Analysis of Intraocular Lens Biofilms and Fluids After Long-Term

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

PALOMA MAZOTERAS, MILENE GONC¸ALVES QUILES, PAULO JOSE´ MARTINS BISPO, ANA LUISA HO¨ FLING-LIMA, ANTONIO C. PIGNATARI, AND RICARDO P. CASAROLI-MARANO

PURPOSE: Postoperative endophthalmitis is a poten- ATARACT REMOVAL AND LENS REPLACEMENT tially sight-threatening complication of cataract surgery. with an artificial intraocular lens (IOL) is the However, the pathophysiological mechanisms are not most frequent surgery performed by ophthalmolo- C1 completely understood. We sought to study and evaluate gists. The most fearsome complication of cataract surgery the intraocular environment (aqueous and vitreous hu- is postoperative endophthalmitis, which is defined as a se- mors), the capsular tissue, and the intraocular lens vere inflammation of both the anterior and posterior seg- (IOL) surfaces of normal eyes after long-term uncompli- ments of the eye secondary to an infectious agent.2 With cated cataract surgery. incidence rates of 0.05%–0.34%,1,3 postoperative DESIGN: Experimental laboratory investigation. endophthalmitis is clinically classified into acute or METHODS: We studied 69 eyes donated for transplanta- chronic, depending on the onset after surgery. Acute tion that had previously undergone cataract surgery with endophthalmitis presents with pain; photophobia; posterior chamber IOL implantation and that had no floaters; reduced vision; an inflamed anterior segment, recorded clinical history of postoperative inflammation. including a variable hypopyon; and vitritis.4 The predom- We assessed the intraocular environment (DNA traces inant clinical feature of chronic or delayed-onset endoph- and biofilm formation) by microbiological evaluation of thalmitis is white plaques on the lens capsule or the IOL intraocular fluids using conventional microbiology and surface, representing collections of organisms.4,5 molecular techniques, including assessment for the pres- Endophthalmitis is associated with recurring episodes of ence of microbes (biofilm formation) on the IOL surface low-grade inflammation that are initially responsive to by scanning electron microscopy and ultrastructural topical steroid treatment, but that become refractive at capsular remnants by transmission electron microscopy. later stages.6 RESULTS: Isolated or aggregated cocci were probable in 18.8% of IOL optic surfaces (n [ 13) studied by scan- Coagulase-negative staphylococci, especially Staphylo- ning electron microscopy, suggesting the presence of bac- coccus epidermidis, and others, such as S aureus and Strepto- terial biofilm. In 3 intraocular fluid samples for IOLs with coccus spp. are currently the most common etiologic agents 6,7 biofilm, we identified 16S rDNA by polymerase chain re- of acute postoperative endophthalmitis. In contrast, action and sequencing. No microbial contamination was indolent organisms are usually responsible for chronic found in intraocular fluids by conventional microbiolog- endophthalmitis, including Propionibacterium acnes and 4,6 ical methods. Corynebacterium spp. However, it is well known that CONCLUSIONS: Our data suggest the possibility of microorganisms can be introduced into the eye from the 1,8 bacterial biofilm formation on the optic surface of patient’s external microbiota during surgery. In fact, IOLs in normal eyes after long-term uncomplicated molecular typing techniques have been used to confirm cataract surgery even in the absence of clinical or that 68%–82% of cases of postoperative endophthalmitis 9,10 subclinical symptoms. (Am J Ophthalmol 2016;169: are caused by ocular and periocular microbiota. 46–57. Ó 2016 Elsevier Inc. All rights reserved.) 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 Supplemental Material available at AJO.com. 11,12 Accepted for publication Jun 6, 2016. against endophthalmitis. From the Department of Surgery (School of Medicine) & Hospital Endophthalmitis is a potentially sight-threatening infec- Clıńic de Barcelona (IDIBAPS) (R.P.C.-M.) and CellTec-UB (P.M., tion.13 Detection of endophthalmitis and identification of R.P.C.-M.), University of Barcelona, Barcelona. Spain; and Special Laboratory of Clinical Microbiology (LEMC) & Laboratory of Ocular the causative agent are therefore essential to ensure that 14 Microbiology, Federal University of Saõ Paulo (UNIFESP), Saõ Paulo, rational treatment is given, yet routine microbiologic Brazil (M.G.Q., P.J.M.B., A.L.H.-L., A.C.P.). diagnosis relies on time-consuming classical culture tech- Inquiries to Ricardo P. Casaroli-Marano, Instituto Clıńic de Oftalmologıá (Hospital Clıńic de Barcelona), Calle Sabino de Arana 1, niques that offer low sensitivity; indeed, the positivity rates E-08028 Barcelona, Spain; e-mail: [email protected] range from 22% to 30% in the aqueous humor and from

