Different Application Volumes of Ethyl-Cyanoacrylate Tissue Adhesive Can Change Its Antibacterial Effects against Ocular Pathogens In Vitro

This article was downloaded by: [Trinity College Dublin] On: 8 November 2009 Access details: Access Details: [subscription number 785045690] Publisher Informa Healthcare Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Current Eye Research Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713618400 Different Application Volumes of Ethyl-Cyanoacrylate Tissue Adhesive Can Change Its Antibacterial Effects against Ocular Pathogens In Vitro Ivana Lopes Romero a; Tulio Pereira Paiato a; Cely Barreto Silva b; Joao Baptista Nigro Santiago Malta ac; Lycia Mara Jenne Mimica b; H. Kaz Soong c; Richard Yudi Hida ad a Department of Ophthalmology, Santa Casa de Sao Paulo, Sao Paulo, Brazil b Department of Microbiology, Santa Casa de Sao Paulo, Sao Paulo, Brazil c W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA d Department of Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil First Published on: 01 October 2008 To cite this Article Romero, Ivana Lopes, Paiato, Tulio Pereira, Silva, Cely Barreto, Malta, Joao Baptista Nigro Santiago, Mimica, Lycia Mara Jenne, Soong, H. Kaz and Hida, Richard Yudi(2008)'Different Application Volumes of Ethyl-Cyanoacrylate Tissue Adhesive Can Change Its Antibacterial Effects against Ocular Pathogens In Vitro',Current Eye Research,33:10,813 — 818 To link to this Article: DOI: 10.1080/02713680802437692 URL: http://dx.doi.org/10.1080/02713680802437692 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. 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ISSN: 0271-3683 print / 1460-2202 online DOI: 10.1080/02713680802437692 Downloaded By: [Trinity College Dublin] At: 19:13 8 November 2009 Different Application Volumes of Ethyl-Cyanoacrylate Tissue Adhesive Can Change Its Antibacterial Effects against Ocular Pathogens In Vitro Ivana Lopes Romero and Tulio Pereira Paiato Department of Ophthalmology, Santa Casa de Sao Paulo, Sao Paulo, Brazil Cely Barreto Silva Department of Microbiology, Santa Casa de Sao Paulo, Sao Paulo, Brazil Joao Baptista Nigro Santiago Malta Department of Ophthalmology, Santa Casa de Sao Paulo, Sao Paulo, Brazil, and W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA Lycia Mara Jenne Mimica Department of Microbiology, Santa Casa de Sao Paulo, Sao Paulo, Brazil H. Kaz Soong W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, USA Richard Yudi Hida Department of Ophthalmology, Santa Casa de Sao Paulo, Sao Paulo, Brazil, and Department of Ophthalmology, University of Sao Paulo, Sao Paulo, Brazil Received 21 January 2008 Accepted 10 March 2008 Correspondence: Dr. Richard Yudi Hida, Rua Afonso de Freitas, 488 apt 61, Paraiso Sao Paulo, SP Brazil, 04006-052. E-mail: ryhida@mandic.com.br ABSTRACT Purpose: To analyze the antibacterial effects in vitro of ethylcyanoacrylate (EC) tissue adhesive in different application volumes. Methods: Volumes of 4, 6, 8, 10, 12, 14, 25, and 35 μl of EC were applied onto the surface of monolayer cultures of Staphylococcus aureus (ATCC25924), Streptococcus pneumoniae (ATCC49619), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Klebsiella pneumoniae (ATCC13883). The diameter of each EC drop was measured, and the area of the circle of EC (formed after its application onto the monolayer culture) was calculated. The area of the antibacterial inhibitory halo surrounding the drop of EC on the monolayer culture was calculated. The ratio between the area of the EC and of the corresponding inhibitory halo was obtained for each EC volume and for each of the 5 types of bacteria studied. Results: EC volume-dependent inhibitory halos were seen in Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, but not in Pseudomonas aeruginosa or Klebsiella pneumoniae. Conclusion: The in vitro antibacterial effect of EC against Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli varies in a dose-dependent fashion with its volume. No effect was observed against Pseudomonas aeruginosa and Klebsiella pneumoniae. KEYWORDS anti-bacterial agents; bacteria; bacteriological techniques; cyanoacrylate; tissue adhesive INTRODUCTION Cyanoacrylate tissue adhesive solidifies by rapid polymerization in the presence of catalyst, such as water or any weak anionic solution present on surfaces of live tissue.1 With time, the polymer degrades to formaldehyde and the corresponding cyanoacetate. The use of cyanoacrylate in ophthalmology was first described in 1963 in rabbit eyes.2 Since then, main applications of cyanoacrylate have been in the treatment of small sterile or infected corneal perforations,2 corneal thinning,3 corneal melting,4 and in corneal5 or scleral3,6,7 sutureless surgical closure. 