![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/SanjaDUVNJAK.jpg\")
<\/p>\n<\/p>\n
S. Duvnjak<\/strong>, \u017d. Pavlinec<\/strong>, R. Vaser<\/strong>, K. Kri\u017eanovi\u0107<\/strong>, M. \u0160iki\u0107<\/strong>, M. Zdelar-Tuk<\/strong>, I. Reil<\/strong>* and S. \u0160pi\u010di\u0107<\/strong><\/p>\n <\/a><\/p>\n <\/a> ▲<\/a><\/p>\n Brucella<\/em>, 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. Key words:<\/strong> Brucella suis; brucellosis; swine; horses; Nanopore MinION; NGS<\/em><\/p><\/blockquote>\n <\/a> ▲<\/a><\/p>\n Brucellae<\/em> belong to the \u03b1<\/em>-Proteobacteria, a class of bacteria that are extremely diverse and adaptable to new habitat conditions. Brucella<\/em> sp. is a genus currently consisting of 13 species (Ledwaba et al<\/em>., 2019), in contrast to the 6 species known in 2003 (Osterman and Moryion, 2003). Brucellae<\/em> are known to infect wild and domestic animals such as wild boars, cows, sheep, goats, dogs (Godfroid, 2002), but have also recently been cultured from marine animals, common voles, red foxes, baboons, human breast implants and fish (Al Dahouk et al<\/em>., 2007; Cvetnic et al<\/em>., 2017; Scholz et al<\/em>., 2008a, 2008b, 2010, 2016; Whatmore et al<\/em>., 2014). Brucella suis<\/em> was the first pathogen to be used as a bioweapon in the 1950s. This goes to show that this is a genus of highly adaptable bacteria that can realistically be expected anywhere. It is important to note that human neglect and misdiagnosis are two key factors that have facilitated these resourceful bacteria in remaining a globally persistent pathogen for well over a decade, causing significant economic losses and public health issues (Pappas et al<\/em>., 2006).<\/p>\n Brucella suis<\/em> causes the chronic disease known as porcine brucellosis, which manifests as infertility and miscarriage in sows, high mortality of piglets, and orchitis in boars. B. suis<\/em> biovars 1, 2 and 3 appear around the world wherever pigs are bred, with biovars 1 and 3 the most abundant globally (OIE Manual, Porcine brucellosis, 2009).<\/p>\n Brucellosis is an endemic disease in Croatia, and various Brucella<\/em> sp. have been confirmed in swine, wild boars, cows, sheep, goats and humans (Spicic et al<\/em>., 2010). B. suis<\/em> in Croatia is present in both domestic swine and wild boar populations in all counties where pigs are bred. Croatia was the first European country where B. suis<\/em> biovar 3 infection was detected in horses, swine and wild boars. Swine in extensive production are most affected, given the high density of animals and proximity of wild boar that facilitate the spread of the disease (Cvetnic et al<\/em>., 2003, 2005; Spicic et al<\/em>., 2010). The prevalence of B. suis<\/em> bv. 2 and the established link between wildlife and outdoor breeding has been reported in other European countries, such as Hungary, Poland, and others (Szulowski et al<\/em>., 2013; Kreizinger et al<\/em>., 2014). The most prevalent brucellosis in Croatia is swine brucellosis, though Croatia is one of few countries having a favourable brucellosis status. However, financially and professionally well-supported control and eradication programmes should be implemented to prevent this disease from becoming an even more serious problem than in the past. The omnipresence of brucellosis can only be combatted through accurate and detailed pathogen identification. Brucella<\/em> genomes are highly conserved and show a high degree of similarity with less than 6% nucleotide sequence variation, which is attributed to the recent origin of the genus (Bergey\u2019s Manual, 2018). They have two chromosomes and no plasmids. They have ribosomal gene clusters carrying around 3200 protein- coding genes according to Sanchez- Jimenez et al<\/em>. (2015). Also, Meyer and Shaw (Bergey\u2019s Manual, 2018) noted that Brucella suis bv. 3 is unique, with a single chromosome 3.2 Mbp long.