46 © 2016 ELSEVIER INC.ALL RIGHTS RESERVED. 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2016.06.012 40% to 69% in the vitreous humor.15 Modern molecular discarded. Donated eyes were evaluated and processed be- techniques—such as polymerase chain reaction (PCR) tween 12 and 36 hours postmortem. Eyes were maintained combined with different post-PCR analyses—now allow in a 0.1 M phosphate-buffered solution (pH 7.4) containing for greater sensitivity and more rapid results7,14,16 and are antibiotics and antifungals (penicillin 100 IU/mL, strepto- particularly useful when only a small amount of material mycin 10 mg/mL, and amphotericin B 2.5 mg/mL) at 4 C is available (eg, aqueous humor) or if the patient has for 8–12 hours and then decontaminated by immersion in previously received antibiotic treatment.14 In addition, 40 mL of 5% povidone-iodine solution for 3 minutes under molecular techniques are promising for the detection of or- a laminar-flow hood. ganisms associated with chronic postoperative endophthalmitis because the samples are often small, difficult to DECONTAMINATION EFFICACY OF 5% POVIDONE- culture, fastidious, or slow growing.16 IODINE: The procedure was performed under a laminar- The mechanism through which postoperative endoph- flow hood. Initially, the eye globe was removed from the thalmitis develops has not been completely elucidated to antimicrobial maintenance solution and rinsed with date. Nevertheless, it is recognized that bacterial adhesion 500 mL of sterile saline solution. A mark was made to to IOLs and intraocular tissues during implantation is divide the globe into 2 equal halves and a sterile swab probably the primary step in the pathogenesis. Then, was taken from half of the limbus and cornea. Next, the cell-to-cell adhesion and the production of extracellular globe was fully immersed in 40 mL of 5% povidone- polysaccharide glycocalyx (slime) on the lens surface, iodine for 3 minutes, removed, and rinsed with 500 mL of followed by the formation of a confluent biofilm structure, sterile saline to remove excess decontaminating agent appear to be relevant.13 Indeed, S epidermidis produces few that could inhibit microorganism growth. A postdeconta- virulence factors, but its success as an opportunistic path- mination sterile swab was then taken from the second ogen is related to its ability to adhere to, and then form a half of the limbus and cornea, taking care not to swab mature biofilm on, the surface of different medical de- the same area as in the pretreatment swab to avoid false- vices.17 Finally, although numerous in vitro studies have negative results. Pre- and postdecontamination swabs demonstrated that bacterial adhesion may be influenced were rotated and soaked onto different culture media. by IOL biomaterials, differences in experimental condi- The incubation conditions are summarized in tions and protocols have meant that results differ.13 Supplemental Table 1 (Supplemental Material available Despite advances in our knowledge, little is known about at AJO.com). Isolates were identified by standard microbi- the intraocular environment and behavior of the IOL sur- ological methods. The effectiveness of decontamination face in the long term after uncomplicated cataract surgery. was determined by calculating the following: (1) the per- Thus, we aimed to analyze the intraocular fluids (aqueous centage reduction of positive ocular surfaces after decon- and vitreous humors), capsular tissues, and IOL surfaces tamination and (2) the percentage reduction in the of normal eyes donated for corneal transplantation after number of each bacterial species after decontamination. long-term uncomplicated cataract surgery. To our knowledge, this is the first time that biofilm formation has been MICROBIOLOGICAL ANALYSIS OF INTRAOCULAR studied in this setting on a large scale. 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 mL) were collected by anterior chamber limbic paracentesis with a 30 G needle, whereas the vitreous sam- METHODS ples (500 mL) were obtained by pars plana puncture 4 mm EYE BANK SPECIMENS: In this study, we analyzed 69 from the limbic region with a 19 G needle. Samples were donated eyes that had previously undergone cataract sur- divided into 2 aliquots: 1 aliquot per sample was kept gery and posterior chamber IOL implantation. Globes at 80 C for further analysis and the other was inoculated were obtained from the Eye Bank for the Treatment of onto different culture media. Microbiological analysis and Blindness (Centro de Oftalmologıá Barraquer, Barcelona, procedures were carried out as described (Supplemental Spain) after obtaining consent from the family and in Table 1). accordance with the Standardized Rules for Development and Applications of Tissues and Organ Transplants, as LIGHT AND ELECTRON MICROSCOPY STUDIES: Posterior defined in Spanish law. Eyes were donated for corneal chamber IOLs and their corresponding capsule remnants transplantation, but were discarded because they did not were explanted from ocular globes under aseptic conditions. meet the criteria for penetrating or lamellar keratoplasty. A portion of lens capsule and IOLs were immediately fixed in According to the recorded family interview documentation 2% paraformaldehyde þ 2.5% glutaraldehyde solution in a for donation, all eyes underwent cataract surgery without 0.1 M phosphate buffer. Ocular globes with IOL implanted postoperative complications. Globes with suspected or into the sulcus or anterior chamber were excluded. After confirmed histories of postoperative complications were chemical fixation over 12 hours, IOLs were examined with

