Antibacterial activity of donkey’s milk against clinical isolate of Klebsiella pneumoniae

The purpose of this study was to investigate the antibacterial activity of raw donkey milk toward the clinical isolate of Klebsiella pneumoniae at 9, 15 and 38 °C as well as to clarify the role of lysozyme, lactoferrin and calcium content in the antibacterial activity of donkey’s milk. The effects of contamination level and incubation period on this antibacterial activity were also examined. Antibacterial assays were performed by determing the bacterial count in intentionally contaminated (102, 103, 104 cfu/mL) donkey milk samples during 8 and 96 hours. Lab-on-a-chip electrophoresis and atomic absorption spectrometry were used for the determination of lysozyme, lactoferrin and calcium contents in donkey milk, respectively. The donkey milk samples showed varying degrees of antibacterial activity against the tested strain K. pneumoniae. The antibacterial synergism of lysozyme and lactoferrin was proven for this clinical strain as the samples with higher lactoferrin amount showed a stronger antibacterial activity. The correlation between calcium content and antibacterial activity of donkey milk samples was not established. Donkey milk showed stronger antibacterial potential at 15 °C compared to 9 °C, but this was limited by a higher growth rate of K. pneumoniae at 15 °C. The higher level of contamination resulted in a faster consumption of the antibacterial capacity of donkey milk. The artificial neural network model for prediction of K. pneumoniae count gave acurate fit to experimental data, showing a reasonably good (overall r2 for donkey milk was 0.986, with training error 4.67∙10-4, while r2 for nutrient broth was 0.982 and training error was 0.002).
Svrha ovog istraživanja bila je utvrditi antibakterijsko djelovanje sirovog mlijeka magarice prema kliničkom izolatu Klebsiella pneumoniae pri 9, 15 i 38 °C, te razjasniti ulogu lizozima, laktoferina i kalcija u antibakterijskom djelovanju mlijeka magarice. Također su ispitani učinci razine kontaminacije i razdoblja inkubacije na navedenu antibakterijsku aktivnost. Antibakterijski testovi provedeni su određivanjem broja bakterija u ciljano kontaminiranim (102, 103, 104 cfu/ mL) uzorcima mlijeka magarice tijekom 8 i 96 sati. Lab-on-a-chip elektroforeza i atomska apsorpcijska spektrometrija korištene su za određivanje sadržaja lizozima, laktoferina i kalcija u mlijeku magarice. U ispitivanim uzorcima mlijeka magarice utvrđeni su različiti stupnjevi antibakterijske aktivnosti prema ispitivanom soju K. pneumoniae. Za ovaj klinički soj dokazan je antibakterijski sinergizam lizozima i laktoferina, jer su uzorci s većom količinom laktoferina pokazali jače antibakterijsko djelovanje. Korelacija između sadržaja kalcija i antibakterijske aktivnosti uzoraka mlijeka magarice nije utvrđena. Mlijeko magarice pokazalo je jači antibakterijski potencijal na 15 °C u usporedbi s antibakterijskim potencijalom na 9 °C, ali je on bio ograničen većom stopom rasta K. pneumoniae na 15 °C. Viša razina kontaminacije uzrokovala je bržu potrošnju antibakterijskog kapaciteta mlijeka magarice. Model umjetne neuronske mreže za predviđanje broja K. pneumoniae dobro se slagao s eksperimentalnim podacima (ukupni r2 za mlijeko magarice bio je 0,986, s pogreškom učenja od 4,67 10-4, dok je r2 za hranjivi medij bio 0,982, a pogreška učenja bila je 0,002).