S. KIRMUSAOĞLU, S. YURDUGÜL, M. E. KOÇOĞLU Turk J Med Sci 2012; 42 (4): 689-694 © TÜBİTAK Original Article E-mail: [email protected] doi:10.3906/sag-1103-4

Th e eff ect of N-acetylcysteine on growth and biofi lm formation in Staphylococcus epidermidis strains

Sahra KIRMUSAOĞLU1, Seyhun YURDUGÜL1, M. Esra KOÇOĞLU2

Aim: To examine the possible preventive eff ect of N-acetylcysteine (NAC) on biofi lm formation in Staphylococcus epidermidis strains, which cause very important problems such as implant-associated infections in hospitalized and immunocompromised patients worldwide. Materials and methods: Th e eff ects of varying concentrations of NAC on the formation of biofi lm by 28 strains of Staphylococcus epidermidis was investigated spectrophotometrically by micro-ELISA assay. Results: Signifi cant diff erences in biofi lm formation in the presence of various NAC concentrations were found in 15 methicillin-resistant Staphylococcus epidermidis and 13 methicillin-sensitive Staphylococcus epidermidis strains (P < 0.05). Conclusion: It was found that slime formation was decreased due to the increased concentrations of the NAC treatments. Based on these fi ndings, it is possible to use NAC as an antibiofi lm agent on medical devices or in catheter lock solutions to prevent colonization and implant-associated infections.

Key words: Staphylococcus epidermidis, biofi lm formation, indwelling device-associated infections, N-acetylcysteine

N-asetilsisteinin Staphylococcus epidermidis suşlarında üreme ve biyofi lm oluşturma üzerine etkisi

Amaç: Bu çalışmanın amacı N-asetilsisteinin (NAC) hastaneye yatmış ve bağışıklık sistemi baskılanmış hastalarda, tüm dünyada implant ile ilişkili infeksiyonlar gibi çok önemli sorunlara neden olan Staphylococcus epidermidis suşlarında biyofi lm oluşumundaki olası önleyici etkilerini incelemekti. Yöntem ve gereç: NAC’nin çeşitli konsantrasyonlarının 28 Staphylococcus epidermidis suşu tarafından oluşturulan biyofi lm üzerine etkisi mikroelisa yöntemiyle spektrofotometrik olarak test edildi. Bulgular: NAC’nin çeşitli konsantrasyonlarının varlığında 15 metisilin dirençli Staphylococcus epidermidis ve 13 metisilin duyarlı Staphylococcus epidermidis suşlarında biyofi lm oluşturma üzerinde anlamlı farklılıklar bulundu (P < 0,05). Sonuç: NAC’nin artan konsantrasyonlarından dolayı yapışkan tabaka oluşumunda azalma görüldü. Bu bulgulara göre, kolonizasyonu ve yabancı cisim infeksiyonlarını önlemek için NAC’ in medikal aletlerde ya da kateter solüsyonlarında antibiyofi lm ajan olarak kullanımı mümkündür.

Anahtar sözcükler: Staphylococcus epidermidis, biyofi lm oluşumu, yabancı cisim infeksiyonları, N-asetilsistein

Received: 02.03.2011 – Accepted: 22.07.2011 1 Department of Biology, Faculty of Science, Abant İzzet Baysal University, 14280 Bolu - TURKEY 2 Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Abant İzzet Baysal University, 14280 Bolu - TURKEY Correspondence: Sahra KIRMUSAOĞLU, Department of Biology, Faculty of Science, Abant İzzet Baysal University, 14280 Bolu - TURKEY E-mail: [email protected]