VOL. 169 BIOFILMS AND CATARACT SURGERY 47 a binocular magnifying loupe, photographed, and classified Amplified products were purified using the Wizard SV into polymethylmethacrylate (PMMA), silicone, and hydro- Gel and PCR Clean-Up System (Promega Corporation, phobic or hydrophilic acrylic lenses. Madison, Wisconsin, USA) and sequenced using the For scanning electron microscopy (SEM), fixed IOLs previously described primers on an automated sequencer were dehydrated in ethanol–water mixtures with (ABI-3730 DNA Analyzer Sequencer System; Applied increasing concentrations of ethanol (50%, 70%, 90%, Biosystems, Thermo Fisher Scientific, London, UK) at 96%, and 100% ethanol by volume). After critical point CRAG (Centre de Recerca Agrogeno`mica, Generalitat drying, samples were stuck on metal holders with double- de Catalunya, Barcelona, Spain). The sequences ob- sided adhesive tape and coated with gold. The IOL surface tained were compared with those available in the observations were performed at 15 kV with an SEM (JEOL GenBank, EMBL, and DDBJ databases with the gapped JSM-840; JEOL Ltd, Tokyo, Japan). The entire IOL surface BLASTN 2.0.5 program obtained from the NCBI infor- was examined for the presence of adherent microorganisms mation server (http://www.ncbi.nlm.nih.gov/BLAST). or biofilm. The minimal percentages to identify genus and species For transmission electron microscopy (TEM), capsule were used as recommended by the CLSI document remnants were abundantly rinsed in phosphate-buffered sa- MM18-A.19 line, postfixed in 1% osmium tetroxide in 0.1 M phosphate- To confirm the 16S rDNA PCR results, molecular anal- buffered solution (pH 7.4) for 1 hour, dehydrated in ysis by real-time quantitative PCR (qPCR) was performed increasing graded concentrations of acetone, and then pro- at LEMC (Escola Paulista de Medicina, Universidad Fed- gressively embedded in resin (Spurr technique) for poly- eral de Sao Paulo, Sao Paulo, Brazil), as previously merization at 60 C. We obtained semi-thin (0.5–1.0 mm) described.20 The universal primers NT-341Fw and 16S- and ultrathin (50–75 nm) sections by conventional ultra- 522Rv were used to amplify a fragment of approximately microtomy (OmU2; Reichert-Jung, Wein, Austria). The 192 bp containing the V3 region (Supplemental semi-thin sections were placed on coverslips and stained Table 2). A primer set and a probe for the b-globin gene with toluidine blue solution, then observed by light micro- was used as the internal control. The qPCR mix (Power scopy (BX61; Olympus R-FTL-T; Olympus America Inc, SYBR Green PCR master mix; Life Technologies) was Center Valley, Pennsylvania, USA) coupled with the pretreated using 0.3 U of RQ1 RNAse-free DNAse (Prom- Olympus DP Controller Program for digital image acquisi- ega) for 30 minutes at 37 C to remove contaminating bac- tion. Ultrathin sections were placed on copper grids (200 terial DNA, followed by 50 minutes at 95 C to inhibit the mesh) and stained with uranyl acetate and lead citrate DNAse activity. Amplification was performed on a real- for conventional TEM (Hitachi 800 MT; Hitachi, Tokyo, time PCR system (model 7500; ABI) with the following Japan). 