Ocular Immunology & Inflammation, Early Online, 1–5, 2014 ! Informa Healthcare USA, Inc. ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2014.971973
ORIGINAL ARTICLE
An in Vitro Experimental Study on the Antimicrobial Activity of Silicone Oil against Anaerobic Bacteria Ceyhun Arici,
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1
MD
1
, Cengiz Aras, MD1, Hrisi Bahar Tokman, Muzeyyen Mamal Torun, PhD, 3
PhD
2
, and
Cerrahpasa Medical Faculty, Department of Ophthalmology, 2Cerrahpasa Medical Faculty, Department of Medical Microbiology, Istanbul University, Istanbul, Turkey, 3Medical Faculty, Department of Medical Microbiology, Bahcesehir University, Istanbul, Turkey
ABSTRACT Purpose: To investigate the in vitro antimicrobial activity of silicone oil against anaerobic agents, specifically Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium. Method: A 0.5 McFarland turbidity of Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium was prepared, and 0.1 mL was inoculated into 0.9 mL of silicone oil. Control inoculations were performed in anaerobic blood agar and fluid thioglycollate medium without silicone oil. Results: Propionibacterium acnes retained their viability on the 3rd day in the presence of silicone oil. In total, 9.7 106 colonies were enumerated from 1 mL of silicone oil. After a prolonged incubation of 7 days, the number of colonies observed was 9.2 106. The other bacteria disappeared after the 3rd day of incubation in silicone oil. Conclusions: Propionibacterium acnes, which is the most common chronic postoperative endophthalmitis agent, is thought to be resistant to silicone oil. Keywords: Anaerobic bacteria, antimicrobial resistance, antimicrobial susceptibility, endophthalmitis, silicone oil
Endophthalmitis is an uncommon but serious sightthreatening intraocular inflammation caused by a noninfectious process or the introduction of contaminating microorganisms following trauma, surgery, or hematogenous spread from a distant infection site. According to the Endophthalmitis Vitrectomy Study, postoperative endophthalmitis is generally divided into two types, acute and chronic. Acute postoperative endophthalmitis is defined as infections within 6 weeks of surgery, while chronic postoperative endophthalmitis is defined as infections occurring more than 6 weeks following surgery.1 A variety of organisms have been implicated in chronic postoperative endophthalmitis, with
Propionibacterium species accounting for the majority of cases (41–63%).2–4 Propionibacterium acnes is also the most frequently isolated anaerobe in posttraumatic endophthalmitis.5,6 Propionibacterium acnes, a commensal, anaerobic, gram-positive, nonmotile pleomorphic rod, is found on the eyelid skin or on the conjunctiva in healthy individuals.7 Silicone oils are linear, synthetic, organic–inorganic polymers that have a common chain composed of repetitive siloxane (–Si–O–) units. Silicone oil is used in vitreoretinal surgery for internal tamponade, especially in complex cases of rhegmatogenous retinal detachment, if the condition is complicated by proliferative vitreoretinopathy and giant retinal tears.8,9
Received 13 June 2014; revised 17 September 2014; accepted 29 September 2014; published online 29 October 2014 Correspondence: Ceyhun Arici, MD, Olgunlar sokak, No: 6\10, Bakirkoy – Istanbul, Turkey 34145. E-mail: [email protected]
1
2
C. Arici et al.
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The in vitro antimicrobial activity of silicone oil has been tested against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus spp., which are common causative agents of acute endophthalmitis.10,11 To our knowledge, there have been no previous studies on the antimicrobial activity of silicone oil against anaerobic agents. Therefore, in this study, we aimed to investigate the in vitro antimicrobial activity of silicone oil against anaerobic agents, specifically Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium.