689 Eff ect of N-acetylcysteine on Staphylococcus epidermidis

Introduction in chronic obstructive pulmonary disease and Th e placement of indwelling medical devices into the nephropathy has been reported (22). NAC, which human body for supporting and restoring function is a glutathione precursor, provides neuroprotection has become common practice in medicine (1). by preventing oxidative damage (22,23). In addition, Indwelling device-associated infections demonstrate NAC is also used in the treatment of chronic serious complications of high signifi cance with bronchitis, cancer, and paracetamol intoxication related high morbidity and mortality rates, and also (24,25). Acetylcysteine has been used to treat aspergilloma by local installation (26). NAC has with high associated healthcare costs (2). been found to reduce biofi lm formation by a variety S. epidermidis is one of the most frequent causes of bacteria (21,27,28) and promote the disruption of of nosocomial infections (3) and the most common mature biofi lm (27,28). source of infections of indwelling medical devices such as intravascular catheters, cerebrospinal fl uid Materials and methods shunts, prosthetic heart valves, peritoneal dialysis catheters, and prosthetic joints. S. epidermidis, Bacteria which is a colonizer of human skin, can cause Fift een MRSE and 13 methicillin-sensitive S. contamination of medical devices during insertion epidermidis (MSSE) isolates from samples from (4). Th e biofi lm is broadly protective against host patients who visited the microbiology laboratory defenses (1). Infections of medical prosthetic devices of Abant İzzet Baysal University Hospital (Faculty in trauma and orthopedic surgery, which are diffi cult of Medicine, Abant İzzet Baysal University, Bolu, to treat with conventional therapies like antibiotics Turkey) were used as test microorganisms. and surgery (5), may lead to removal of the implant All of the isolates were identifi ed as S. epidermidis to destroy biofi lm-associated infections (6) and may according to colonial and microscopic morphology, lead to removal of the implant with function loss of positive catalase, and negative coagulase in the the aff ected limb (7,8). Th e prevalence of methicillin- laboratory. All of the isolates were tested for resistant S. epidermidis (MRSE) strains (9-11) and the methicillin resistance. Th e disk diff usion method was emergence of vancomycin-resistant S. epidermidis used with a 1-μg oxacillin disk (Oxoid, Basingstoke, (VRSE) further complicate the treatment of medical UK). Isolates with zone sizes of ≤10 mm were prosthetic device infections (12,13). considered methicillin-resistant. Antibiofi lm activities of antibiotics or biocides Treatment with NAC combined with biofi lm-specifi c agents such as NAC was added to tryptic soy broth (TSB) (Merck, N-acetylcysteine (NAC) (14), protamine sulfate (15), Whitehouse Station, NJ, USA) at concentrations of and dispersin B (16), which primarily target the 0.03, 0.12, 0.5, and 2.0 mg/mL. Each concentration extracellular matrix of biofi lms, have been examined. of NAC was analyzed separately. Isolates were Many drugs and compounds have been tested as inoculated into NAC-treated TSB and nontreated biofi lm inhibitors and some of these (e.g., silver, TSB as a control, which were then incubated at 37 °C minocycline, rifampin, platinum, nitrofurantoin, for 24 h in the incubator. Th e process was repeated chlorhexidine, and sulfadiazine) are used to coat in triplicate. catheters (17-20). Qualitative determination of the slime NAC, which is a nonantibiotic drug, has antibacterial properties. It is a mucolytic agent that Congo red agar method disrupts disulfi de bonds in mucus and, for this reason, For screening biofi lm formation by S. aureus, bacteria it reduces the viscosity of secretions (21). In recent were grown on Congo red agar (Merck) as described years, the actions of NAC in clinical applications have by Freeman et al. (1989) (29). Colony morphology also broadened. NAC is widely used as a mucolytic was examined aft er holding at 37 °C for 24 h. A agent and in the treatment of HIV, and its effi cacy positive result was indicated by black colonies.