<\/p>\n The global leader in next-generation sequencing technology is the Illumina platform (Illumina, Inc., San Diego, CA, USA), which uses synthesis-based sequencing (SBS) (www.illumina.com). MinION\u2122 (Oxford Nanopore Technologies (ONT), Oxford, UK) uses different technology based on E. coli<\/em> nanopores. As the DNA molecule passes through the membrane it causes a change in membrane current, which is recorded and translated into base pairs. This study was aimed at introducing fourth-generation sequencing into our lab and for typing 11 Brucella suis<\/em> strains to the biovar level. MinION is small, fast, simple and cost-effective since barcoding is possible. It takes less hands-on time during preparation than typing analyses currently in use if one has good software solutions. It also produces data that can be used for complete genome sequencing since it sequences long DNA molecules. <\/a> ▲<\/a><\/p>\n The tested strains are listed in Table 1. All strains were also tested biochemically and using classical PCR-based molecular methods.<\/p>\n All strains were microbiological, cultural and biochemically tested according to Corbel et al<\/em>. (1983) and Alton et al<\/em>. (1988). This involved colony morphology (size, convexity, transparency, roughness), CO2<\/sub> growth requisite, susceptibility to thionine and fuchsine, production of H2<\/sub>S, agglutination with specific monosera, ability to hydrolyse urea and reaction with Wb, Iz, Tb and R\/C phages.<\/p>\n Bruce-ladder was used as the reference method to determine Brucella<\/em> species (Lopez-Goni et al<\/em>., 2008, 2011); and restriction fragment length polymorphism (RFLP) (Cloeckaert et al<\/em>., 1995; Vizcaino et al<\/em>., 1997) and multiplex-suis (Suis-ladder) were used to determine Brucella suis<\/em> biovars (Lopez-Goni et al<\/em>., 2011). MLVA-16 genotyping was performed on a total of 16 gene loci (Al Dahouk et al<\/em>., 2005; Le Fl\u00e8che et al<\/em>., 2006). B. melitensis<\/em> 16M was used as the reference strain for comparison and verification of test quality.<\/p>\n Strains were grown from cryobeads on Brucella<\/em> agar plates for 72 hours at 37\u00b0C. For PCR-based molecular methods, DNA was isolated using the QIAcube DNA Mini Kit and the QIAcube system (QIAGEN, Hilden, Germany) according to the manufacturer\u2019s instructions.<\/p>\n The supernatant (2 or 5 \u00b5L) was used in DNA-based tests. The same PCR reaction mixture was used for all molecular tests: 20 \u03bcL reaction mixtures consisting of 10 \u03bcL HotStarTaq Master Mix (Qiagen, Hilden, Germany), 6 \u03bcL water (Rnase-free water, Qiagen, Hilden, Germany), 0.5 \u03bcM each primer pair specific for the target locus (Macrogen, Netherlands) and 2 \u03bcL template DNA. MLST and epidemiological analyses were performed using the BioNumerics software scheme (version 8.0; BioMerieux, Applied Maths, Belgium).<\/p>\n Genomic DNA isolation for sequencing started with centrifugation of the bacterial cell suspension at 5000 g for 10 minutes. The bacterial cell precipitate was treated according to the manufacturer\u2019s instructions using Genomic-tip 500\/G (Qiagen). DNA quality and concentration were defined on DS-11 Spectrophotometer (DeNovix), Qubit using Qubit dsDNA BR Assay Kit (Invitrogen) and Tapestation 2200 using Genomic DNA Screen Tape and Reagents (Agilent). All samples were tested in triplicate on each device. We prepared a suspension of 9 \u00b5L DNA (\u2248 1 \u00b5g genomic DNA) and 3 \u00b5L fragmentation mix and extended the incubation with AMPure XP beads to 10 minutes. Beads were washed with 80% ethanol and incubated for 30 seconds before removing the ethanol. After the second ethanol wash, beads were air-dried for 1 minute and resuspended in 12 \u00b5L Tris-Hcl pH 7.85 without NaCl and incubated for 10 minutes. Sequencing was performed on the MinION device using the FLO-MIN106 R9.4 flowcell. The sequencing run lasted 46 hours and produced 3.29 million reads with approximately 12.98 billion bases (for 12 samples).