Lactobacillus acidophilus ve Lactobacillus casei’nin, Metisilin dirençli ve metisilin duyarlı Staphylococcus aureus biyofilm genlerine in-vitro etkisinin incelenmesi

Amaç: Staphylococcus aureus’un icaA ve icaR genleri üzerindendüzenlediği biyofilm yeteneği, oluşturdukları enfeksiyonlar ilemücadeleyi zorlaştırmaktadır. Bu çalışmada, Lactobacillus acidophilusve Lactobacillus casei’nin, metisilin duyarlı (MSSA) vemetisilin dirençli (MRSA) S. aureus suşlarının icaA ve icaR genekspresyonlarına etkisinin in-vitro olarak incelenmesi amaçlanmıştır.Gereç ve Yöntem: IcaA ve icaR gen ekspresyonlarının kontrolüiçin 6 tüp hazırlandı. 37oC’da %5 CO2’li ortamda inkübe edildi.İnkübasyonun 6., 12., ve 24. saatinde RNA izolasyonları yapıldı.cDNA sentezi sonrasında,icaA, icaR ve 16SrRNA genlerine spesifikprimerler ile real-time PCR’la çalışıldı. Delta delta Ct yönteminegöre sonuçlar analiz edildi.Bulgular: S. aureus ATCC 43300 (MRSA) ve S. aureus ATCC29213 (MSSA) icaA gen ve icaR ekspresyonları, L. acidophilusve L. casei ile karşılaşma sonrası sırasıyla down regülasyon veupregulasyon göstermiştir. 12. saat ve 24. saatte saptanan icaAve icaR gen ekspresyon seviyelerinin hem MRSA hem de MSSAsuşlarında benzer olduğu gözlenmiştir.Öneri: Sonuç olarak, çalışmamız, probiyotik etkili L. acidophilusve L. casei suşlarının S. aureus suşlarının biyofilm etkinlikleriniicaA ve icaR gen ekspresyonlarını real-time PCR yöntemiile araştıran ilk çalışmadır. L. acidophilus ve L. casei’nin in-vitroortamda MRSA ve MSSA suşlarını etkileyerek icaA ve icaR genekspresyonlarını değiştirebildikleri saptanmıştır. Probiyotiketkili bu suşların antibiyofilm özelliklerinin S. aureus enfeksiyonlarınakarşı yeni bir mücadele seçeneği olabileceği düşünülmektedir.

Investigation of the in-vitro effect of Lactobacillus acidophilus and Lactobacillus casei on biofilm genes of methicillin-resistant and methicillin-sensitive Staphylococcus aureus

Aim: Staphylococcus aureus’s biofilm ability which control via icaA and icaR gene is a serious problem in the treatment of infections. Aim of this study was to investigate in-vitro effects of important probiotic L. acidophilus and L. casei on the icaA and icaR genes expressions of the MRSA and MSSA strains. Materials and Methods: In this study, to determine the icaA and icaR gene expressions, 6 tubes in probiotics were prepared. Incubated at 37°C in 5% CO2 medium. RNA isolations were performed at the 6th, 12th, and 24th hours of incubation. After cDNA synthesis, real-time PCR with primers specific for icaA, icaR and 16S rRNA genes were studied. The results were calculated with delta delta Ct method. Results: S. aureus ATCC 43300 (MRSA) and S. aureus ATCC 29213 (MSSA) icaA gene and icaR gene expressions showed down regulation and upregulation respectively after encountering L. acidophilus and L. casei. The icaA and icaR gene expression ratios determined at 12 hours and 24 hours were found to be similar in both MRSA and MSSA strains. Conclusion: This study was the first study to investigate effect of L. acidophilus and L. casei strains on biofilm gene expression of the S. aureus strains by the real-time PCR method. It was determined that L. acidophilus and L. casei were able to alter icaA and icaR gene expression by affecting the MRSA and MSSA strains in-vitro. We believe that the antibiotic properties of these probiotic strains may be a new treatment option against S. aureus biofilm infections.