MATERIALS AND METHODS Bacterial suspensions with a turbidity of 0.5 McFarland units (1 108 CFU/mL) were prepared with each of our clinical isolates, specifically, Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium, in Schaedler broth enriched with vitamin K1. Then, 0.1 mL of these suspensions was inoculated into tubes with 0.9 mL of silicone oil (1300 centistokes) (Oxane, Chauvin Opsia S.A., France). Additionally, another 0.1 mL was inoculated into tubes containing 10 mL of fluid thioglycollate medium supplemented with hemin as control broth medium, and 0.01 mL was inoculated onto anaerobic blood agar prepared with Brucella agar supplemented with hemin, vitamin K1, and 5% sheep blood as control agar medium. P. acnes (ATCC 11828) and Bacteroides fragilis (ATCC 25285) were used as control strains.12 All tubes containing bacteria and silicone oil or control media were immediately incubated under anaerobic conditions (Anaero-Gen, Oxoid & Mitsubishi Gas Company) in anaerobic jars (Oxoid USA, Columbia, MD) at 37 C for a minimum of 72 h.13 After this incubation period, 0.1 mL was taken from the tubes containing silicone oil and the anaerobic bacteria and was immediately seeded over the surface of anaerobic blood agar prepared with Schaedler agar enriched with vitamin K1 and 5% sheep blood. The agar was then incubated for 72 h at 37 C under anaerobic conditions. Additionally, another 0.1 mL was taken from the same tubes, seeded onto chocolate agar, and incubated for 24 h at 37 C under aerobic conditions to verify and confirm the absence of aerobic bacteria and contaminants. Finally, 0.01 mL was taken from the control tubes of fluid thioglycollate medium without silicone oil, seeded onto anaerobic blood agars, and incubated for 72 h at 37 C under anaerobic conditions. After these incubation periods, if no growth was detected on the chocolate agar, the colony-forming units (CFUs) in anaerobic agars were enumerated.13,14
TABLE 1. CFU/mL of each anaerobic bacteria in three media on the 3rd day. Organisms and media
Mean CFU/3rd day
Propionibacterium acnes, n = 22 SO ABA (control) FTM (control) Peptostreptococcus anaerobius, n = 5 SO ABA (control) FTM (control) Peptostreptococcus spp., n = 19 SO ABA (control) FTM (control) Bacteroides fragilis, n = 3 SO ABA (control) FTM (control) Fuobacterium spp., n = 6 SO ABA (control) FTM (control) Clostridium tertium, n = 6 SO ABA (control) FTM (control)
9.7 106 9.5 106 6.7 108 0 2.1 106 8.1 108 0 1.7 106 7.6 108 0 6.1 106 1.2 108 0 4.1 103 1.3 104 0 4.6 104 2.3 105
SO, silicone oil; ABA, anaerobic blood agar; FTM, fluid thiyoglycolate medium.
RESULTS As shown in Table 1, 22 strains of Propionibacterium acnes retained their viability on the 3rd day in the presence of silicone oil and control media (anaerobic blood agar and fluid thiyoglycolate medium) (Figure 1). A total of 9.7 106 colonies were enumerated from 1 mL of silicone oil. After a prolonged incubation of 7 days, 9.2 106 colonies were observed (Figure 2). Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium disappeared after 3 days of incubation in silicone oil, which was a shorter period than that observed after incubation in anaerobic blood agar and fluid thiyoglycolate medium (Table 1).
DISCUSSION Anaerobes are predominant in the bacterial flora of normal human skin and mucous membranes and are a common endogenous cause of bacterial infections. Because of their fastidious nature, they are difficult to isolate and are often overlooked. The failure to direct therapy against these organisms often leads to clinical failures. Their isolation requires appropriate methods of collection transportation and specimen cultivation. The treatment of anaerobic bacterial infection is complicated by the slow growth of these organisms, Ocular Immunology & Inflammation
Silicone Oil against Anaerobic Bacteria in Vitro
Ocul Immunol Inflamm Downloaded from informahealthcare.com by Nyu Medical Center on 04/23/15 For personal use only.
FIGURE 1. Propionibacterium acnes grew on anaerobic blood agar without silicone oil.
FIGURE 2. Propionibacterium acnes colonies after incubation in silicone oil.
which makes laboratory diagnosis possible only after several days. Additionally, bacterial infections are often polymicrobial in nature, and there is a growing resistance of anaerobic bacteria to antimicrobial agents.15 In the conjunctiva, anaerobic bacteria are as prevalent as aerobic bacteria and normal commensal flora. A chronic, low-grade, delayed, and often recurrent postoperative granulomatous uveitis is the typical presentation of Propionibacterium acnes endophthalmitis. These slow-growing, pleomorphic, gram-positive bacilli often become sequestered in the equatorial regions of the lens capsule, where they may not be accessible by routine culture of intraocular fluids. The most common bacteria isolated was Propionibacterium spp., while Bacteroides spp., anaerobic streptococci, and Clostridium spp. were isolated to a lesser extent.16 Propionibacterium acnes is the most commonly detected (63%2) agent in chronic postoperative endophthalmitis and can also cause acute postoperative endophthalmitis. During the acute stage of infections, bacteria are generally in planktonic form and with accurate and early diagnosis they are generally treatable with antibiotics. In cases where the bacteria succeed in forming a biofilm, the infection can transform into a chronic state due to the resistance of the biofilm to the antimicrobial agent.17 Lohmann !
