Določanje bakterij rodu Staphylococcus pri okužbah sklepnih protez z uporabo bakteriofaga K in sonikacijo sklepnih protez

Šuster, Katja (author), Cör, Andrej (mentor), Podgornik, Aleš (comentor)

V okviru naloge smo načrtovali raziskovanje možnosti uporabe bakteriofaga K za določanje bakterij rodu Staphylococcus pri okužbah sklepnih protez (OSP). S preverjanjem njegove specifičnost smo dokazali, da je visoko specifičen za povzročitelje OSP iz rodu Staphylococcus in primeren za uporabo za detekcijo stafilokokov v vzorcih. Optimalni čas za bakterijsko detekcijo z metodo lize stafilokokov z bakteriofagom K in detekcijo sproščenega ATP (metoda ATP) je bil 120 min po okužbi z bakteriofagom K, za detekcijo bakteriofagne DNK z metodo qPCR (metoda qPCR) pa 180 min. Meja detekcije je bila, ob uporabi metode ATP, v območju 10^3 CFU/mL, pri uporabi metode qPCR pa smo detektirali 6,8 × 10^2 CFU/mL bakterij, kar predstavlja manj kot 1 bakterijo na µL vzorca, ki ga testiramo v qPCR. Po uspešni vzpostavitvi živega, vendar nekultivabilnega (VBNC) stanja bakterij, smo jih poizkusili dokazati z optimiziranima metodama detekcije. Z metodo ATP bakterij v stanju VBNC nismo bili sposobni zaznati, saj bakterije niso lizirale. Z metodo qPCR pa je detekcija VBNC bakterij mogoča na podlagi adsorpcije dodanih bakteriofagov. Z uporabo metod za detekcijo bakterij pri 104 kliničnih sonikatih smo dosegli enako občutljivost določitve stafilokokov v vzorcih sonikatov v primerjavi s standardnimi mikrobiološkimi preiskavami, in 100 % specifičnost. Pomembno pa se je zmanjšal čas detekcije. Rezultati odpirajo možnost za nadaljnje raziskovanje bakteriofagov in razširitve spektra detekcije metod s čimer bi se približali k razvoju hitre in specifične metode za natančno in poceni diagnostiko OSP.

• Language: Slovenian
• Keywords: okužbe sklepnih protez, diagnostika, bakteriofag K, stafilokoki, sonikat, bakterije
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-108098
• COBISS.SI-ID: 932471
• Publication date in RUL: 16.06.2019

Secondary language

• Language: English
• Title: Detection of Staphylococcus spp. in prosthetic joint infections with the use of Bacteriophage K and implant sonication

Abstract

The aim of the research was to explore possibilities for the use of bacteriophage K for the detection of Staphylococcus spp. in sonicate fluid (SF) of infected prosthetic joints (PJI). Specificity testing results of bacteriphage K showed that it is able to recognize and lyse only staphylococci. The optimal time for bacterial detection with the method of ATP detection after staphylococcal lysis with bacteriophage (ATP method) was 120 min after infection with bacteriophage K and 180 min with the method of bacteriophage K DNK detection with qPCR (qPCR method). The limit of detection for the ATP method was in the range of 10^3 CFU/mL and 6.8 × 10^2 CFU/mL for the qPCR method. After the induction of the viable but non-culturable (VBNC) state of bacteria, the optimized methods were tested for the possibility of detection. The ATP method did not allow the detection of VBNC bacteria, because lysis did not occur. However, the detection of VBNC bacteria with the qPCR method was successful based on the adsorption of added bacteriophages. With the use of bacteriophage K for the detection of bacteria within 104 tested SF, the same sensitivity of staphylococcal determination was achieved compared to standard microbiological methods. However, the detection time was significantly reduced. The results open up the possibility for further exploration of bacteriophages in order to expand the detection spectrum of methods and thus approach the development of a fast and specific method for accurate and inexpensive diagnosis of PJI.

• Keywords: prosthetic joint infections, diagnosis, bacteriophage K, staphylococci, sonicate fluid, bacteria