813 Downloaded By: [Trinity College Dublin] At: 19:13 8 November 2009 Also, its secondary effects, biocompatibility, and toxicity have been widely studied.8−10 Cyanoacrylate provides structural support and promotes epithelialization of corneal tissue, inhibits inflammatory cell migration,11 promotes vascularization,4−9 and has intrinsic antimicrobial properties.1,9,12 A major use of cyanoacrylate tissue adhesive at our institution has been in the treatment of infected corneal ulcers with stromal melting; therefore, it is important that we quantitatively analyze its antibacterial properties. According to prior studies,1,3,13,14 tissue adhesives with different cyanoacetate chains consistently possess bactericidal effects against gram-positive microorganisms but have, at best, only variable effects against gramnegative microorganisms. Unfortunately, these studies were qualitative and did not attempt to quantitatively establish a dose-response curve. The purpose of this study is to quantitatively correlate the antibacterial effect in vitro of ethylcyanoacrylate (EC) on five types of bacteria, using controlled volumes of EC. MATERIALS AND METHODS The following bacterial strains from the American Type Culture Collection (ATCC) were analyzed: Staphylococcus aureus (ATCC25924), Streptococcus pneu- moniae (ATCC49619), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), and Klebsiella pneumoniae (ATCC13883). They were primarily incubated in a nutrient broth at a temperature of 35◦ C until reaching 0.5 on the McFarland scale (turbidity of bacterial suspension at a population of approximately 1.5 × 108 organisms). The bacteria were then transferred ¨ as monolayer cultures to Muller-Hinton media following the Kirby-Bauer technique, except for the Streptococcus pneumoniae, which was transferred to blood agar media. Volumes of 4, 6, 8, 10, 12, 14, 25, and 35 μl of R EC (Superbonder ; Loctite, Sao Paulo, Brazil) were applied onto the surface of the bacterial cultures using R micropipettes (Eppendorf ) with sterile tips. For each microorganism, thirty samples of a single drop for each volume studied were analyzed, i.e., a total of 150 samples for each volume were analyzed. After 24 hr of incubation of the cultures at 35◦ C, the ratio of the EC (r) and of the inhibitory halo (R) was measured in the specimens (Fig. 1). The radius (R) of the bactericidal inhibitory halo was measured in millimeters from the center of the circle formed by the applied EC, and the halo area (AIH ) was calculated using AIH = πR2 (Fig. 1C). For each volume of EC studied, 110 samples (73.33%) of that particular size drop were chosen FIGURE 1 (A) Muller-Hinton media plate showing inhibitory halos around EC 24 hr after application. (B) Measurement of inhibitory halo ¨ diameter (yellow arrows) and diameter of one drop of 10 μl of EC (black arrow). (C) Inhibitory halo radius (R). White shade shows area of inhibitory halo in mm2 . (D) Radius of one drop of EC (r ). White shade shows the area of one drop of EC in mm2 . I. Lopes Romero et al. 814 Downloaded By: [Trinity College Dublin] At: 19:13 8 November 2009 FIGURE 2 Relationship between inhibitory halo area and EC volume for bacteria studied. randomly for analysis of EC radius (in millimeters) and for calculation of its area (AEC ) using AEC = πr 2 (Fig. 1D). The ratio AIH /AEC was calculated and used as the response parameter in the dose-response graphs (Figs. 2–4). After an additional 24 hr of incubation, bacteria from all specimens that showed inhibitory halos were re-inoculated onto fresh, bacteriafree culture media and re-incubated at 35◦ C for bactericidal analysis. Bactericidal activity was measured for each type of microorganism by calculating the percentage of number of plates with no bacterial growth 48 hr after re-inoculation (Fig. 4). The same individual performed all microbiological procedures. The results were analyzed with the Kruskal-Wallis statistical test (p < 0.05). RESULTS Figure 2 shows the mean of AIH for different volumes applied for each microorganism studied. The average of AIH increase with higher volumes of EC applied in culture media containing Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli. No inhibitory halo was observed on the culture media containing Pseudomonas aeruginosa and Klebsiella pneumoniae, indicating no bactericidal effects of EC in the volumes studied. 