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Vaser, Robert; Spičić, Silvio; Reil, Irena; Zdelar-Tuk, Maja; Šikić, Mile; Križanović, Krešimir; Pavlinec, Željko; Duvnjak, Sanja
Veterinarska stanica, 08/2021, Volume: 53, Issue: 1Journal Article, Paper
Brucella , an extremely diverse but yet genetically highly homogenous genus of bacteria, has been a puzzle for scientists for many decades. These bacteria remain a prominent public health issue, particularly in the Balkan region. Correctly identifying and understanding the pathogen is a vital step in the epidemiology and epizootiology of any bacteria. Identification can be challenging, especially in the case of zoonotic species. This study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis strains kept in our archive and to compare this method to the classical and non-sequencing based molecular methods used to date. Classical biotyping is highly subjective and gave inconclusive results for 3 strains. Molecular methods used were multiplex PCR and RFLP methods since no one method can identify both species and biovar which is vital in the case of Brucella suis infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results. Oxford Nanopore long-read sequencing was used on a MinION device for next-generation sequencing (NGS). Various algorithms were implemented for genome assembly and BioNumerics 8.0 software was used for MLST identification and analysis. MLST 21 was used for biovar identification and epidemiological comparison of tested strains. The assembled genomes were 3,2 Mb in size and assembled into two chromosomes. MLST 21 analysis placed our strains into species and biovar clusters in concordance with other molecular tests used. To the extent of our knowledge, this is the first documented use of long-read sequencing in Brucella suis identification in this region. We conclude that bacteriological biotyping is outdated and host-specific identification in this genus is incorrect and that molecular characterisation is always the safer, faster and more suitable option. MinION sequencing proved to be a strong, accessible solution for species determination. Future study is required to determine how detailed genome information it can give, considering the error rate. Rod Brucella biološki je iznimno raznolik, ali genetski vrlo homogen rod bakterija te je već desetljećima znanstvenicima nepoznanica. Ove bakterije su veliki javno-zdravstveni problem, a osobito na Balkanu. Pravilno prepoznavanje i razumijevanje patogena ključan je korak u epidemiologiji i epizootiologiji bilo koje bakterijske vrste, čija identifikacija može biti izazovna, osobito u slučaju zoonotskih vrsta. Cilj je ovog rada bio implementirati sekvenciranje četvrte generacije u tipizaciji 11 sojeva Brucella suis koje se čuvaju u našoj arhivi te ovu metodu usporediti s klasičnim i molekularnim metodama koje se trenutačno primjenjuju, a ne zasnivaju se na sekvenciranju. Klasično je biotipiziranje vrlo subjektivno i dalo je podvojene rezultate za 3 soja. Od molekularnih metoda koristili smo višestruku lančanu reakciju polimerazom (engl. Polymerase Chain Reaction, PCR) i polimorfizam duljine restrikcijskih fragmenata (engl. Restriction Fragment Lenght Polymorphism, RFLP) budući da niti jedna od metoda ne može zasebno identificirati i vrstu i biovar, a što je važno u slučaju Brucella suis infekcije. Vrsta i biovar svih sojeva uspješno su potvrđene i u skladu s rezultatima biotipizacije. Sekvenciranje sljedeće generacije (engl. Next Generation Sequencing, NGS) provodili smo na Oxford Nanopore MinION uređaju koji sekvencira duge lance DNK. Za sastavljanje genoma rabljeni su različiti algoritmi, a za identifikaciju i analizu rezultata MLST-a korišten je softver BioNumerics 8.0. MLST 21 je korišten za identifikaciju biovara i epidemiološku usporedbu ispitivanih sojeva. Genomi su bili veličine 3,2 Mb i sastavljeni u dva kromosoma. Analiza MLST 21 smjestila je naše sojeve u vrsne i biovarne skupine u skladu s drugim korištenim molekularnim testovima. Koliko je nama poznato, ovo je prva dokumentirana uporaba sekvenciranja dugih lanaca DNK u identifikaciji Brucella suis u jugoistočnoj Europi. Zaključujemo da je bakteriološka biotipizacija zastarjela i da je identifikacija biovara u ovom rodu, ovisno o domaćinu, netočna te da je molekularna karakterizacija uvijek sigurnija, brža i prikladnija opcija. MinION sekvenciranje pokazalo se kao vrlo pristupačno rješenje za određivanje vrste i biovara Brucella suis . Daljnja su istraživanja potrebna da bi se ustvrdilo koliko detaljne informacije o genomu može dati, imajući u vidu značajniji postotak pogreške prilikom sekvenciranja.
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