Biyofilm pozitif Staphylococcus epidermidis icaA gen ekspresyonuna, Lactobacillus acidophilus ATCC 4356 kökeninin etkisinin in-vitro incelenmesi

Abstract

Amaç: S. epidermidis biyofilm oluşumu, bu bakterilerin antibakteriyel ilaçlara ve bağışıklık sistemi savunmasına karşı koruyan önemli bir bariyer görevi oluşturmaktadır. IcaA geni bunun oluşumu için önemlidir. Lactobacillus türlerinin probiyotik etkileri bilinmektedir. Bizde çalışmamızda, CRA yöntemi ile biyofilm pozitifliği saptanan S. epidermidis kökenlerinin icaA gen ekspresyonuna, L. acidophilus ATCC 4356 kökeninin in-vitro etkisinin incelenmesi amaçladık.

Materyal ve metod: Haziran – Aralık 2017 döneminde klinik örneklerden izole edilmiş olan ve CRA metodu ile biyofilm üretimi pozitif olarak saptanan yirmi (20) S. epidermidis kökeni çalışmamıza dahil edildi. CRA’da siyah pozitif kolonilerden, 2 McFarland’lık süspansiyonundan 250 mL alınarak, L. acidophilus içeren ve içermeyen 2 tüpe ilave edildi ve %5CO₂’li ortamda 37^oC’da inkübasyona bırakıldı. Inkübasyonun 6. saatinde, tüplerden RNA izolasyonları gerçekleştirildi. cDNA sentezi sonrası, spesifik primerler ile LightCycler480 sisteminde real-time PCR yöntemi ile çalışıldı. Gruplara ait sonuçlar delta delta Ct yöntemi ile oluşturularak, Mann-Whitney U testi ile istatistiksel olarak incelendi.

Bulgular: L. acidophilus ATCC 4356 etkileşimi sonrasında S. epidermidis kökenlerinde icaA gen ekspresyon seviyesinde gözlenen upregulasyon, istatistiksel olarak anlamlı düzeyde bulundu (p<0.0001).

Sonuç: Sonuç olarak, çalışmamızda, in-vitro olarak, biyofilm yeteneği olan S. epidermidis kökenlerinin probiyotik etkili L. acidophilus ile etkileşimi sonrası icaA gen ekspresyon düzeyinde upregulasyon gösterdiğini saptadık. Diğer probiyotikleri de kapsayacak şekilde daha geniş kapsamlı bakteri-bakteri etkileşimi çalışmalarının yapılarak yeni stratejilerin geliştirilebileceği kanaatindeyiz.

Keywords

• Lactobacillus acidophilus,biyofilm,Staphyloccoccus epidermidis,icaA gen ekspresyonu

Investigation of in-vitro effects of L. acidophilus ATCC 4356 strain on icaA gene expression of biofilm positive Staphylococcus epidermidis

Abstract

Background: The formation of S. epidermidis biofilm is an important barrier to protecting these bacteria against antibacterial drugs and defense of the immune system. IcaA gene is important for the formation of this. The probiotic effects of Lactobacillus species are known. Aim of this study was to investigate the in-vitro effect of L. acidophilus ATCC 4356 genotype on the expression of the icaA gene in S. epidermidis strains, which were found to be positively biofilm with CRA method.

Methods: Between June and December 2017, Twenty (20) S. epidermidis strains isolated from clinical specimens and detected as positive for biofilm production by CRA method were included the study. 250 μL of 2 McFarland suspension of black-positive colonies from the CRA were added to 2 vials containing with and without L. acidophilus and incubation was carried out at 37°C in 5% CO2. At the 6^th hour of incubation, RNA was isolated from these tubes. After cDNA synthesis, real-time PCR was performed on the LightCycler480 system with specific primers. The results of groups by delta delta Ct method were statistically analyzed by Mann-Whitney U test.

Results: The upregulation at the level of icaA gene expression in S. epidermidis strains after L. acidophilus ATCC 4356 interaction was found statistically significant (p <0.0001) according to without L. acidophilus.

Conclusions: In conclusion, our study showed that in-vitro biofilm strains of S. epidermidis showed upregulation of icaA gene expression ratio after interaction with probiotic L. acidophilus. We believe that new strategies can be developed by conducting broader, bacterial-bacterial interaction studies, including other probiotics.