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et al. emphasized that delayed postoperative endophthalmitis appear from months to years after surgery and Propionibacterium acnes is one of the predominant causative microorganisms that adhere to the intraocular lens and produce biofilm.18 Additionally in a 2011 study it was confirmed by anaerobic culture and histopathology that P. acnes was the cause of a chronic recurrent endophthalmitis in a post-cataract surgery patient.19 However, other factors, such as host characteristics or inoculum size, may affect the clinical course of the disease.7,20 Additionally, anaerobic bacteria also cause a large number of cases of post-traumatic endophthalmitis. The most common bacteria isolated was Propionibacterium acnes, while Bacteroides spp. and Peptostreptococcus spp. were isolated to a lesser extent.5,6,21 The in vitro antimicrobial activity of silicone oil against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus spp. was first tested and reported by Ozdamar et al.10 The authors demonstrated that silicone oil (1300 centistokes) inhibited the growth of bacteria and fungi. Yan and Li11 reported a similar study with silicone oil (5000 centistokes) against the same agents, except Aspergillus spp., and showed that bacteria and fungi survived for a shorter time in silicone oil than in the physiological saline and culture media. The authors hypothesized that the antimicrobial effects of the silicone oil observed in vitro could be due to a reduction in the availability of nutrients necessary for microbial growth, as well as its toxicity.10,11 The antimicrobial effect of silicone oil against anaerobic agents has not been reported in the literature. In our in vitro study, we showed that silicone oil has an antimicrobial activity against Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium. However, silicone oil showed poor antimicrobial activity against Propionibacterium acnes. Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium did not retain their viability after 3 days of incubation with silicone oil. This result may have been caused by nutritional deprivation and/or the toxicity of silicone oil against these bacteria. The lack of nutrients in silicone oil may lead to the growth inhibition and death of the microorganisms. Furthermore, the low-molecular-weight components, which are common impurities in silicone oil,22 as well as the inactivated catalysts remaining in the silicone oil, may contribute to its toxicity.23 Additionally, the affinity of silicone oil to oxygen may have played an additional role in the suppression of the growth of anaerobic bacteria.24 Propionibacterium acnes showed a decrease in CFUs in silicone oil and anaerobic blood agar on the 3rd day
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but retained their viability. Therefore, the incubation period was extended to 7 days. No change in CFUs in the silicone oil was observed, which may have been due to biofilm formation by Propionibacterium acnes. Additionally, Propionibacterium acnes produces propionic acid as a metabolic product, and the chemical effect of propionic acid on silicone oil is not known. This chemical interaction may have contributed to the retention of bacterial viability in the silicone oil. Biofilm formation of Propionibacterium acnes was demonstrated in 2003. Propionibacterium acnes isolates in the biofilm phase of growth have been demonstrated to have considerably greater resistance to various antimicrobial agents than bacteria in the planktonic growth phase.25 Both in vitro and in vivo biofilm formation has been detected,26,27 and the production of lipase, a putative virulence determinant, has been found to be upregulated in the biofilm phase of bacterial growth.27 While biofilm has been visualized by electron microscopy,26 neither its chemical composition, the genes encoding it, nor the factors governing its regulation are currently known. Silicone oil resistance of Propionibacterium acnes was observed in vitro. The pharmacokinetics of intravitreal and systemically administered antibiotics in silicone oil-filled eyes would therefore be of interest to the clinician. Talwar et al.28 evaluated intraocular ciprofloxacin levels after oral administration in silicone oil– filled eyes. Two 750-mg ciprofloxacin doses every 12 h, with the last dose 12 h before surgery, were given to 10 vitrectomized eyes with silicone oil endotamponade and another 10 patients scheduled for vitrectomy for the first time. Although both groups exceeded the minimal inhibitory concentration for 90% of isolates (MIC90) for Propionibacterium acnes, antibiotic levels in silicone oil-filled eyes was higher than levels reached in the vitreous in nonvitrectomized eyes (p = 0.001). Perkins et al.29 found that the vitreous ganciclovir levels at days 21 and 42 were similar in the saline-filled and silicone oil-filled eyes, but