Isolation, cultivation, and in vitro susceptibility testing of Borrelia burgdorferi sensu lato: A review

Lyme borreliosis is the most common vector-borne disease in the northern hemisphere. The agents of Lyme borreliosis are borrelia, bacteria of the family Spirochaetaceae, which are grouped in Borrelia burgdorferi sensu lato species complex. Borreliae are fastidious, slow-growing and biochemically inactive bacteria that need special attention and optimal conditions for cultivation. The isolation of Borrelia from clinical material and their cultivation is a time-consuming and demanding procedure. Cultivation lasts from 9 up to 12 weeks, which is much longer than is necessary to grow most other human bacterial pathogens. Although B. burgdorferi sensu lato is susceptible to a wide range of antimicrobial agents in vitro, up to now the susceptibility of individual Borrelia species to antibiotics is defined only partially. [Projekat Ministarstva nauke Republike Srbije, br. 175011

Influence of cultivation conditions on growth, reproduction, survival and morphology of Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto

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..

A review on antibiotic resistance: origin and mechanisms of bacterial resistance as biological phenomenon

Antimicrobial resistance is a natural biological phenomenon in their struggle for existence and represents a major health care issue, associated with high mortality and morbidity. The first cases of mass emergence of resistant strains were observed in the middle of the 20th century, and since then cases of resistance have been reported all over the world, and in the last two decades even more frequently are reported multiple bacterial resistance. Factors that contribute to the development of bacteria resistance are abuse in the use of antibacterial agents, in humans or livestock, and releases of antibacterial agents into the environment. Moreover, the development of new effective antibiotics is decreasing, contrary to increasing the overall effort for the synthesis of new ones. Identification and reporting of bacterial resistance, as well as monitoring of the use of antibiotics in the outpatient and inpatient setting, today is the obligation of all countries

Divlje kanide kao domaćini za vektorima prenosive zoonozne patogene u Srbiji

Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum, was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia.Divlje kanide su prepoznate kao važni domaćini rezervoari za nekoliko vektorima prenosivih zoonotskih patogena i predstavljaju dobre sentinel vrste za epidemiološki nadzor. Identifikacija domaćina rezervoara za pojedine zoonotske patogene na određenom području omogućava primenu odgovarajućih mera kako bi se sprečila pojava zoonoza kod ljudi i domaćih životinja. Cilj ove studije bio je da se predstavi uloga divljih kanida u epidemiologiji zoonoza koje se prenose vektorima u Srbiji na temelju prethodno objavljenih radova i da se dostupni podaci prodiskutuju sa epidemiološkog stanovišta. Na osnovu dosadašnjih molekularnih istraživanja u Srbiji, divlje kanide su prepoznate kao potencijalni rezervoari za nekoliko krpeljima prenosivih zoonotskih patogena. Kod šakala je potvrđeno prisustvo Anaplasma phagocytophilum, dok je prisustvo Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii i Borrelia lusitaniae molekularnim metodama potvrđeno kod lisica. Rezultati pokazuju da su divlje kanide potencijalni domaćini rezervoari i izvor zoonotskih patogena u Srbiji i da predstavljaju dobre domaćine za više vrsta krpelja koji su potvrđeni kompetentni vektori pojedinih patogena. Potrebno je kontinuirano epidemiološko praćenje bolesti uzrokovanih patogenima čije je prisustvo prethodno potvrđeno kod divljih kanida. Takođe, potrebna su dodatna istraživanja kako bi se otkrila uloga drugih divljih i domaćih životinja u epidemiologiji zoonoza koje se prenose vektorima u našoj zemlji

Borrelia lusitaniae and Borrelia valaisiana – potential for causing Lyme borreliosis in Serbia