690 S. KIRMUSAOĞLU, S. YURDUGÜL, M. E. KOÇOĞLU

Tube method of 0.03, 0.12, 0.5, and 2.0 mg/mL to determine the Th e case study was verifi ed by an assay, in which eff ects of NAC on the slime production of the isolates. the biofi lm formation by bacteria was additionally Determination of the slime index detected by another method described by Christensen Following the 24-h incubation of the isolates, which et al. (1985). S. epidermidis isolates were inoculated in were treated with diff erent concentrations of NAC, a polystyrene test tube that contained TSB and were the growth of S. epidermidis was confi rmed with a then incubated at 37 °C for 24 h in the incubator (30). micro-ELISA reader instrument (Th ermo Fisher). Th e biofi lms that formed on the walls of the polystyrene Th e OD value of the biofi lm corresponded with test tube were washed twice with phosphate buff ered saline (PBS) to remove the planktonic cells. Th e the OD value of the bacterial growth determined cells were then stained with safranin for 1 h. Aft er spectrophotometrically, before the aspiration of discarding the safranin, the polystyrene test tube was the culture, in order to compensate for the partial washed twice with PBS and the process of air-drying inhibition in growth caused by the NAC, and this was the polystyrene test tube was conducted. If a visible called the slime index (SI). Th e result was expressed fi lm had lined the walls of the tube, slime production as a percentage relative to the control without NAC. could be confi rmed (30). Th e adherent bacterial fi lms For this purpose, the following formula was applied: were measured spectrophotometrically at 540 nm in SI = 100 × (mean density of biofi lm with treatment a microplate reader (Th ermo Fisher, Waltham, MA, / mean growth with treatment) / (mean density of USA). Th is process was repeated with NAC-treated biofi lm without treatment / mean growth without TSB at concentrations of 0.03, 0.12, 0.5, and 2.0 mg/ treatment) (21). mL to determine the eff ects of the NAC on the slime Statistical analysis production of the isolates. Th e procedures were repeated in triplicate. Th e Friedman test was used to determine the existence of diff erences in the diff erent concentrations of NAC. Quantitative determination of the slime Th e comparison between the diff erent concentrations Nontreated TSB was used for the control. TSB of NAC was carried out with the 2 related sample supplemented with the diff erent concentrations tests (Wilcoxon test) where signifi cant diff erences of NAC was also used. Th e optical density (OD) were present. At the beginning of the study, P < 0.05 value of the inoculum was approximately 0.600 nm, was set as statistically signifi cant. as calculated with a spectrophotometer (Hitachi, Tokyo, Japan), and 200 μL of bacterial suspension that contained TSB was inoculated into a 96-well fl at- Results bottomed sterile polystyrene microplate (LP Italiana, In this study, 28 investigated strains of S. epidermidis Milan, Italy). Some wells were left free of NAC as were found to be biofi lm-producing. Th e results controls and the wells were incubated for 24 h at 37 determined by spectrophotometric assay for growth °C. and biofi lm formation in the presence of diff erent Th e biofi lm formation by bacteria was detected by concentrations of NAC are shown in Table 1. the method described by Christensen et al., as follows (30). Th e biofi lms that formed on the plates were It was found that there were signifi cant diff erences washed twice with PBS to remove the planktonic in biofi lm formation and growth among the diff erent cells. Th e cells were then stained with safranin concentrations of NAC (P < 0.05). In addition, there (EMD Chemicals Inc., Gibbstown, NJ, USA) for 1 h. were signifi cant diff erences in the biofi lm formation of Aft er removal of safranin from the microplate, the MSSE and MRSE among the diff erent concentrations microplate was washed twice with PBS, followed by of NAC. Th e decrease in the OD of the biofi lms was the air-drying of the wells. Adherent bacterial fi lms probably in direct relation to the NAC concentration were measured spectrophotometrically at 540 nm in a (Table 1). NAC applied at 4 diff erent concentrations microplate reader (Th ermo Fisher). Th is process was showed the same eff ect on the biofi lm formation and repeated with NAC-treated TSB at concentrations growth of MSSE and MRSE (P < 0.05).

691 Eff ect of N-acetylcysteine on Staphylococcus epidermidis

Table 1. Friedman test results showing the eff ects of diff erent concentrations of NAC on the growth and biofi lm formation of 28 isolates.

NAC (mean ± SD) df N P 0.03 mg/mL 0.12 mg/mL 0.5 mg/mL 2.0 mg/mL SI 84.22 ± 26.04 76.90 ± 22.41 72.00 ± 2.42 76.96 ± 3.88 3 28 0.019* Slime 79.72 ± 21.50 73.65 ± 26.14 66.00 ± 2.11 56.47 ± 2.07 3 28 0.000* Growth 98.35 ± 26.83 95.57 ± 23.61 88.24 ± 1.75 77.02 ± 2.05 3 28 0.000*