that at day 70 the drug levels in the saline-filled eyes were significantly lower than in the silicone oilfilled eyes. Overall, although silicone oil resistance of Propionibacterium acnes was observed in vitro, higher antibiotic levels in silicone oil-filled eyes may be more effective in endophthalmitis treatment, whereas injection of antibiotics into a silicone oil-filled eye may cause retinal toxicity due to unacceptably high concentration in the retrosilicone/preretinal space because antibiotic solutions are water based and immiscible with silicone oil.30 In conclusion, antimicrobial activity of silicone oil against Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium was observed in this study. The resistance of Propionibacterium acnes, which is the major chronic postoperative endophthalmitis agent, against silicone oil even after 7 days of incubation
may lead to suspect associated with the treatment of endophthalmitis with vitrectomy combined with silicone oil filling. Because Propionibacterium acnes showed resistance against the antimicrobial effect of silicone oil, it might be logical not to expect antimicrobial activity when we inject silicone oil in vitrectomy for the treatment of chronic endophthalmitis. Prospective, randomized, and long-term in vitro studies are needed to evaluate the role of silicone oil against Propionibacterium acnes in endophthalmitis management.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES 1. Johnson MW, Doft BH, Kelsey SF, et al. The Endophthalmitis Vitrectomy Study. Relationship between clinical presentation and microbiologic spectrum. Ophthalmology. 1997;104:261–272. 2. Fox GM, Joondeph BC, Flynn HW, et al. Delayed-onset pseudophakic endophthalmitis. Am J Ophthalmol. 1991;111: 163–173. 3. Al-Mezaine HS, Al-Assiri A, Al-Rajhi AA. Incidence, clinical features, causative organisms, and visual outcomes of delayed-onset pseudophakic endophthalmitis. Eur J Ophthalmol. 2009;19:804–811. 4. Shirodkar AR, Pathengay A, Flynn Jr HW, et al. Delayedversus acute-onset endophthalmitis after cataract surgery. Am J Ophthalmol. 2012;153:391–398. 5. Ormerod LD, Paton BG, Haaf J, et al. Anaerobic bacterial endophthalmitis. Ophthalmology. 1987;94:799–808. 6. Sharma T, Gopal L, Parikh S, et al. Endophthalmitis caused by anaerobic bacteria. Indian J Ophthalmol. 1995; 43:191–194. 7. Samson CM, Foster CS. Chronic postoperative endophthalmitis. Int Ophthalmol Clin. 2000;40:57–67. 8. Scott IU, Flynn Jr HW, Azen SP, et al. Silicone oil in the repair of pediatric complex retinal detachments: a prospective, observational, multicenter study. Ophthalmology. 1999;106:1399–1407. 9. Azen SP, Scott IU, Flynn Jr HW, et al. Silicone oil in the repair of complex retinal detachments: a prospective observational multicenter study. Ophthalmology. 1998;105: 1587–1597. 10. Ozdamar A, Aras C, Ozturk R, et al. In vitro antimicrobial activity of silicone oil against endophthalmitis-causing agents. Retina. 1999;19:122–126. 11. Yan H, Li J. An experimental study on antimicrobial activity of silicone oil in vitro. Ophthalmologica. 2008;222: 245–248. 12. Shames R, Satti F, Vellozzi EM, et al. Susceptibilities of Propionibacterium acnes ophthalmic isolates to ertapenem, meropenem, and cefepime. J Clin Microbiol. 2006;44: 4227–4228. 13. Koneman EW, Allen SD, Janda WM, et al. Color Atlas and Text Book of Diagnostic Microbiology, 6th ed. Philadelphia: Lippincott; 2006:893–901. Ocular Immunology & Inflammation
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Silicone Oil against Anaerobic Bacteria in Vitro 14. Jousimies-Somer HR, Summanen P, Citron D, et al. Wadsworth KTL Anaerobic Bacteriology Manual, 6th ed. Belmont, CA: Star; 2002:43–54. 15. Goldstein EJC, Citron DM. Resistance trends in antimicrobial susceptibility of anaerobic bacteria, part I. Clin Microbiol Newslett. 2011;33:1–8. 16. Hall GS, York MK, Church DL. Ocular cultures. In: Garcia LS, Isenberg HD, eds. Clinical Microbiology Procedure Handbook, 3rd ed. Washington, DC: ASM; 2011: 3.10.1–3.10.9. 17. Bjarnsholt T. The role of bacterial biofilms in chronic infections. APMIS Suppl. 2013;121:1–51. 18. Lohmann CP, Linde HJ, Reischl U. Improved detection of microorganisms by polymerase chain reaction in delayed endophthalmitis after cataract surgery. Ophthalmology. 2000;107:1047–1051; discussion 1051–1052. 19. Kao AA, Miller D, Dubovy SR, Flynn HW. Chronic endophthalmitis caused by Propionibacterium acnes: a clinicopathologic report. Retinal Physician, 2011 March. 20. Kresloff MS, Castellarin AA, Zarbin MA. Endophthalmitis. Surv Ophthalmol. 1998;43:193–224. 21. El-Asrar AM, Al-Obeidan SA, Yeboah EA. Late onset posttraumatic Propionibacterium acnes endophthalmitis. Eur J Ophthalmol. 2004;14:442–444. 22. Nakamura K, Refojo MF, Crabtree DV, et al. Ocular toxicity of low-molecular-weight components of silicone and fluorosilicone oils. Invest Ophthalmol Vis Sci. 1991;32: 3007–3020.