PDF

___

Abdulgader SM, Shittu AO, Nicol MP, Kaba M, 2015. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Africa: a systematic review. Front Microbiol, 6, 348.
Conlon KM, Humphreys H, O'Gara JP, 2002. Regulation of icaR gene expression in Staphylococcus epidermidis. FEMS Microbiol Lett, 216(2), 171-177.
Crosby HA, Kwiecinski J, Horswill AR, 2016. Staphylococcus aureus aggregation and coagulation mechanisms, and their function in host-pathogen interactions. Adv Appl Microbiol, 96, 1-41.
Cue D, Lei MG, Luong TT, Kuechenmeister L, Dunman PM, O'Donnell S, Lee CY, 2009. Rbf promotes biofilm formation by Staphylococcus aureus via repression of icaR, a negative regulator of icaADBC. J Bacteriol, 191, 6363-6373.
Eggers S, Barker AK, Valentine S, Hess T, Duster M, Safdar N, 2018. Effect of Lactobacillus rhamnosus HN001 on carriage of Staphylococcus aureus: results of the impact of probiotics for reducing infections in veterans study. BMC Infect Dis, 18, 129.
Kashefi pasandideh N, Habibi MR, Tahmasebi H, Arabestani MR, 2018. Activity of biofilm genes icaA and icaR in methicillin- resistant Staphylococcus aureus treated with vitamin K in wound specimens. Koomesh, 20, 588-593.
Liévin-Le Moal V, Servin AL, 2014. Anti-infective activities of Lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents. Clin Microbiol Rev, 27, 167-199.
Lin MH, Shu JC, Lin LP, yu Chong K, Cheng YW, Du JF, Liu, ST, 2015. Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis. PLoS One, 10(4), 1-13.
Livak KJ, Schmittgen TD, 2001.Analysis of relative gene expression data usingreal-time quantitative PCR andthe 2(-Delta Delta C(T)) Method. Methods, 25(4),402-408.
Loewen K, Schreiber Y, Kirlew M, Bocking N, Kelly L, 2017. Community-associated methicillin-resistant Staphylococcus aureus infection: Literature review and clinical update. Can Fam Physician, 63, 512-520.
Mashruwala AA, Guchte AV, Boyd JM, 2017. Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus. Elife, 6, 23845.
Melo TA, Dos Santos TF, de Almeida ME, Junior LAGF, Andrade EF, Rezende RP. Romano C C, 2016. Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa. BMC Microbiol, 16(1), 250.
Möller MS, Fredslund F, Majumder A, Nakai H, Poulsen JCN, Leggio LL, Hachem MA, 2012. Enzymology and structure of the GH13_31 glucan 1,6-α-glucosidase that confers isomaltooligosaccharide utilization in the probiotic Lactobacillus acidophilus NCFM. J Bacteriol. 2012, 194(16), 4249-4259.
Nguyen D, Huynh T, Nguyen T, 2016. Anti-biofilm activities of Lactobacillus acidophilus against Staphylococcus aureus ATCC 25923. J Chem Pharm Res, 8, 464-467.
Nicholson TL, Shore SM, Smith TC, Frana TS, 2013. Livestockassociated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of swine origin form robust biofilms. PLoS One, 8(8),
Nuryastuti T, Van der Mei HC, Busscher HJ, Iravati S, Aman AT, Krom BP, 2009. Effect of cinnamon oil on icaA expression and biofilm formation by Staphylococcus epidermidis. Appl Environ Microbiol, 75, 6850-6855.
Orlin I, Rokney A, Onn A, Glikman D, Peretz A, 2017. Hospital clones of methicillin-resistant Staphylococcus aureus are carried by medical students even before healthcare exposure. Antimicrob Resist Infect Control, 6, 15.
Perl TM, Cullen JJ, Wenzel RP, Zimmerman MB, Pfaller MA, Sheppard D, 2002. Intranasal mupirocin to prevent postoperative Staphylococcus aureus infections. N Engl J Med, 346, 1871-1877.
Sakr A, Brégeon F, Mège JL, Rolain JM, Blin O, 2018. Staphylococcus aureus nasal colonization: an update on mechanisms, epidemiology, risk factors, and subsequent infections. Front Microbiol, 9, 2419.
Savage JW, Anderson PA, 2013. An update on modifiable factors to reduce the risk of surgical site infections. Spine J, 13, 1017-1029.
Shrestha L, Kayama S, Sasaki M, et al, 2016. Inhibitory effects of antibiofilm compound against Staphylococcus aureus biofilms. Microbiol Immunol, 60, 148-59.
Stefania DM, Miranda P, Diana M, Claudia Z, Rita P, 2017. Antibiofilm and antiadhesive activities of different synbiotics. J Prob Health, 5 (182), 2.
Tiptiri-Kourpeti A, Spyridopoulou K, Santarmaki V, Aindelis G, Tompoulidou E, Lamprianidou E E, Kotsianidis I, 2016. Lactobacillus casei exerts anti-proliferative effects accompanied by apoptotic cell death and up-regulation of trail in Colon Carcinoma Cells. PLoS One, 11(2).