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 MICROBIOLOGICAL ANALYSIS BY MOLECULAR TECH- 60 seconds. Melting curve analysis was performed after NIQUES: To minimize the possibility of cross-DNA amplification, by heating samples gradually from 68 C to contamination, we performed DNA extraction, PCR 95 C at a rate of 0.5 C/s. The amplification of clinical sam- mixture preparation, and post-PCR analyses in separate ples was performed in the presence of a negative control, rooms, using designated equipment. Total DNA was including all PCR reagents, except the DNA template extracted from intraocular fluids with a DNA purifica- and positive control samples, with 10 ng of S epidermidis tion kit (QIAamp DNA mini kit; Qiagen, Hilden, Ger- DNA. After the reaction, a 15 mL PCR aliquot was purified many) according to the tissue protocol, with a few with a PCR purification kit (QIAquick; Qiagen). DNA 18 modifications. The oligonucleotide primers designed molecules were sequenced in both directions with dye- for panbacterial and panfungal detection have been termination chemistry (Big Dye Terminator; 3000 Genetic 18 described previously, and they produced 590- and analyzer; ABI). The sequences obtained were edited 654-base-pair fragments of 16S and 18S ribosomal (SeqMan; DNASTAR, Madison, Wisconsin, USA) and DNA, respectively (Supplemental Table 2; Supple- searched for similarity in GenBank using the BLAST mental Material available at AJO.com). Positive control (BLASTN algorithm) program with automatically adjusted consistedof10ngofpurifiedEscherichia coli or Candida parameters. The minimal percentages needed to identify albicans DNA for the 16S and 18S reactions, respec- genus and species were used, as recommended by CLSI tively. Sterile purified water was used as a negative con- document MM18-A.19 trol. PCR amplification was carried out in a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer, Emery- STATISTICAL ANALYSIS: Results were compared glob- ville, California, USA) as follows: 35 cycles of 30 sec- ally using the nonparametric Kruskal-Wallis test (SPSS onds at 94 C; 30 seconds at 52 C or 50 C, for for Windows, version 22.0; SPSS Inc, Chicago, Illinois, bacterial and fungal DNA, respectively; and 40 or 45 sec- USA). Then the Mann-Whitney nonparametric test was onds at 68 C for bacterial and fungal DNA, respectively; used to compare pairs of materials. Two-sided P values followed by 7 minutes of final elongation step at 68 C. <_.05 were considered statistically significant.

