinically classified into acute or chronic, depending on the onset after surgery. Acute endophthalmitis presents with pain; photophobia; floaters; reduced vision; an inflamed anterior segment, including a variable hypopyon; and vitritis. The predominant clinical feature of chronic or delayed-onset endophthalmitis is white plaques on the lens capsule or the IOL surface, representing collections of organisms. Endophthalmitis is associated with recurring episodes of low-grade inflammation that are initially responsive to topical steroid treatment, but that become refractive at later stages.

Coagulase-negative staphylococci, especially Staphylococcus epidermidis , and others, such as S aureus and Streptococcus spp. are currently the most common etiologic agents of acute postoperative endophthalmitis. In contrast, indolent organisms are usually responsible for chronic endophthalmitis, including Propionibacterium acnes and Corynebacterium spp. However, it is well known that microorganisms can be introduced into the eye from the patient’s external microbiota during surgery. In fact, molecular typing techniques have been used to confirm that 68%–82% of cases of postoperative endophthalmitis are caused by ocular and periocular microbiota. Reducing the number of organisms present on the ocular surface with the preoperative application of topical povidone-iodine in the conjunctival sac and periocular skin is the only measure with proven prophylactic value against endophthalmitis.

Endophthalmitis is a potentially sight-threatening infection. Detection of endophthalmitis and identification of the causative agent are therefore essential to ensure that rational treatment is given, yet routine microbiologic diagnosis relies on time-consuming classical culture techniques that offer low sensitivity; indeed, the positivity rates range from 22% to 30% in the aqueous humor and from 40% to 69% in the vitreous humor. Modern molecular techniques—such as polymerase chain reaction (PCR) combined with different post-PCR analyses—now allow for greater sensitivity and more rapid results and are particularly useful when only a small amount of material is available (eg, aqueous humor) or if the patient has previously received antibiotic treatment. In addition, molecular techniques are promising for the detection of organisms associated with chronic postoperative endophthalmitis because the samples are often small, difficult to culture, fastidious, or slow growing.

The mechanism through which postoperative endophthalmitis develops has not been completely elucidated to date. Nevertheless, it is recognized that bacterial adhesion to IOLs and intraocular tissues during implantation is probably the primary step in the pathogenesis. Then, cell-to-cell adhesion and the production of extracellular polysaccharide glycocalyx (slime) on the lens surface, followed by the formation of a confluent biofilm structure, appear to be relevant. Indeed, S epidermidis produces few virulence factors, but its success as an opportunistic pathogen is related to its ability to adhere to, and then form a mature biofilm on, the surface of different medical devices. Finally, although numerous in vitro studies have demonstrated that bacterial adhesion may be influenced by IOL biomaterials, differences in experimental conditions and protocols have meant that results differ.

Despite advances in our knowledge, little is known about the intraocular environment and behavior of the IOL surface in the long term after uncomplicated cataract surgery. Thus, we aimed to analyze the intraocular fluids (aqueous and vitreous humors), capsular tissues, and IOL surfaces of normal eyes donated for corneal transplantation after long-term uncomplicated cataract surgery. To our knowledge, this is the first time that biofilm formation has been studied in this setting on a large scale.


Eye Bank Specimens

In this study, we analyzed 69 donated eyes that had previously undergone cataract surgery and posterior chamber IOL implantation. Globes were obtained from the Eye Bank for the Treatment of Blindness (Centro de Oftalmología Barraquer, Barcelona, Spain) after obtaining consent from the family and in accordance with the Standardized Rules for Development and Applications of Tissues and Organ Transplants, as defined in Spanish law. Eyes were donated for corneal transplantation, but were discarded because they did not meet the criteria for penetrating or lamellar keratoplasty. According to the recorded family interview documentation for donation, all eyes underwent cataract surgery without postoperative complications. Globes with suspected or confirmed histories of postoperative complications were discarded. Donated eyes were evaluated and processed between 12 and 36 hours postmortem. Eyes were maintained in a 0.1 M phosphate-buffered solution (pH 7.4) containing antibiotics and antifungals (penicillin 100 IU/mL, streptomycin 10 mg/mL, and amphotericin B 2.5 μg/mL) at 4 C for 8–12 hours and then decontaminated by immersion in 40 mL of 5% povidone-iodine solution for 3 minutes under a laminar-flow hood.