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23. Gabel VP, Kampik A, Burkhardt J. Analysis of intraocularly applied silicone oils of various origins. Graefes Arch Clin Exp Ophthalmol. 1987;225:160–162. 24. Dumont E, Andre`s Y, Cloirec PL. Mass transfer coefficients of styrene and oxygen into silicone oil emulsions in a bubble reactor. Chem Eng Sci. 2006;61:5612–5619. 25. Ramage G, Tunney MM, Patrick S, et al. Formation of Propionibacterium acnes biofilms on orthopaedic biomaterials and their susceptibility to antimicrobials. Biomaterials. 2003;24:3221–3227. 26. Bayston R, Ashraf W, Barker-Davies R, et al. Biofilm formation by Propionibacterium acnes on biomaterials in vitro and in vivo: impact on diagnosis and treatment. J Biomed Mater Res A. 2007;81:705–709. 27. Coenye T, Peeters E, Nelis HJ. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Microbiol. 2007;158: 386–392. 28. Talwar D, Kulkarni A, Azad R, et al. Intraocular ciprofloxacin levels after oral administration in silicone oil-filled eyes. Invest Ophthalmol Vis Sci. 2003;44:505–509. 29. Perkins SL, Yang CH, Ashton PA, Jaffe GJ. Pharmacokinetics of the ganciclovir implant in the silicone-filled eye. Retina. 2001;21:10–14. 30. Zimmer-Galler IE, Santos A, Haller JA, Campochiaro PA. Management of endophthalmitis in a silicone oil-filled eye. Retina. 1997;17:507–509.
ORIGINAL ARTICLE
An in Vitro Experimental Study on the Antimicrobial Activity of Silicone Oil against Anaerobic Bacteria Ceyhun Arici,
Ocul Immunol Inflamm Downloaded from informahealthcare.com by Nyu Medical Center on 04/23/15 For personal use only.