<\/p>\n Base-calling was done with Guppy version 3.6.0 using \u201cdna_r9.4.1_450bps_hac\u201d as the configuration file and \u201cSQK-RBK004\u201d as the barcoding kit.<\/p>\n For assembly, we used Raven version 1.1.5 (Vaser and Sikic, 2020), Wtdbg2 version 2.5 (Ruan and Li, 2019) and Flye version 2.7.1 (Kolmogorov et al<\/em>., 2019). Ten of the eleven barcodes were successfully assembled with Raven. A complete assembly for barcode 105 was possible only with Wtdbg2. Due to the lowest coverage, the assembly of barcode 7 is a combination of the Raven and Flye assemblies, as each of them completely assembled only one contig. All barcodes were later polished with Medaka version 1.0.1 (Medaka) using the model \u201cr941_min_high_g360\u201d and rotated with Circulator version 1.5.5 (Hunt et al<\/em>., 2015). The evaluation was performed using BUSCO version 4.0.6 (Seppey et al<\/em>., 2019), Quast version 5.0.2 (Gurevich et al<\/em>., 2013) and Mauve version 2.4.0 (Darling et al<\/em>., 2004).<\/p>\n <\/a> ▲<\/a><\/p>\n Biotyping identified the tested strains as follows: samples CVI_58, 59 and 60 inconclusive as Brucella suis<\/em> biovar 1 or 3, samples CVI_50, 76, 105, 196 and 213 as biovar 2, sample CVI_71 as biovar 3; sample CVI_72 as biovar 4 and sample CVI_73 as biovar 5.<\/p>\n Bruce-ladder identified all strains as Brucella suis<\/em>. Since Bruce-ladder cannot be used to assign biovars, we used multiplex-suis and RFLP methods to identify the biovar. Both methods identified the strains in accordance with biotyping, except for strains 58, 59 and 60 that were identified solely as B. suis<\/em> biovar 1.<\/p>\n The MLST 21 results are presented in Figure 1 as calculated via BioNumerics using the PubMLST Brucella<\/em> database.<\/p>\n The epidemiological analysis of the samples according to the MLST 21 results compared with international Brucella suis<\/em> samples in the PubMLST Brucella<\/em> database is presented in Figures 2, 3 and 4 as a dendrogram, planar network and minimum spanning tree.<\/p>\n DNA was isolated focusing on reads being as long as possible and DNA concentrations had to be just right to saturate enough pores but not to block them. After isolation, we used about 600 ng for barcoding and library preparation. In total, 1.69 million reads were successfully base-called across twelve barcodes. The statistics of each barcode are outlined in Table 2.<\/p>\n Bioinformation analyses resulted in complete, circular sequences of two chromosomes. The analysis revealed a BUSCO score of >96% and QUAST average accuracy >99.9%. Chromosome sizes differ among biovars. GC% was around 57.2%. Genomes were deposited in NCBI under accession numbers CP054945 \u2013 CP054964.<\/p>\n <\/a> ▲<\/a><\/p>\n The virulence and epidemiology of Brucella<\/em> sp. depend most on the species involved, particularly the species biovar. Bacterial genome sequencing has opened endless questions and possibilities. The key point in the sequencing itself is the correct isolation of DNA. The kit we used was a good choice. The genome structure of the strains tested here is consistent with previous findings: all strains from all biovars tested have two chromosomes, approximately 3.2 Mbp in size. GC content was, as expected, around 57.2% per chromosome. MLST 21 identified new MLST 21 genotypes not yet present in the database (CVI_50, 60, 71, 72, 73, 105 and 196). Species identification, once compared with other isolates in the PubMLST database, gave the expected results. Strains clustered by biovar except for CVI_60 that clustered separately (Figures 2 and 3). Also, biovar 3 and 4 strains cluster closely together but can still be differentiated. Except for referent strains 71, 72, 73 and 213, all strains were regionally specific and unique in the diagnostic sense (Figures 3 and 4). There may be a possibility that this is connected to the unique virulent capabilities that allow them to circulate and survive more easily in this region, though this is merely an assumption. This study was aimed at more detailed identification of strains using new, recently available techniques.