Decontamination Efficacy of 5% Povidone-Iodine

The procedure was performed under a laminar-flow hood. Initially, the eye globe was removed from the antimicrobial maintenance solution and rinsed with 500 mL of sterile saline solution. A mark was made to divide the globe into 2 equal halves and a sterile swab was taken from half of the limbus and cornea. Next, the globe was fully immersed in 40 mL of 5% povidone-iodine for 3 minutes, removed, and rinsed with 500 mL of sterile saline to remove excess decontaminating agent that could inhibit microorganism growth. A postdecontamination sterile swab was then taken from the second half of the limbus and cornea, taking care not to swab the same area as in the pretreatment swab to avoid false-negative results. Pre- and postdecontamination swabs were rotated and soaked onto different culture media. The incubation conditions are summarized in Supplemental Table 1 (Supplemental Material available at ). Isolates were identified by standard microbiological methods. The effectiveness of decontamination was determined by calculating the following: (1) the percentage reduction of positive ocular surfaces after decontamination and (2) the percentage reduction in the number of each bacterial species after decontamination.

Microbiological Analysis of Intraocular Fluids by Classical Culture Techniques

Samples of aqueous and vitreous humor were collected aseptically under a laminar-flow hood. The aqueous humor samples (100–150 μL) were collected by anterior chamber limbic paracentesis with a 30 G needle, whereas the vitreous samples (500 μL) were obtained by pars plana puncture 4 mm from the limbic region with a 19 G needle. Samples were divided into 2 aliquots: 1 aliquot per sample was kept at −80 C for further analysis and the other was inoculated onto different culture media. Microbiological analysis and procedures were carried out as described ( Supplemental Table 1 ).

Light and Electron Microscopy Studies

Posterior chamber IOLs and their corresponding capsule remnants were explanted from ocular globes under aseptic conditions. A portion of lens capsule and IOLs were immediately fixed in 2% paraformaldehyde + 2.5% glutaraldehyde solution in a 0.1 M phosphate buffer. Ocular globes with IOL implanted into the sulcus or anterior chamber were excluded. After chemical fixation over 12 hours, IOLs were examined with a binocular magnifying loupe, photographed, and classified into polymethylmethacrylate (PMMA), silicone, and hydrophobic or hydrophilic acrylic lenses.

For scanning electron microscopy (SEM), fixed IOLs were dehydrated in ethanol–water mixtures with increasing concentrations of ethanol (50%, 70%, 90%, 96%, and 100% ethanol by volume). After critical point drying, samples were stuck on metal holders with double-sided adhesive tape and coated with gold. The IOL surface observations were performed at 15 kV with an SEM (JEOL JSM-840; JEOL Ltd, Tokyo, Japan). The entire IOL surface was examined for the presence of adherent microorganisms or biofilm.

For transmission electron microscopy (TEM), capsule remnants were abundantly rinsed in phosphate-buffered saline, postfixed in 1% osmium tetroxide in 0.1 M phosphate-buffered solution (pH 7.4) for 1 hour, dehydrated in increasing graded concentrations of acetone, and then progressively embedded in resin (Spurr technique) for polymerization at 60 C. We obtained semi-thin (0.5–1.0 μm) and ultrathin (50–75 nm) sections by conventional ultramicrotomy (OmU2; Reichert-Jung, Wein, Austria). The semi-thin sections were placed on coverslips and stained with toluidine blue solution, then observed by light microscopy (BX61; Olympus R-FTL-T; Olympus America Inc, Center Valley, Pennsylvania, USA) coupled with the Olympus DP Controller Program for digital image acquisition. Ultrathin sections were placed on copper grids (200 mesh) and stained with uranyl acetate and lead citrate for conventional TEM (Hitachi 800 MT; Hitachi, Tokyo, Japan).