Kompleks Borrelia burgdorferi sensu lato trenutno obuhvata dvadest i jednu opisanu vrstu borelija a karakteriše ih značajna varijabilnost u pogledu geografske distribucije kao i specifičnosti na relaciji domaćini rezervoari- kompetentni vektor. Poznato je da samo određeni sojevi vrsta borelija izazivaju bolest kod ljudi. U Severnoj Americi, lajmsku boreliozu uzrokuje samo jedna vrsta borelija-Borrelia burgdorferi sensu stricto (s.s.), dok u Evropi infekciju kod ljudi može uzrokovati nekoliko različitih vrsta borelija-Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii i Borrelia burgdorferi s.s. Vrste Borrelia valaisiana, Borrelia lusitaniae i Borrelia bissettii identifikovane su samo u pojedinačnim slučajevima i prepoznate su kao potencijalni patogeni. Različite vrste borelija povezane su sa različitim kliničkim manifestacijama lajmske borelioze; B. afzelii obično ostaje lokalizovana u koži, B. garinii i B. bavariensis se obično povezuju s poremećajima nervnog sistema, dok se B. burgdorferi s.s. često povezuje sa razvojem artritisa. Iako postoje registrovani slučajevi lajmske borelioze u Srbiji, ne postoje podaci o tome koje vrste borelija izazivaju određene kliničke manifestacije lajmske borelioze. Region Srbije karakteriše visoka rasprostranjenost i raznovrsnost vrsta borelija u krpeljima i njihovim domaćinima. Istraživanja prisustva borelija u krpeljima kao i mikrobiološka izolacija sojeva borelija iz krpelja u Srbiji, ukazala su na dominaciju vrste B. lusitaniae, slede vrste B. afzelii, B. bavariensis, B. garinii, B. valaisiana i B. burgdorferi s.s. Takođe, B. lusitaniae je detektovana u krpeljima sakupljenim sa guštera i u uzorcima slezine lisica, dok je B. valaisiana detektovana u krpeljima sakupljenim sa šakala i ptica. Ispitivanje in vitro osetljivosti lokalnih sojeva borelija (izolovanih iz krpelja vrste Ixodes ricinus sakupljenih sa vegetacije) na ljudski serum, pokazalo je da su sojevi B. valaisiana otporni na komplement, dok su određeni sojevi B. lusitaniae (u prisustvu komplementa) podjednako pokretljvi kao i jedini postojeći soj B. lusitaniae izolovan iz hronične kožne lezije, poreklom iz Portugala. Iako su lokalni sojevi borelija osetljivi na antibiotike in vitro, ostaje nepoznato koje kliničke manifestacije lajmske borelioze mogu izazvati vrste B. valaisiana i B. lusitaniae. Potrebno je da se razjasne mehanizami rezistencije sojeva B. valaisiana i B. lusitaniae (koji cirkulišu u ovoj oblasti) u prisustvu komplementa.The twenty-one described Borrelia species with considerable variability in host and vector associations, and geographical distribution have been classified to the Borrelia burgdorferi sensu lato complex. Only a subset of Borrelia species is known to cause human illness. In North America, Lyme borreliosis (LB) is predominantly caused by the Borrelia burgdorferi sensu stricto (s.s.), while in Europe, the overall diversity of Borrelia species is higher and LB can be caused by Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, and Borrelia burgdorferi s.s., while Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have been identified in single cases only and are recognized as potential pathogens. Different Borrelia species are involved in distinct clinical manifestations of LB; B. afzelii usually remains localized in the skin, B. garinii and B. bavariensis are usually associated with nervous system disorders, while B. burgdorferi s.s. is commonly associated with development of the arthritis. Although there are registered cases of LB in Serbia, there is no data on which Borrelia species cause certain clinical manifestations of LB. Region of Serbia is characterized by high prevalence and diversity of Borrelia species in ticks and their vertebrate hosts. The studies on the presence and isolation of Borrelia from ticks from Serbia pointed to the domination of B. lusitaniae followed by B. afzelii, B. bavariensis, B. garinii, B. valaisiana and B. burgdorferi s.s. Also, B. lusitaniae was detected in ticks collected from lizards and in spleen samples collected from red foxes, while B. valaisiana was detected in ticks collected from golden jackals and birds. In vitro susceptibility of local Borrelia strains isolated from ticks to human complement, showed B. valaisiana strains resisted complement-mediated killing, while certain B. lusitaniae strains (in the presence of complement) were equally motile as the only existing one B. lusitaniae human isolate from Portugal. Although local Borrelia strains are susceptible to antibiotics in vitro, it remains unknown what clinical manifestations of LB can be caused by B. valaisiana and B. lusitaniae. It is needed to elucidate the mechanism behind B. valaisiana and B. lusitaniae strains (circulating in the area) survival in the presence of human complement