48 AMERICAN JOURNAL OF OPHTHALMOLOGY SEPTEMBER 2016 RESULTS TABLE 1. Prevalence of Suspected Bacterial Biofilm DECONTAMINATION PROCEDURE EFFICACY: The effi- Formation on the Intraocular Lens Surface by Scanning cacy of chemical decontamination with 5% povidone- Electron Microscopy iodine was controlled (n ¼ 20). Initial treatment with Total Absent Probable the antimicrobial maintenance solution eliminated the external microbiota in half of the globes (n ¼ 10) selected IOL Material N N (%) N (%) for decontamination control efficacy. The microbial flora PMMA 20 13 (65.0) 7 (35.0) found on the ocular surface before treatment was similar Hydrophilic acrylic 26 23 (88.5) 3 (11.5) to the microbial flora found in the conjunctiva and eyelids Hydrophobic acrylic 16 15 (93.8) 1 (6.3) of healthy eyes.21 More than 1 species was isolated in most Silicone 7 5 (71.4) 2 (28.6) studied globes; commensal species predominated, with 7 TOTAL 69 56 (81.2) 13 (18.8) isolates of coagulase-negative staphylococci (33.3%), 6 IOL ¼ intraocular lens; PMMA ¼ polymethylmethacrylate. 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 spherical- and rod-shaped structures (Figure 4). In addi- solution completely decontaminated 90.0% of ocular tion, some IOLs presented with a few isolated bacteria globes (n ¼ 9), with an efficacy of 95.2% in eliminating while others were part of microcolonies or clusters of commensal species, including the most prevalent ones different sizes (Figure 4). Finally, some of these struc- (Supplemental Table 3; Supplemental Material available tures exhibited a more or less compacted organization at AJO.com). The 5% povidone-iodine was very effective associated to a probable glycocalyx matrix shrouding at decontaminating the ocular surfaces of globes donated the bacteria (Figure 4). In contrast to the results of for transplantation. in vitro studies, the characteristics of the biofilm could not be attributed to a specific IOL biomaterial. Howev- MICROBIOLOGICAL ANALYSIS OF INTRAOCULAR er, as we had previously observed,22 biofilm was predom- FLUIDS: Microorganisms were not detected in either the inantly detected at the periphery of the IOL, regardless ¼ aqueous or vitreous humor samples (n 69 per intraocular of the biomaterial studied. fluid) cultured by conventional microbiologic techniques. TEM was only performed on capsule remnants corresponding to IOLs with surface structures compatible with LIGHT AND ELECTRON MICROSCOPY STUDIES: IOLs biofilm on SEM (n ¼ 13). This approach was less effective observed by binocular magnifying loupe were classified ac- for localizing bacterial structures, certainly for ultrastruc- cording to the biomaterial of the lens (PMMA, silicone, tural evaluation with ultrathin sections. TEM studies hydrophobic acrylic, or hydrophilic acrylic). When showed some areas of compacted and vacuolated cell analyzed by SEM, most of the IOLs showed cellular epithe- debris, with abundant sections of interdigitated lens fibers lial aggregates and crystalline fiber remnants. Bacterial bio- (Figure 1). Fragments of lens capsule, cell remnants, and ¼ film was absent in 81.2% (n 56) and probable in 18.8% epithelial-like cells were also observed, probably corre- ¼ (n 13) of IOL surfaces studied (Table 1). IOLs classified sponding to fragments of anterior lens capsule (Figures 2 as having probable biofilm showed isolated or aggregated and 3). Several of these fragments were associated with m cocci (about 1 m) on the surfaces of 35.0% of PMMA electron-dense spherical structures (Figures 2 and 3). In ¼ ¼ IOLs (n 7), 28.6% of silicone IOLs (n 2), 11.5% of the semi-thin sections, these structures were better identi- ¼ acrylic hydrophilic IOLs (n 3), and 6.3% of acrylic hy- fied, firmly adhered to lens capsule, and were occasionally ¼ drophobic IOLs (n 1) (Figures 1–4). Significant associated with pigmented granules (Figures 2 and 3). In difference in the prevalence of cocci on acrylic some localized areas (posterior lens capsule and lens fiber < hydrophobic biomaterial and PMMA (P .042) was remnants) deposits of electron-dense structures were pre- present. Interestingly, probable biofilm formation was also sent with high affinity to toluidine blue (Figures 1–3). more evident in the periphery of all IOL optic surfaces (Figures 1–4). In 4 of the 13 IOLs, the cocci were MOLECULAR ANALYSIS OF INTRAOCULAR FLUIDS AND embedded by some extracellular debris (Figure 4). The CAPSULES: Molecular analysis was carried out in samples prevalence of suspected biofilm formation on the surface of intraocular fluids (aqueous and vitreous humors) for all of IOLs is summarized in Table 2. IOLs that showed structures compatible with probable bio- Different characteristics were observed in the struc- film (n ¼ 13) on SEM and for a couple of IOLs in which tures compatible with biofilm, including the composi- biofilm was absent. The 18S PCR showed no DNA ampli- tion, number of microorganisms, and degree of fication, but the 16S PCR detected microorganisms in 3 adhesion. Some of them were composed of only 1 samples in which the IOLs had structures compatible apparent microbial morphology while others had mixed with biofilm on their surface by SEM. Specifically, P acnes