*P < 0.05

At 4 diff erent concentrations of NAC, 28 strains growth were the same at the concentrations of 0.03 were found to decrease the biofi lm formation, and and 0.12 mg/mL, and were less than the inhibitory NAC diminished the biofi lm formation by 44% at a eff ects of the concentrations of 0.5 and 2.0 mg/mL concentration of 2.0 mg/mL. Th e mean percentage (Tables 1 and 2). of biofi lm of all of the strains relative to the control at a concentration of 0.03, 0.12, 0.5, and 2.0 mg/mL Discussion of NAC was 79.72 ± 21.50%, 73.65 ± 26.14%, 66.00 ± 2.11%, and 56.47 ± 2.07% (P < 0.05), respectively Th e prevalence of MRSE strains (9-11) and the (Table 1). NAC demonstrated a dose-dependent emergence of vancomycin-resistance in these species slime reducing activity at the 4 concentrations of further complicate the treatment of biomaterial NAC (Tables 1 and 2). infections (12,13). Nosocomial staphylococcal foreign-body infections related to biofi lm formation Th e SI indicates that there is a signifi cant diff erence are a serious threat, demanding new and eff ective in biofi lm formation between the concentrations of therapeutic and preventive strategies. 0.03 and 0.5 mg/mL and between the concentrations Several studies have been conducted to decrease of 0.03 and 2.0 mg/mL (P < 0.05) (Table 2). Th erefore, the adherence of several bacteria, such as coagulase- the inhibitory eff ects of NAC on biofi lm formation negative Staphylococcus, on catheters by coating them are the same at the concentrations of both 0.5 and 2.0 with antiseptics and silver (18,20). Th e antibiofi lm mg/mL (P < 0.05) (Table 2), and are higher than the activities of antibiotics combined with NAC (14), inhibitory eff ect of the concentration of 0.03 mg/mL protamine sulfate (15), and dispersin B (16) have (Table 1). been examined in recent studies. NAC was found to At 4 diff erent concentrations, NAC decreased the decrease biofi lm formation by a variety of bacteria growth of 28 strains; this was found to be statistically (21,27,28) and to promote the disruption of mature signifi cant (Table 1). Th e inhibitory eff ects of NAC on biofi lm (27,28).

Table 2. Wilcoxon test results showing the eff ects of diff erent concentrations of NAC on the growth and biofi lm formation of 28 isolates.

Concentrations of NAC (mg/mL-mg/mL) 0.12-0.03 0.5-0.03 2.0-0.03 0.5-0.12 2.0-0.12 2.0-0.5 SI 0.076 0.011* 0.022* 0.200 0.203 0.615 P Slime 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* Growth 0.295 0.002* 0.000* 0.018* 0.000* 0.002*

*P < 0.05

692 S. KIRMUSAOĞLU, S. YURDUGÜL, M. E. KOÇOĞLU

In our study using 0.03, 0.12, 0.5, and 2.0 mg/ indwelling devices to prevent the adhesion of S. mL of NAC, 28 strains lost 20%, 26%, 34%, and epidermidis on the devices. It is very diffi cult to 44% of their capacity to produce biofi lms. Juda et remove an infected device, and the necessity of this al. (19) studied the eff ect of EDTA on the formation may be prevented with the use of NAC. Indwelling of biofi lm by S. epidermidis and showed that the device-associated infections, even by MRSE, may adhesion and formation of the S. epidermidis biofi lm be prevented without using antibiotics or other on polychloride vinyl Nelaton and Th orax catheters chemicals, which may cause the development of was inhibited by EDTA at low concentrations resistance in bacteria. (between 1 and 2 mmol/L) (19). In conclusion, our results suggest that NAC could Our results show that NAC decreases growth- prevent the formation of biofi lms and adherence of S. independent formation of biofi lm, which is a major epidermidis. NAC is a specifi c antibiofi lm compound virulence factor of staphylococcal infections. For and might be used as an antibiofi lm coating on this reason, NAC may be an eff ective alternative for medical devices or in catheter lock solution to preventing indwelling prosthetic infections by S. prevent colonization and infection by biofi lm epidermidis. Th is study has demonstrated that higher pathogens. When incurable indwelling device- doses of NAC lead to lower levels of biofi lm formation. associated infections arise from S. epidermidis, NAC In the presence of NAC at concentrations of 0.03 could be an alternative treatment to antibiotics. In mg/mL or higher, the results become statistically this way, resistance to antibiotics in bacteria could be signifi cant. Four diff erent concentrations of NAC decreased. showed the same eff ect on the biofi lm formation and growth of MSSE and MRSE. It would be appropriate to confi rm these results Acknowledgments by animal and clinical experiments. NAC could Th e authors are grateful to Assist Prof Dr Aysu Kıyan be administered through direct instillation, either for the statistical studies and to Assist Prof Dr Alper intravenously or orally. However, it may be possible Karakaş for the opportunity to use micro-ELISA to obtain useful concentrations by local application in his laboratory. Th e fi rst author is grateful to her to prevent the formation of biofi lms and adherence mother, Nurhan Kırmusaoğlu, for her support in the of S. epidermidis. NAC may be incorporated into laboratory at nights.

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