1
MD
1
, Cengiz Aras, MD1, Hrisi Bahar Tokman, Muzeyyen Mamal Torun, PhD, 3
PhD
2
, and
Cerrahpasa Medical Faculty, Department of Ophthalmology, 2Cerrahpasa Medical Faculty, Department of Medical Microbiology, Istanbul University, Istanbul, Turkey, 3Medical Faculty, Department of Medical Microbiology, Bahcesehir University, Istanbul, Turkey
ABSTRACT Purpose: To investigate the in vitro antimicrobial activity of silicone oil against anaerobic agents, specifically Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium. Method: A 0.5 McFarland turbidity of Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium was prepared, and 0.1 mL was inoculated into 0.9 mL of silicone oil. Control inoculations were performed in anaerobic blood agar and fluid thioglycollate medium without silicone oil. Results: Propionibacterium acnes retained their viability on the 3rd day in the presence of silicone oil. In total, 9.7 106 colonies were enumerated from 1 mL of silicone oil. After a prolonged incubation of 7 days, the number of colonies observed was 9.2 106. The other bacteria disappeared after the 3rd day of incubation in silicone oil. Conclusions: Propionibacterium acnes, which is the most common chronic postoperative endophthalmitis agent, is thought to be resistant to silicone oil. Keywords: Anaerobic bacteria, antimicrobial resistance, antimicrobial susceptibility, endophthalmitis, silicone oil
Endophthalmitis is an uncommon but serious sightthreatening intraocular inflammation caused by a noninfectious process or the introduction of contaminating microorganisms following trauma, surgery, or hematogenous spread from a distant infection site. According to the Endophthalmitis Vitrectomy Study, postoperative endophthalmitis is generally divided into two types, acute and chronic. Acute postoperative endophthalmitis is defined as infections within 6 weeks of surgery, while chronic postoperative endophthalmitis is defined as infections occurring more than 6 weeks following surgery.1 A variety of organisms have been implicated in chronic postoperative endophthalmitis, with
Propionibacterium species accounting for the majority of cases (41–63%).2–4 Propionibacterium acnes is also the most frequently isolated anaerobe in posttraumatic endophthalmitis.5,6 Propionibacterium acnes, a commensal, anaerobic, gram-positive, nonmotile pleomorphic rod, is found on the eyelid skin or on the conjunctiva in healthy individuals.7 Silicone oils are linear, synthetic, organic–inorganic polymers that have a common chain composed of repetitive siloxane (–Si–O–) units. Silicone oil is used in vitreoretinal surgery for internal tamponade, especially in complex cases of rhegmatogenous retinal detachment, if the condition is complicated by proliferative vitreoretinopathy and giant retinal tears.8,9
Received 13 June 2014; revised 17 September 2014; accepted 29 September 2014; published online 29 October 2014 Correspondence: Ceyhun Arici, MD, Olgunlar sokak, No: 6\10, Bakirkoy – Istanbul, Turkey 34145. E-mail: [email protected]
1
2
C. Arici et al.
Ocul Immunol Inflamm Downloaded from informahealthcare.com by Nyu Medical Center on 04/23/15 For personal use only.
The in vitro antimicrobial activity of silicone oil has been tested against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus spp., which are common causative agents of acute endophthalmitis.10,11 To our knowledge, there have been no previous studies on the antimicrobial activity of silicone oil against anaerobic agents. Therefore, in this study, we aimed to investigate the in vitro antimicrobial activity of silicone oil against anaerobic agents, specifically Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium.
MATERIALS AND METHODS Bacterial suspensions with a turbidity of 0.5 McFarland units (1 108 CFU/mL) were prepared with each of our clinical isolates, specifically, Propionibacterium acnes, Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium, in Schaedler broth enriched with vitamin K1. Then, 0.1 mL of these suspensions was inoculated into tubes with 0.9 mL of silicone oil (1300 centistokes) (Oxane, Chauvin Opsia S.A., France). Additionally, another 0.1 mL was inoculated into tubes containing 10 mL of fluid thioglycollate medium supplemented with hemin as control broth medium, and 0.01 mL was inoculated onto anaerobic blood agar prepared with Brucella agar supplemented with hemin, vitamin K1, and 5% sheep blood as control agar medium. P. acnes (ATCC 11828) and Bacteroides fragilis (ATCC 25285) were used as control strains.12 All tubes containing bacteria and silicone oil or control media were immediately incubated under anaerobic conditions (Anaero-Gen, Oxoid & Mitsubishi Gas Company) in anaerobic jars (Oxoid USA, Columbia, MD) at 37 C for a minimum of 72 h.13 After this incubation period, 0.1 mL was taken from the tubes containing silicone oil and the anaerobic bacteria and was immediately seeded over the surface of anaerobic blood agar prepared with Schaedler agar enriched with vitamin K1 and 5% sheep blood. The agar was then incubated for 72 h at 37 C under anaerobic conditions. Additionally, another 0.1 mL was taken from the same tubes, seeded onto chocolate agar, and incubated for 24 h at 37 C under aerobic conditions to verify and confirm the absence of aerobic bacteria and contaminants. Finally, 0.01 mL was taken from the control tubes of fluid thioglycollate medium without silicone oil, seeded onto anaerobic blood agars, and incubated for 72 h at 37 C under anaerobic conditions. After these incubation periods, if no growth was detected on the chocolate agar, the colony-forming units (CFUs) in anaerobic agars were enumerated.13,14
TABLE 1. CFU/mL of each anaerobic bacteria in three media on the 3rd day. Organisms and media
Mean CFU/3rd day
Propionibacterium acnes, n = 22 SO ABA (control) FTM (control) Peptostreptococcus anaerobius, n = 5 SO ABA (control) FTM (control) Peptostreptococcus spp., n = 19 SO ABA (control) FTM (control) Bacteroides fragilis, n = 3 SO ABA (control) FTM (control) Fuobacterium spp., n = 6 SO ABA (control) FTM (control) Clostridium tertium, n = 6 SO ABA (control) FTM (control)
9.7 106 9.5 106 6.7 108 0 2.1 106 8.1 108 0 1.7 106 7.6 108 0 6.1 106 1.2 108 0 4.1 103 1.3 104 0 4.6 104 2.3 105
SO, silicone oil; ABA, anaerobic blood agar; FTM, fluid thiyoglycolate medium.