VOL. 169 BIOFILMS AND CATARACT SURGERY 49 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 mm for Top right, upper image; 25 mm for Top right, lower image; 10 mm for Middle left, Bottom right, and Bottom left images; 1 mm for Middle right image.

50 AMERICAN JOURNAL OF OPHTHALMOLOGY SEPTEMBER 2016 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 mm for Top right, upper image; 10 mm for Top right, lower image; 10 mm for Middle left, Middle right upper, and Bottom left images; and 5 mm for Bottom right images; 1 mm for Middle right lower image.

VOL. 169 BIOFILMS AND CATARACT SURGERY 51 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 bire- fringent 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) Trans- mission 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 mm for Top right, upper image; 25 mm for Top right, bottom image; 10 mm for Middle left and Bottom left; 5 mm for Bottom right; 1 mm for Middle right.

52 AMERICAN JOURNAL OF OPHTHALMOLOGY SEPTEMBER 2016 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 mm for Top center, Top right, and Middle center; 1 mm for Middle right, Bottom center, and Bottom right.

was detected in 2 samples of vitreous humor and Moraxella lecular analysis approaches. However, because of the high catarrhalis in a sample of aqueous humor (Table 2). sensitivity of PCR, and despite extensive measures to avoid Furthermore, Massilia timonae was identified in the 2 nonspecific microbial DNA amplification, the bacteria samples (aqueous and vitreous) from IOLs with probable were considered to represent environmental contamina- biofilm. These results were comparable between both mo- tion introduced at the time of extraction.

VOL. 169 BIOFILMS AND CATARACT SURGERY 53 54

TABLE 2. Classiﬁcation of Intraocular Lens by Scanning Electron Microscopy and Molecular Analysis

PCR q-PCR Bioﬁlm on IOL Microbial-like Structures on IOL by SEM IOL IOL Material by SEM Observation Observation Sample PCR Sample 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 5 Aqueous NA NA NA ocular tissue remains 6 Vitreous NA NA NA Massilia timonae Massilia timonae

A 16 PMMA Probable Mainly cocci surrounded by 7 Aqueous NA

MERICAN ocular tissue remains 8 Vitreous NA Massilia timonae Massilia timonae 19 Acrylic hydrophobic Absent No 9 Aqueous NA NA NA 10 Vitreous NA NA NA J

UNLOF OURNAL 21 Acrylic hydrophilic Probable Mainly cocci surrounded by 11 Aqueous NA NA NA ocular tissue remains 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 Moraxella catharralis Moraxella catharralis O 27 PMMA Probable Mainly cocci and few rods 15 Aqueous NA

PHTHALMOLOGY 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 19 Aqueous NA Massilia timonae Massilia timonae ocular tissue remains 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 S