Microbiological Analysis by Molecular Techniques

To minimize the possibility of cross-DNA contamination, we performed DNA extraction, PCR mixture preparation, and post-PCR analyses in separate rooms, using designated equipment. Total DNA was extracted from intraocular fluids with a DNA purification kit (QIAamp DNA mini kit; Qiagen, Hilden, Germany) according to the tissue protocol, with a few modifications. The oligonucleotide primers designed for panbacterial and panfungal detection have been described previously, and they produced 590- and 654-base-pair fragments of 16S and 18S ribosomal DNA, respectively ( Supplemental Table 2 ; Supplemental Material available at ). Positive control consisted of 10 ng of purified Escherichia coli or Candida albicans DNA for the 16S and 18S reactions, respectively. Sterile purified water was used as a negative control. PCR amplification was carried out in a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer, Emeryville, California, USA) as follows: 35 cycles of 30 seconds at 94 C; 30 seconds at 52 C or 50 C, for bacterial and fungal DNA, respectively; and 40 or 45 seconds at 68 C for bacterial and fungal DNA, respectively; followed by 7 minutes of final elongation step at 68 C. Amplified products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, Wisconsin, USA) and sequenced using the previously described primers on an automated sequencer (ABI-3730 DNA Analyzer Sequencer System; Applied Biosystems, Thermo Fisher Scientific, London, UK) at CRAG (Centre de Recerca Agrogenòmica, Generalitat de Catalunya, Barcelona, Spain). The sequences obtained were compared with those available in the GenBank, EMBL, and DDBJ databases with the gapped BLASTN 2.0.5 program obtained from the NCBI information server ( ). The minimal percentages to identify genus and species were used as recommended by the CLSI document MM18-A.

To confirm the 16S rDNA PCR results, molecular analysis by real-time quantitative PCR (qPCR) was performed at LEMC (Escola Paulista de Medicina, Universidad Federal de Sao Paulo, Sao Paulo, Brazil), as previously described. The universal primers NT-341Fw and 16S-522Rv were used to amplify a fragment of approximately 192 bp containing the V3 region ( Supplemental Table 2 ). A primer set and a probe for the β-globin gene was used as the internal control. The qPCR mix (Power SYBR Green PCR master mix; Life Technologies) was pretreated using 0.3 U of RQ1 RNAse-free DNAse (Promega) for 30 minutes at 37 C to remove contaminating bacterial DNA, followed by 50 minutes at 95 C to inhibit the DNAse activity. Amplification was performed on a real-time PCR system (model 7500; ABI) with the following conditions: 50 C for 2 minutes and 95 C for 10 minutes followed by 40 cycles at 95 C for 15 seconds and 60 C for 60 seconds. Melting curve analysis was performed after amplification, by heating samples gradually from 68 C to 95 C at a rate of 0.5 C/s. The amplification of clinical samples was performed in the presence of a negative control, including all PCR reagents, except the DNA template and positive control samples, with 10 ng of S epidermidis DNA. After the reaction, a 15 μL PCR aliquot was purified with a PCR purification kit (QIAquick; Qiagen). DNA molecules were sequenced in both directions with dye-termination chemistry (Big Dye Terminator; 3000 Genetic analyzer; ABI). The sequences obtained were edited (SeqMan; DNASTAR, Madison, Wisconsin, USA) and searched for similarity in GenBank using the BLAST (BLASTN algorithm) program with automatically adjusted parameters. The minimal percentages needed to identify genus and species were used, as recommended by CLSI document MM18-A.

Statistical Analysis

Results were compared globally using the nonparametric Kruskal-Wallis test (SPSS for Windows, version 22.0; SPSS Inc, Chicago, Illinois, USA). Then the Mann-Whitney nonparametric test was used to compare pairs of materials. Two-sided P values ≤.05 were considered statistically significant.


Decontamination Procedure Efficacy

The efficacy of chemical decontamination with 5% povidone-iodine was controlled (n = 20). Initial treatment with the antimicrobial maintenance solution eliminated the external microbiota in half of the globes (n = 10) selected for decontamination control efficacy. The microbial flora found on the ocular surface before treatment was similar to the microbial flora found in the conjunctiva and eyelids of healthy eyes. More than 1 species was isolated in most studied globes; commensal species predominated, with 7 isolates of coagulase-negative staphylococci (33.3%), 6 isolates of Corynebacterium species (28.6%), and 2 isolates of Micrococcus spp. (9.5%). Other noncommensal microorganisms, including S aureus and Streptococcus spp., were present less frequently. The 5% povidone-iodine solution completely decontaminated 90.0% of ocular globes (n = 9), with an efficacy of 95.2% in eliminating commensal species, including the most prevalent ones ( Supplemental Table 3 ; Supplemental Material available at ). The 5% povidone-iodine was very effective at decontaminating the ocular surfaces of globes donated for transplantation.

Microbiological Analysis of Intraocular Fluids

Microorganisms were not detected in either the aqueous or vitreous humor samples (n = 69 per intraocular fluid) cultured by conventional microbiologic techniques.