RESULTS As shown in Table 1, 22 strains of Propionibacterium acnes retained their viability on the 3rd day in the presence of silicone oil and control media (anaerobic blood agar and fluid thiyoglycolate medium) (Figure 1). A total of 9.7 106 colonies were enumerated from 1 mL of silicone oil. After a prolonged incubation of 7 days, 9.2 106 colonies were observed (Figure 2). Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium disappeared after 3 days of incubation in silicone oil, which was a shorter period than that observed after incubation in anaerobic blood agar and fluid thiyoglycolate medium (Table 1).
DISCUSSION Anaerobes are predominant in the bacterial flora of normal human skin and mucous membranes and are a common endogenous cause of bacterial infections. Because of their fastidious nature, they are difficult to isolate and are often overlooked. The failure to direct therapy against these organisms often leads to clinical failures. Their isolation requires appropriate methods of collection transportation and specimen cultivation. The treatment of anaerobic bacterial infection is complicated by the slow growth of these organisms, Ocular Immunology & Inflammation
Silicone Oil against Anaerobic Bacteria in Vitro
Ocul Immunol Inflamm Downloaded from informahealthcare.com by Nyu Medical Center on 04/23/15 For personal use only.
FIGURE 1. Propionibacterium acnes grew on anaerobic blood agar without silicone oil.
FIGURE 2. Propionibacterium acnes colonies after incubation in silicone oil.
which makes laboratory diagnosis possible only after several days. Additionally, bacterial infections are often polymicrobial in nature, and there is a growing resistance of anaerobic bacteria to antimicrobial agents.15 In the conjunctiva, anaerobic bacteria are as prevalent as aerobic bacteria and normal commensal flora. A chronic, low-grade, delayed, and often recurrent postoperative granulomatous uveitis is the typical presentation of Propionibacterium acnes endophthalmitis. These slow-growing, pleomorphic, gram-positive bacilli often become sequestered in the equatorial regions of the lens capsule, where they may not be accessible by routine culture of intraocular fluids. The most common bacteria isolated was Propionibacterium spp., while Bacteroides spp., anaerobic streptococci, and Clostridium spp. were isolated to a lesser extent.16 Propionibacterium acnes is the most commonly detected (63%2) agent in chronic postoperative endophthalmitis and can also cause acute postoperative endophthalmitis. During the acute stage of infections, bacteria are generally in planktonic form and with accurate and early diagnosis they are generally treatable with antibiotics. In cases where the bacteria succeed in forming a biofilm, the infection can transform into a chronic state due to the resistance of the biofilm to the antimicrobial agent.17 Lohmann !