PEBR2016 EPTEMBER NA ¼ no specific amplification or sequencing; PCR ¼ polymerase chain reaction; PMMA ¼ polymethylmethacrylate; q-PCR ¼ quantitative polymerase chain reaction; 18S ¼ rDNA, panfungal; 16S ¼ rDNA, panbacterial. DISCUSSION active and high turnover rates, whereas bacteria can remain protected within the biofilm on the IOL surface. THE AIM OF THIS STUDY WAS TO ANALYZE THE INTRAOC- TEM failed to provide much information about either ular environment and the IOL surface after long-term un- biofilm formation or rod- and spherical-shaped cells in complicated cataract surgery. Our large-scale study was the lens capsules. This could be explained by the known made possible by the availability of ocular globes that did limitations of TEM approaches and the low amount of mi- not fulfill the eye bank criteria for biosubstitutive purposes, crobial inoculum. However, TEM has been successfully because of the low endothelial count density or the pres- used in cases of acute or chronic inflammation with high ence of structural alterations and changes in corneal trans- concentrations of microorganisms that are adherent to parency. Most IOLs analyzed by SEM had evidence of surgical-related structures.5 cellular epithelial aggregates and crystalline fiber remnants. In the present study, we also analyzed the microbial flora Interestingly, isolated or aggregated cocci were also on the ocular surface of eyes donated for transplantation observed in 18.8% of the IOLs studied. These structures but that did not fulfill the criteria for keratoplasty. This suggest the presence of a microorganism biofilm colonizing confirmed the efficacy of our decontamination protocol the surface of the IOL, even though no symptoms or epi- with 5% povidone-iodine, which is frequently used in daily sodes of intraocular inflammation were reported by the practice. This approach also guaranteed our further molec- donor families. ular analyses of intraocular fluids by PCR. As previously re- In numerous in vitro studies,8,23–26 it has been shown ported,21 even though globes were kept in an antimicrobial that the characteristics of a biofilm are dependent on the maintenance solution, a high incidence of contamination biomaterial of the IOL. Although the results of these on the surface of donor eyes was observed prior to decon- studies often conflict with each other, the differences tamination. The microbial flora on the surface of ocular may be explained by variations in experimental globes before decontamination was similar to that found conditions, including the strain, time, incubation on the conjunctivae and eyelids of healthy eyes.28 From temperature, and methods used.13 In this study, SEM the 21 microbial isolates that were identified before decon- allowed us to observe different patterns of adhesion, but tamination, only 1 was not effectively removed; thus, the the biofilm characteristics could not be attributed to a spe- effectiveness of decontamination was 95.2%. Several in- cific IOL biomaterial because the conditions differed for vestigators have hypothesized that the patient’s external each patient. Interestingly, the presence of biofilm was flora cause most cases of postoperative endophthalmitis,1,8 mainly detected at the periphery of the IOL optic surfaces emphasizing the need for pre- and postoperative of most specimens. As previously mentioned, we confirmed decontamination procedures to prevent infection.2 The in vitro that there was a significant predisposition for bio- high effectiveness of povidone-iodine in decontaminating film formation at the edges of IOL optic surfaces, indepen- the ocular surface suggests that the solution could be suit- dent of the biomaterial.22 Thus, the inner periphery of the able not only for disinfection of the conjunctival sac prior lens capsule bag could provide protection against biofilm to cataract surgery but also for decontamination of ocular formation (quiescent or not). globes donated for transplantation. Although none of the intraocular fluid samples showed Numerous investigations support the hypothesis that mi- microbial growth by conventional culture methods, ampli- croorganisms routinely enter the anterior chamber during fication of bacterial DNA—or at least the DNA rem- surgery, with irrigation or adherence to the IOL as it is nants—was possible in 21.4% of globes (intraocular inserted through the incision being reported vectors.1 fluids) with probable biofilm formation. This confirms Despite positive culture rates of 0%–46% from aqueous hu- that molecular techniques improve bacterial DNA detec- mor aspirates,1 the incidence of endophthalmitis is very tion by providing a more sensitive and rapid assessment low at 0.05%–0.34%.1,3 Several mechanisms combine to compared with conventional microbiology.7,14,16 This explain why the presence of bacteria does not may be especially so with small samples (especially for automatically lead to clinical endophthalmitis: aqueous humor), with those subject to antibiotic use microorganisms may be present in a low inoculum, may before sampling, where the microorganism has fastidious have a low virulence, or may be poorly adherent to growth requirements, and, of course, where there is polymeric surfaces, making them incapable of colonizing sequestration of bacteria on solid surfaces.14,27 However, the IOL. In most cases, the host immune response is in 2 of the ocular globes analyzed, DNA amplification, therefore sufficient to overcome bacterial infections in sequencing, and database comparison suggested that the their initial phases.1 Indeed, the anterior chamber is identified microorganism was a probable environmental capable of clearing a low bacterial inoculum after cataract contaminant. Nevertheless, the presence of bacteria was surgery without progressing to endophthalmitis, and the less evident in intraocular fluids by PCR than on IOL integrity of the posterior lens capsule itself represents an surfaces by SEM. This might be explained by bacterial important protective barrier against progression. In addi- DNA being removed from the aqueous humor through tion, postoperative topical steroids and antibiotics are

