Uticaj uslova kultivisanja na rast, razmnožavanje, preživljavanje i morfološke odlike vrsta Borrelia afzelii, Borrelia garinii i Borrelia burgdorferi sensu stricto

Abstract

Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstom B. afzelii a ređe vrstama B. garinii i B. burgdorferi sensu stricto. Borelije mogu biti izolovane iz različitog kliničkog materijala kao što su koža, krv i cerebrospinalna tečnost (CST). Modifikovana Kelly-Pettenkofer-ova (MKP) podloga je jedna od najčešće korišćenih hranljivih podloga za izolaciju i kultivaciju borelija. Prvi cilj ove doktorske disertacije je bio proceniti da li pojedinačne vrste borelija (B. afzelii, B. garinii i B. burgdorferi sensu stricto) imaju sposobnost da rastu u MKP podlogama koje su prethodno bile čuvane u frižideru na +4 °C u vremenskom periodu od manje od jednog meseca do 12 meseci, i kako dugotrajno čuvanje podloga može uticati na rast i morfologiju borelija. Rast borelija je procenjen posle 5 dana inkubacije na 33 °C; procenjen je broj borelija/mL, morfologija i pokretljivost. Rezultati su pokazali, da je starost MKP podloga imala statistički značajan uticaj na rast B. afzelii (P=0,021) i B. garinii (P=0,004) ali ne i na rast B. burgdorferi sensu stricto (P=0,204), dok starost podloga nije imala uticaj na morfologiju borelija i njihovu pokretljivost. U ovom radu je pokazano, da hranljive podloge starije od jednog meseca pa sve do 12 meseci, mogu podržati rast borelija. Optimalna temperatura za rast borelija u in vitro uslovima, predstavljena je u širokom rasponu od 30 do 37 °C. Borelije obično rastu na temperaturi 30-34 °C, a ponekad i na 35- 37 °C. Drugi cilj ove doktorske disertacije je bio proceniti i uporediti rast trideset jednog različitog soja vrsta B. afzelii, B. garinii i B. burgdorferi sensu stricto na pet različitih temperatura (4, 23, 28, 33 i 37 ºC) kako bi se procenio uticaj temperature na rast i preživljavanje borelija i definisala optimalna temperatura za njhovu kultivaciju. Rast je definisan kao konačni broj borelija/mL posle 3 dana inkubacije. Sve tri vrste borelija najbolje su rasle na 33 °C, sledele su temperature 37, 28 i 23 °C, dok na 4 °C rast nije detektovan. Na svim temperaturama, rast vrste B. afzelii bio je slabiji u poređenju sa druge dve vrste borelija. Nije postojala statistički značajna razlika između rasta B. garinii i B. burgdorferi sensu stricto na 23 °C (P=0,807), 28 °C (P=0,123), 33 °C (P=0,868) i 37 °C(P=0,490)...

Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, it is predominantly caused by B. afzelii and less frequently by B. garinii and B. burgdorferi sensu stricto. Borrelia can be isolated from different clinical material, such as skin, blood and cerebrospinal fluid (CSF). Modified Kelly-Pettenkofer (MKP) medium is one of several media used for isolation and cultivation of Borrelia. The first aim of this doctoral dissertation was to assess whether particular Borrelia species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) have the ability to grow in MKP medium stored at +4 °C for periods for one moth up to one year, and how prolonged storage may influences borrelia growth and morphology. The growth of Borrelia was evaluated after 5 days of incubation at 33 °C: cell count per mL, morphology and motility were assessed. The results of the present study showed that the duration of storage of MKP medium had statistically significant influence on growth of B. afzelii (P=0.021) and B. garinii (P=0.004) but not on growth of B. burgdorferi sensu stricto (P=0.204), while duration of storage of the medium had no impact on Borrelia morphology and motility. The results of the study indicate that medium stored for more than one and up to 12 months support borrelia growth. The optimum temperature for growth of Borrelia in vitro was presented widely as 30-37 °C. Borreliae are usually grown at 30-34 °C but sometimes at 35 or 37 °C. A second aim of this doctoral dissertation was to evaluate and compare growth of thirty-one different Borrelia strains (B. afzelii, B. garinii, and B. burgdorferi sensu stricto) at five different temperatures (4, 23, 28, 33, and 37 ºC) in order to find the influence of temperature on borrelia growth and survivance and to define the optimal temperature for cultivation of Borrelia. For all three Borrelia species, the best growth (growth was defined as final number of cells/mL after three days of incubation) was found at 33 °C followed by 37, 28, and 23 °C, while no growth was detected at 4 °C. The growth of B. afzelii species was weaker in comparison to the other two species at 23, 28, 33 and 37 °C, respectively. There was no statistically significant difference between growth of B. garinii and B. burgdorferi sensu stricto at 23 °C (P=0.807), 28 °C (P=0.123), 33 °C (P=0.868), and 37 °C (P=0.490), respectively...