Light and Electron Microscopy Studies

IOLs observed by binocular magnifying loupe were classified according to the biomaterial of the lens (PMMA, silicone, hydrophobic acrylic, or hydrophilic acrylic). When analyzed by SEM, most of the IOLs showed cellular epithelial aggregates and crystalline fiber remnants. Bacterial biofilm was absent in 81.2% (n = 56) and probable in 18.8% (n = 13) of IOL surfaces studied ( Table 1 ). IOLs classified as having probable biofilm showed isolated or aggregated cocci (about 1 μm) on the surfaces of 35.0% of PMMA IOLs (n = 7), 28.6% of silicone IOLs (n = 2), 11.5% of acrylic hydrophilic IOLs (n = 3), and 6.3% of acrylic hydrophobic IOLs (n = 1) ( Figures 1–4 ). Significant difference in the prevalence of cocci on acrylic hydrophobic biomaterial and PMMA ( P < .042) was present. Interestingly, probable biofilm formation was also more evident in the periphery of all IOL optic surfaces ( Figures 1–4 ). In 4 of the 13 IOLs, the cocci were embedded by some extracellular debris ( Figure 4 ). The prevalence of suspected biofilm formation on the surface of IOLs is summarized in Table 2 .

Table 1

Prevalence of Suspected Bacterial Biofilm Formation on the Intraocular Lens Surface by Scanning Electron Microscopy

IOL Material Total Absent Probable
N N (%) N (%)
PMMA 20 13 (65.0) 7 (35.0)
Hydrophilic acrylic 26 23 (88.5) 3 (11.5)
Hydrophobic acrylic 16 15 (93.8) 1 (6.3)
Silicone 7 5 (71.4) 2 (28.6)
TOTAL 69 56 (81.2) 13 (18.8)

IOL = intraocular lens; PMMA = polymethylmethacrylate.

Figure 1

Probable biofilm formation for Propionibacterium acnes by DNA sequencing after polymerase chain reaction amplification (Top left: intraocular lens 24; hydrophilic acrylic). (Top right, upper and lower images) Semi-thin sections of capsular tissue with localized areas of rounded structures accumulating with high affinity to toluidine blue dye, which stains acidic tissue components, such as nucleic acids (DNA and RNA remnants), in dark blue. (Middle) Scanning electron microscopy showing (left) scattered areas of biofilm formation at the peripheral edge of the intraocular lens optic surface, characterized by (right) microcolonies of mixed structures with spherical- and rod-shaped cells grouped in a monolayer to the compacted low-stickiness biofilm. (Bottom, left and right) Transmission electron microscopy showing areas of compacted and vacuolated cell debris (r) with abundant residues of interdigitated lens fibers (lf). Bar = 50 μm for Top right, upper image; 25 μm for Top right, lower image; 10 μm for Middle left, Bottom right, and Bottom left images; 1 μm for Middle right image.

Figure 2

Probable biofilm formation positive for Moraxella catarrhalis by DNA sequencing after polymerase chain reaction amplification (Top left: intraocular lens 27; polymethylmethacrylate). (Top right) Semi-thin sections showed anterior capsule remnants with epithelial cells (c) and fragments of posterior lens capsule (lc). The most superficial layers of the posterior lens capsule exhibit linear deposits of rounded structures with high affinity for toluidine blue dye (Top right, bottom image). (Middle left) Scanty aggregates of spherical- and rod-shaped structures appear to accumulate at the periphery of the intraocular lens optic surface. (Middle right) A mucoid layer of glycocalyx (slime; upper image) composed of low-stickiness coccoid aggregates and a few rod-shaped cells (lower image), demonstrating the possibility of contamination by mixed microcolonies. (Bottom) Transmission electron microscopy analysis showing fragments of lens capsule (lc) related to (Bottom left) cell remnants (c) and (Bottom right) epithelial-like cells (c). Bar = 25 μm for Top right, upper image; 10 μm for Top right, lower image; 10 μm for Middle left, Middle right upper, and Bottom left images; and 5 μm for Bottom right images; 1 μm for Middle right lower image.