Keywords

• Lactobacillus acidophilus,biofilm,Staphyloccoccus epidermidis,icaA gene expression

References

1. Büttner H, Mack D, Rohde H. Structural basis of Staphylococcus epidermidis biofilm formation: mechanisms and molecular interactions. Frontiers in Cellular and Infection Microbiology. 2015; 5:14. doi:10.3389/fcimb.2015.00014.
2. Otto M. Molecular basis of Staphylococcus epidermidis infections. Seminars in Immunopathology. 2012; 34(2):201-214. doi:10.1007/s00281-011-0296-2.
3. Sabaté Brescó M, Harris LG, Thompson K, et al. Pathogenic Mechanisms and Host Interactions in Staphylococcus epidermidis Device-Related Infection. Frontiers in Microbiology. 2017; 8:1401. doi:10.3389/fmicb.2017.01401.
4. Rogers KL, Fey PD, Rupp ME. Coagulase-negative staphylococcal infections. Infect Dis Clin North Am. 2009; 23(1):73-98. doi: 10.1016/j.idc.2008.10.001.
5. Hogan S, Stevens NT, Humphreys H, O'Gara JP, O'Neill E. Current and future approaches to the prevention and treatment of staphylococcal medical device-related infections. Curr Pharm Des. 2015; 21(1):100-13.
6. Salgueiro VC, Iorio NLP, Ferreira MC, Chamon RC, dos Santos KRN. Methicillin resistance and virulence genes in invasive and nasal Staphylococcus epidermidis isolates from neonates. BMC Microbiology. 2017; 17:15. doi:10.1186/s12866-017-0930-9.
7. Gerke C, Kraft A, Süssmuth R, Schweitzer O, Götz F. Characterization of the N-acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesin. J Biol Chem. 1998; 273(29):18586-93.
8. Härtel C, Pagel J, Spiegler J, et al. Lactobacillus acidophilus/Bifidobacterium infantis probiotics are associated with increased growth of VLBWI among those exposed to antibiotics. Scientific Reports. 2017; 7:5633. doi:10.1038/s41598-017-06161-8.

9. Sun B, Hu C, Fang H, Zhu L, Gao N, Zhu J. The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model. Hu W, ed. PLoS ONE. 2015; 10(6):e0128214. doi:10.1371/journal.pone.0128214.
10. Hou W, Sun X, Wang Z, Zhang Y. Biofilm-forming capacity of Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa from ocular infections. Invest Ophthalmol Vis Sci. 2012; 53(9):5624-31. doi: 10.1167/iovs.11-9114.
11. Nuryastuti T, van der Mei HC, Busscher HJ, Iravati S, Aman AT, Krom BP. Effect of Cinnamon Oil on icaA Expression and Biofilm Formation by Staphylococcus epidermidis. Applied and Environmental Microbiology. 2009; 75(21):6850-6855. doi:10.1128/AEM.00875-09.
12. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4):402-8.
13. Solati SM, Tajbakhsh E, Khamesipour F, Gugnani HC. Prevalence of virulence genes of biofilm producing strains of Staphylococcus epidermidis isolated from clinical samples in Iran. AMB Express. 2015; 5:47. doi:10.1186/s13568-015-0134-3.
14. Fey PD, Olson ME. Current concepts in biofilm formation of Staphylococcus epidermidis. Future microbiology. 2010; 5(6):917-933. doi:10.2217/fmb.10.56.
15. Crawford EC, Singh A, Metcalf D, Gibson TW, Weese SJ. Identification of appropriate reference genes for qPCR studies in Staphylococcus pseudintermedius and preliminary assessment of icaA gene expression in biofilm-embedded bacteria. BMC Research Notes. 2014; 7:451. doi:10.1186/1756-0500-7-451.
16. Prince T, McBain AJ, O’Neill CA. Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion. Applied and Environmental Microbiology. 2012; 78(15):5119-5126. doi:10.1128/AEM.00595-12.
17. Johansson MA, Björkander S, Mata Forsberg M, et al. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells. Frontiers in Immunology. 2016; 7:273. doi:10.3389/fimmu.2016.00273.
18. Reiter KC, Sant'Anna FH, d'Azevedo PA. Upregulation of icaA, atlE and aap genes by linezolid but not vancomycin in Staphylococcus epidermidis RP62A biofilms. Int J Antimicrob Agents. 2014; 43(3):248-53. doi: 10.1016/j.ijantimicag.2013.12.003.
19. Szczuka E, Jabłońska L, Kaznowski A. Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis. Microbiology. 2017; 163(5):712-718. doi:10.1099/mic.0.000453.
20. Holz C, Benning J, Schaudt M, Heilmann A, Schultchen J, Goelling D, Lang C. Novel bioactive from Lactobacillus brevis DSM17250 to stimulate the growth of Staphylococcus epidermidis: a pilot study. Benef Microbes. 2017; 8(1):121-131. doi: 10.3920/BM2016.0073.