815 Figure 3 shows the mean of different volumes applied for each microorganism studied. For Streptococcus pneumoniae, the AIH /AEC did not show statistically significant differences between the volumes studied (p > 0.05). The bactericidal effect was dose independent for Escherichia coli, and AIH /AEC showed a stepwise difference in effect above 25 and 35 μl of applied EC (p < 0.05) when compared to other volumes applied. For Staphylococcus aureus, AIH /AEC showed differences in 8, 10, 12, and 14 μl of EC when compared to AIH /AEC of 4, 6, 25, and 35 μl (p < 0.05). Figure 4 shows the bactericidal effect of EC against its respective microorganism studied when plotted against different applied volumes. The bactericidal effect for Streptococcus pneumoniae was extremely variable for all volumes of EC studied. For Escherichia coli, this effect was observed in all samples above 25 μl of volume. For Streptococcus pneumoniae, this effect was observed in all volumes above 10 μl. The bactericidal activity was not studied for Pseudomonas aeruginosa and Klebsiella pneumoniae, since no inhibition halo was observed for the volumes studied. DISCUSSION Previous studies showed antimicrobial and toxic effects of cyanoacrylate adhesives;1,3,13−15 however, these Antibacterial Effect of Cyanoacrylate using Different Volumes Downloaded By: [Trinity College Dublin] At: 19:13 8 November 2009 FIGURE 3 Relationship between AIH /AEC and EC volumes. prior studies did not include quantitative volume criteria or dose-response analyses. We believe that application in vivo of cyanoacrylate adhesive enough to cover deep corneal ulcers is approximately between 4 and 8 μl. Our study volumes were chosen to specifically investigate this range. Our study showed antimicrobial effects of EC against Staphylococcus aureus, Streptococcus pneumoniae, FIGURE 4 Bactericidal effect (%) for various EC volumes. I. Lopes Romero et al. 816 Downloaded By: [Trinity College Dublin] At: 19:13 8 November 2009 and Escherichia coli, but did not show any effects against Pseudomonas aeruginosa and Klebsiella pneumoniae, suggesting that EC does not have a widein vitro antibacterial spectrum. This confirms previous studies.5,13,14 Volume-dependent bactericidal activities against Staphylococcus aureus and Escherichia coli were observed for the volumes studied. Variability was reduced by analyzing the normalized ratio of AIH /AEC , rather than the more variable diameter and the respective EC volume (Fig. 3). For Streptococcus pneumoniae, low variation of AIH /AEC can be explained by its culture environment, as this microorganism is dependent on the catalase in blood agar media. Staphylococcus aureus is a resistant microorganism showing a higher AIH /AEC ratio with 10 μl, probably because of its efflux pumping system and its drug capturing system, similar to those described in vancomycin-resistant bacteria.16,17 Escherichia coli, a gram-negative microorganism, has an additional external membrane, which probably explains its resistance at low volumes of EC.16,17 Previous studies suggest that polymerization process of cyanoacrylate adhesives could possibly have an influence in the antimicrobial activity against grampositive bacteria.3,14 High temperatures generated by the exothermic reaction of EC polymerization may possibly induce a temporary bactericidal effect. Other studies, however, suggest that the antimicrobial activity could be related to the cyanoacrylate degradation products.18 Formaldehyde, one of these products, for example, destroys cell membranes by denaturing the membrane proteins.19 Toxic degradation products of cyanoacrylate may additionally retard cell growth, alter the physiology, and change the cell membrane structure by inhibiting a specific receptor.20 Our study did not show any inhibitory halo against Pseudomonas aeruginosa and Klebsiella pneumoniae, thus confirming previous studies.12−14,21 The external membrane present in gram-negative microorganisms may act as a protective barrier against cyanoacrylate products.22 Structural variations of this external membrane in different strains may explain the variable behavior of the Escherichia coli, in contrast to other gram-negative microorganisms.23 Bactericidal activity was observed only in volumes higher than 25 μl. Tissue toxicity of cyanoacrylate is inversely proportional to the number of carbon atoms in the alkyl chain. Short-chain cyanoacrylate (less than 4 esters), like methyl-cyanoacrylate and ethyl-cyanoacrylate, have 817 faster polymerization but greater toxicity9,18 Longchain cyanoacrylates, such as butyl-2-cyanoacrylate R (Histoacryl ; B. Braun, Melsungen, Germany), nR butyl-2-cyanoacrylate (Histoacryl Blue ; B. Braun), R and 2-octyl-cyanoacrylate (Dermabond ; Johnson & Johnson, New Brunswick, NJ, USA), are well tolerated because of their reduced toxicity; however, they polymerize slowly and are reabsorbed slowly by biological tissue.18 Its antimicrobial effect can also be inversely proportional to the number of carbon atoms in the alkyl chain; however, other studies should be conducted to further elucidate this relationship. 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