Figure 3

Probable biofilm formation for Propionibacterium acnes by DNA sequencing after polymerase chain reaction amplification (Top left: intraocular lens 28; polymethyl methacrylate). (Top right) Semi-thin sections showed fragments of lens capsule (lc) with cellular remnants and debris (r). Scattered across the lens capsule (lc), several areas are associated with isolated or grouped birefringent structures and round structures with a high affinity to toluidine blue dye (Top right, bottom image). (Middle left) Scanning electron microscopy analysis revealed a slime layer surrounding isolated or grouped cocci at the peripheral edge of the intraocular lens optic surface. (Middle right) Several small groups of spherical- and rod-shaped structures are shown. (Bottom, left and right) Transmission electron microscopy observation showing fragments of lens capsule (lc) associated with cellular debris (c) plus isolated or grouped electron-dense round structures that are adherent to the lens capsule (lc). Bar = 50 μm for Top right, upper image; 25 μm for Top right, bottom image; 10 μm for Middle left and Bottom left; 5 μm for Bottom right; 1 μm for Middle right.

Figure 4

Intraocular lens (IOL) classified as probable biofilm formation with negative polymerase chain reaction amplification for 16S and 18S sequences. (Top left) IOL 45 (polymethylmethacrylate). There is a compacted multilayer of cocci within a low-stickiness homogeneous biofilm on the IOL surface (Top center), and a mucoid layer of glycocalyx seems to shroud the cocci (Top right). (Middle left) IOL 02 (silicone). There are scanty aggregates of spherical- and rod-shaped structures on the IOL surface (Middle center), and mixed microcolonies of spherical and rod-shaped cells are grouped in a monolayer compacted to the low-stickiness biofilm (Middle right). (Bottom left) IOL 21 (hydrophilic acrylic). Hard aggregates of coccoid cells are seen to accumulate in dome-shaped structures at the edge of the optic region of the IOL surface (Bottom center). Microcolonies or clusters of cocci are associated with cell membrane debris or degenerated cells (Bottom right). Bar = 10 μm for Top center, Top right, and Middle center; 1 μm for Middle right, Bottom center, and Bottom right.

Table 2

Classification of Intraocular Lens by Scanning Electron Microscopy and Molecular Analysis

IOL IOL Material Biofilm on IOL by SEM Observation Microbial-like Structures on IOL by SEM Observation Sample PCR Sample PCR q-PCR
18S 16S 16S
2 Silicone Probable Mainly cocci and few rods 1 Aqueous NA NA NA
2 Vitreous NA NA NA
5 Acrylic hydrophobic Probable Mainly cocci and few rods 3 Aqueous NA NA NA
4 Vitreous NA NA NA
8 PMMA Probable Mainly cocci surrounded by ocular tissue remains 5 Aqueous NA NA NA
6 Vitreous NA NA NA
16 PMMA Probable Mainly cocci surrounded by ocular tissue remains 7 Aqueous NA Massilia timonae Massilia timonae
8 Vitreous NA Massilia timonae Massilia timonae
19 Acrylic hydrophobic Absent No 9 Aqueous NA NA NA
10 Vitreous NA NA NA
21 Acrylic hydrophilic Probable Mainly cocci surrounded by ocular tissue remains 11 Aqueous NA NA NA
12 Vitreous NA NA NA
24 Acrylic hydrophilic Probable Mainly cocci and few rods 13 Aqueous NA NA NA
14 Vitreous NA Propionibacterium acnes Propionibacterium acnes
27 PMMA Probable Mainly cocci and few rods 15 Aqueous NA Moraxella catharralis Moraxella catharralis
16 Vitreous NA NA NA
28 PMMA Probable Mainly cocci and few rods 17 Aqueous NA NA NA
18 Vitreous NA Propionibacterium acnes Propionibacterium acnes
30 PMMA Probable Mainly cocci surrounded by ocular tissue remains 19 Aqueous NA Massilia timonae Massilia timonae
20 Vitreous NA Massilia timonae Massilia timonae
45 PMMA Probable Mainly cocci and few rods 21 Aqueous NA NA NA
22 Vitreous NA NA NA
46 PMMA Probable Mainly cocci and few rods 23 Aqueous NA NA NA
24 Vitreous NA NA NA
49 Silicone Probable Mainly cocci and few rods 25 Aqueous NA NA NA
26 Vitreous NA NA NA
53 PMMA Absent No 27 Aqueous NA NA NA
28 Vitreous NA NA NA
65 Acrylic hydrophobic Probable Mainly cocci and few rods 29 Aqueous NA NA NA
30 Vitreous NA NA NA

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Jan 5, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Analysis of Intraocular Lens Biofilms and Fluids After Long-Term Uncomplicated Cataract Surgery

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