Lajmska borelioza - epidemiologija, klinička slika i terapija

Lyme borreliosis is a multisystem disorder characterized by a wide spectrum of clinical manifestations. In Europe, the main aetiological agents are Borrelia afzelii, Borrelia garinii and B. burgdorferi sensu stricto, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported as rare or potential causes of human disease. In North America, B. burgdorferi sensu stricto is the only human pathogen. The most common clinical manifestation of Lyme borreliosis is a skin lesion, erythema migrans, which may resolve spontaneously without antibiotic treatment. However, the pathogen can spread to other tissues and organs, causing more severe manifestations that can involve a skin, nervous system, joints, and/or heart, requiring antibiotic therapy. The most commonly used antibiotics in the treatment of Lyme borreliosis are: amoxicillin, ceftriaxone, cefuroxime, doxycycline and azithromycin.Lajmska borelioza je multisistemska bolest koju karakteriše širok spektar kliničkih manifestacija. U Evropi je primarno prouzrokovana vrstama B. afzelii, B. garinii i B. burgdorferi sensu stricto, ali Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii se takođe navode kao retki ili potencijalni uzročnici oboljenja kod ljudi. U Severnoj Americi, kao jedini humani patogen, dokazana je vrsta B. burgdorferi sensu stricto. Najčešća klinička manifestacija lajmske borelioze je kožna lezija, erythema migrans, koja može da prođe spontano, bez terapije antibioticima. Međutim, patogen se može širiti do drugih delova kože i organa i tako prouzrokovati teže manifestacije kako na koži, tako i na nervnom sistemu, zglobovima i/ili srcu, pri čemu je terapija antibioticima neophodna. Najčešće korišćeni antibiotici u terapiji lajmske borelioze su: amoksicilin, ceftriakson, cefuroksim, doksiciklin i azitromicin

Evolutionary dynamics and geographical dispersal of Borrelia lusitaniae

Background: Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato, associated with lizards as reservoirs and Ixodes ricinus as its main vector. Borrelia lusitaniae is predominantly distributed in Central and Southeast Europe, and in countries of the Mediterranean basin, such as Portugal, Morocco, Tunisia, and Italy where this spirochete appears to infect vector ticks more frequently than other genospecies. Evolution of this zoonotic tick-borne microparasite is shaped by different environmental factors. Comprehensive phylogenetic analysis may give insight into how B. lusitaniae spreads to new geographic locations. Aim: We applied Bayesian statistical methods to B. lusitaniae multilocus sequence typing (MLST) data to study the migration routes of B. lusitaniae and its potential for further spread. Results: The discrete phylogeographic analysis placed origins of B. lusitaniae in Southeast Europe and identified at least two introductions of B. lusitaniae from Europe to North Africa. Estimated effective reproductive potential (Re), as a key indicator for a pathogen spread, suggested potential for further spread. Conclusion: The results of this study can provide beneficial information about the potential for further spread of B. lusitaniae in Europe and North Africa and estimation of necessity for the development of strategies to monitor and control Lyme borreliosis

First evidence of tick-borne protozoan pathogens, babesia sp. And hepatozoon canis, in red foxes (vulpes vulpes) in Serbia

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Co-infection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes