Killing efficacy and anti-biofilm activity of synthetic human cationic antimicrobial peptide cathelicidin hCAP-18/LL37 against urinary tract pathogens

Abstract

Objectives: Cathelicidin LL37 represents one of the chemical defence components of bladder epithelial cells that include antimicrobial peptides, which also shown to have an important role in the mucosal immunity of the urinary tract by preventing adhesion of bacteria. This study aimed to determine the killing efficacy of LL37 compared to anti-biofilm activity against Staphylococcus aureus and Escherichia coli. Methods: The 96-flat well microtiter plates were used for evaluation of killing rate by estimation of MIC-value to the clinical isolates of E. coli and S. aureus collected from patients with urinary tract infection. S. aureus ATCC 25923 and E.coli ATCC 25922 were investigated in this study. Biofilm formation on polystyrene surface was conducted by growing bacterial isolates on 96-flat well microtiter plates, stained with crystal violet. The bound bacteria were quantified by addition of ethanol 70% and measurement of the dissolved crystal violet absorbance at (OD630 nm) using ELISA reader. Results: LL37 showed minimal inhibitory concentration (MIC) of 32 &mu;g/ml against S. aureus and E. coli. The sub-MIC of LL37 was also able to eliminate about 31% and 34% of both S. aureus and E. coli, respectively. Anti-biofilm activity of LL37 showed biofilm inhibition at 1 &mu;g/ml (1/32 MIC) to 16 &mu;g/ml (1/2 MIC), which exhibited significant difference (p<0.001) against E. coli, whereas LL37 beyond 1 &mu;g/ml showed significant inhibition (p<0.001) of biofilm against S. aureus. Conclusion: The cathelicidin LL37 can be used as a broad-spectrum anti-biofilm agent rather than killing agent.

Keywords

• Cathelicidin LL37, MIC, biofilm, anti-adhesive, killing rate

Killing efficacy and anti-biofilm activity of synthetic human cationic antimicrobial peptide cathelicidin hCAP-18/LL37 against urinary tract pathogens

Öz

Amaç: Cathelicidin LL37 mesane epitel hücrelerinin kimyasal bir savunma komponenti olup antimikrobiyal peptitler arasında idrar yolunda bakterilerin yapışmasını engelleyerek mukozal bağışıklıkta önemli bir role sahiptir. Bu çalışmada LL37’nin öldürücü aktivitesini Staphylococcus aureus ve Escherichia coli’ye karşı oluşan anti-biyofilm aktivitesi ile karşılaştırmayı amaçladık.Yöntemler: İdrar yolu enfeksiyonu olan hastalardan toplanan E. coli ve S. aureus klinik izolatlarının öldürücü oranlarının tahmini MIC değerinin değerlendirilmesi için 96’lık düz kuyucuklu mikropleytler kullanıldı. Bu çalışmada, S. aureus ATCC 25923 ve E. coli ATCC 25922 suşları incelenmiştir. Polistiren yüzey üzerinde biyofilm oluşumu kristal mor ile boyalı 96’lık düz kuyucuklu mikropleytlerde üreyen bakteri izolatı ile gerçekleştirilmiştir. Bağlı bakterinin, % 70’lik etanol ve çözünmüş kristal morunun ilavesinden sonra ELISA okuyucusu kullanılarak (OD630 nm) absorbansı ölçüldü.Bulgular: S. aureus ve E. coli’ye karşı LL37, minimum inhibitör konsantrasyonu (MİK) 32 µg/ml’de gösterildi. LL37’nin alt MİK değerleri ile sırasıyla S. aureus ve E. coli’nin % 31 ve % 34’nü ortadan kaldırmak mümkün oldu. LL37’nin anti-biyofilm aktivitesi 1 µg/ml (1/32 MİK)’dan 16 µg/ml (1/2 MİK)’da biyofilm inhibisyonu gösterdi ve E. coli’ye karşı da belirgin bir fark (p <0.001 ) ortaya koydu. LL37’nin ise 1 µg/ml’nin üzerinde S aureus’a karşı biyofilmi önemli ölçüde inhibe ettiği bulundu (p <0.001).Sonuç: Cathelicidin LL37 geniş spektrumlu anti-biyofilm ajan olarak öldürücü maddeler yerine kullanılabilir

Anahtar Kelimeler

• Cathelicidin LL37, MİK, biyofilm, adezyon önleyici, öldürücü oran

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