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et al. emphasized that delayed postoperative endophthalmitis appear from months to years after surgery and Propionibacterium acnes is one of the predominant causative microorganisms that adhere to the intraocular lens and produce biofilm.18 Additionally in a 2011 study it was confirmed by anaerobic culture and histopathology that P. acnes was the cause of a chronic recurrent endophthalmitis in a post-cataract surgery patient.19 However, other factors, such as host characteristics or inoculum size, may affect the clinical course of the disease.7,20 Additionally, anaerobic bacteria also cause a large number of cases of post-traumatic endophthalmitis. The most common bacteria isolated was Propionibacterium acnes, while Bacteroides spp. and Peptostreptococcus spp. were isolated to a lesser extent.5,6,21 The in vitro antimicrobial activity of silicone oil against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, and Aspergillus spp. was first tested and reported by Ozdamar et al.10 The authors demonstrated that silicone oil (1300 centistokes) inhibited the growth of bacteria and fungi. Yan and Li11 reported a similar study with silicone oil (5000 centistokes) against the same agents, except Aspergillus spp., and showed that bacteria and fungi survived for a shorter time in silicone oil than in the physiological saline and culture media. The authors hypothesized that the antimicrobial effects of the silicone oil observed in vitro could be due to a reduction in the availability of nutrients necessary for microbial growth, as well as its toxicity.10,11 The antimicrobial effect of silicone oil against anaerobic agents has not been reported in the literature. In our in vitro study, we showed that silicone oil has an antimicrobial activity against Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium. However, silicone oil showed poor antimicrobial activity against Propionibacterium acnes. Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium did not retain their viability after 3 days of incubation with silicone oil. This result may have been caused by nutritional deprivation and/or the toxicity of silicone oil against these bacteria. The lack of nutrients in silicone oil may lead to the growth inhibition and death of the microorganisms. Furthermore, the low-molecular-weight components, which are common impurities in silicone oil,22 as well as the inactivated catalysts remaining in the silicone oil, may contribute to its toxicity.23 Additionally, the affinity of silicone oil to oxygen may have played an additional role in the suppression of the growth of anaerobic bacteria.24 Propionibacterium acnes showed a decrease in CFUs in silicone oil and anaerobic blood agar on the 3rd day
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C. Arici et al.
but retained their viability. Therefore, the incubation period was extended to 7 days. No change in CFUs in the silicone oil was observed, which may have been due to biofilm formation by Propionibacterium acnes. Additionally, Propionibacterium acnes produces propionic acid as a metabolic product, and the chemical effect of propionic acid on silicone oil is not known. This chemical interaction may have contributed to the retention of bacterial viability in the silicone oil. Biofilm formation of Propionibacterium acnes was demonstrated in 2003. Propionibacterium acnes isolates in the biofilm phase of growth have been demonstrated to have considerably greater resistance to various antimicrobial agents than bacteria in the planktonic growth phase.25 Both in vitro and in vivo biofilm formation has been detected,26,27 and the production of lipase, a putative virulence determinant, has been found to be upregulated in the biofilm phase of bacterial growth.27 While biofilm has been visualized by electron microscopy,26 neither its chemical composition, the genes encoding it, nor the factors governing its regulation are currently known. Silicone oil resistance of Propionibacterium acnes was observed in vitro. The pharmacokinetics of intravitreal and systemically administered antibiotics in silicone oil-filled eyes would therefore be of interest to the clinician. Talwar et al.28 evaluated intraocular ciprofloxacin levels after oral administration in silicone oil– filled eyes. Two 750-mg ciprofloxacin doses every 12 h, with the last dose 12 h before surgery, were given to 10 vitrectomized eyes with silicone oil endotamponade and another 10 patients scheduled for vitrectomy for the first time. Although both groups exceeded the minimal inhibitory concentration for 90% of isolates (MIC90) for Propionibacterium acnes, antibiotic levels in silicone oil-filled eyes was higher than levels reached in the vitreous in nonvitrectomized eyes (p = 0.001). Perkins et al.29 found that the vitreous ganciclovir levels at days 21 and 42 were similar in the saline-filled and silicone oil-filled eyes, but that at day 70 the drug levels in the saline-filled eyes were significantly lower than in the silicone oilfilled eyes. Overall, although silicone oil resistance of Propionibacterium acnes was observed in vitro, higher antibiotic levels in silicone oil-filled eyes may be more effective in endophthalmitis treatment, whereas injection of antibiotics into a silicone oil-filled eye may cause retinal toxicity due to unacceptably high concentration in the retrosilicone/preretinal space because antibiotic solutions are water based and immiscible with silicone oil.30 In conclusion, antimicrobial activity of silicone oil against Peptostreptococcus spp., Peptostreptococcus anaerobius, Bacteroides fragilis, Fuobacterium spp., and Clostridium tertium was observed in this study. The resistance of Propionibacterium acnes, which is the major chronic postoperative endophthalmitis agent, against silicone oil even after 7 days of incubation
may lead to suspect associated with the treatment of endophthalmitis with vitrectomy combined with silicone oil filling. Because Propionibacterium acnes showed resistance against the antimicrobial effect of silicone oil, it might be logical not to expect antimicrobial activity when we inject silicone oil in vitrectomy for the treatment of chronic endophthalmitis. Prospective, randomized, and long-term in vitro studies are needed to evaluate the role of silicone oil against Propionibacterium acnes in endophthalmitis management.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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