VOL. 169 BIOFILMS AND CATARACT SURGERY 55 routinely prescribed, further reducing the chance of pro- also help to explain the absence of clear symptoms by im- gression to endophthalmitis.1 pairments in the immune response.1 We suggest that a re- Based on the presence of cocci on the surface of IOLs by sidual but inactive biofilm can persist for many years on the SEM and PCR, we suggest that biofilms can develop on surface of the IOL, lens remnant, or capsular tissue. IOLs after cataract surgery without clinical symptoms. To our knowledge, no other studies have investigated This could be explained by microbiological factors. One the intraocular fluids and IOL surfaces of eyes exhaustively possibility is that the inoculum size or virulence may be after long-term uncomplicated cataract surgery from a insufficient to trigger the immune response, allowing bacte- microbiological perspective. Our data suggest the possibil- ria to remain in the eye without leading to clinically rele- ity of bacterial biofilm formation on the surfaces of IOLs vant symptoms. Another possibility is the capacity for explanted from normal eyes after long-term uncomplicated the strain to adhere to the IOL, where intermediately cataract surgery, despite the absence of clinical or subclin- adherent strains persist because they are either too strongly ical symptoms. Further data on bacterial molecular adherent to be cleared by the aqueous humor or insuffi- behavior and anterior chamber environmental circum- ciently adherent to colonize the IOL. These intermediately stances will be necessary for a more accurate and rational adherent microorganisms could therefore be sequestered in understanding of this phenomenon. It is hoped that such a limited and quiescent biofilm on the IOL surface that is progress can ultimately lead to further reductions in the maintained in a balance, thereby avoiding endophthalmi- prevalence of intraocular infection and its consequences af- tis. Patient factors, such as immune status and age, might ter cataract surgery.

FUNDING/SUPPORT: PART OF THIS RESEARCH WAS FUNDED BY A GRANT FROM FONDOS DE INVESTIGACIONES SANITA´ RIAS (PI09/0992), Instituto de Salud Carlos III (Madrid, Spain). Financial disclosures: The following authors have no financial disclosures: Paloma Mazoteras, Milene Gonçalves Quiles, Paulo Jose´ Martins Bispo, Ana Luisa Ho¨fling-Lima, Antonio C. Pignatari, and Ricardo P. Casaroli-Marano. All authors attest that they meet the current ICMJE criteria for authorship. The authors thank Mrs Olga Riera, Mrs Carme Torrecillas, and Mr Jordi Prats (Eye Bank for Blindness Treatment, Centro de Oftalmologıá Barraquer, Barcelona, Spain); Mrs Eva Prats (Centros Cientı´ficos y Tecnolo´gicos de la Universidad de Barcelona); and Dr Jordi Vila, Dr Jordi Bosch, Mrs Marisa Lo´pez, and Dr Cristina Esteban (CDC, Hospital Clıńic de Barcelona) for providing expert assistance.

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