<\/p>\n MinION sequencing still has a higher error rate than Illumina, though researchers are improving this platform weekly. Combining Illumina and MinION reads is a perfect solution, rendering long stretches of DNA with MinION that are polished by very accurate Illumina sequences. However, Guppy version 3.6.0 greatly improved base-calling and the results were substantially better and comparable to the available Illumina sequences (Amaradinghe et al<\/em>., 2020).<\/p>\n We conclude that MinION sequencing is a must-have in bacterial species identification. It is faster, highly specific and produces a huge amount of data that can be used for chromosomal investigation, antibiotic susceptibility, strain specificities and more, especially in combination with short-read sequencing like Illumina. Since this is the first documented use of long-read sequencing in Brucella suis identification in this region, the results are very promising, though a future detailed study is required.<\/p>\n Repositories: The assembled genomes have been deposited in NCBI under accession numbers CP054945 \u2013 CP054964.<\/p>\n <\/a> ▲<\/a><\/p>\n This work was supported through the project \u201eBrucella suis CRO TYPE \u2013 genome analysis\u201d financed by Croatian Veterinary Institute\u2019s Fund for dedicated financing of scientific activities in 2018 (authors from CVI) and by the Croatian Science Foundation under the project \u201cSingle genome and metagenome assembly (IP-2018-01-5886)\u201d (authors from FEEC). <\/a> 1.\tAL DAHOUK, S., H. TOMASO, E. PRENGER- BERNINGHOFF et al. (2005): Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR\u2013RFLP). Crit. Rev. Microbiol. 31, 191-196. <\/a> ▲<\/a><\/p>\n Rod Brucella<\/em> biolo\u0161ki je iznimno raznolik, ali genetski vrlo homogen rod bakterija te je ve\u0107 desetlje\u0107ima nepoznanica znanstvenicima. Ove bakterije su veliki javno-zdravstveni problem, a osobito na Balkanu. Klju\u010dne rije\u010di:<\/strong> Brucella suis, bruceloza, svinje, konji, Nanopore MinION, sekvenciranje sljede\u0107e generacije<\/em><\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":" S. Duvnjak, \u017d. Pavlinec, R. Vaser, K. Kri\u017eanovi\u0107, M. \u0160iki\u0107, M. Zdelar-Tuk, I. Reil* and S. \u0160pi\u010di\u0107 Sanja DUVNJAK, BSc,<\/p>\n","protected":false},"author":8,"featured_media":0,"menu_order":11,"comment_status":"closed","ping_status":"open","template":"","format":"standard","meta":{"footnotes":""},"categories":[21],"tags":[1082,1083,37,1084,1085,624],"issuem_issue":[989],"ppma_author":[1065,802,1086,1087,1088,1064,1062,976],"class_list":["post-3639","article","type-article","status-publish","format-standard","hentry","category-original-scientific-articles","tag-brucella-suis","tag-bruceloza","tag-horses","tag-nanopore-minion","tag-sekvenciranje-sljedece-generacije","tag-svinje","issuem_issue-53-1"],"yoast_head":"\n
\nSanja DUVNJAK<\/strong>, BSc, PhD, \u017deljko PAVLINEC<\/strong>, BSc, PhD, Croatian Veterinary Institute, Zagreb, Croatia; Robert VASER<\/strong>, BSc, PhD, Kre\u0161imir KRI\u017dANOVI\u0106<\/strong>, BSc, PhD, Assistant Professor, Faculty of Electrical Engineering and Computing, Zagreb, Croatia; Mile \u0160IKI\u0106<\/strong>, BsC, PhD, Full Professor, Genome institute Singapore, A*STAR, 60 Biopolis St, Singapore; Maja ZDELAR-TUK<\/strong>, DVM, PhD, Irena REIL<\/strong>*, DVM, PhD, (Corresponding author, e-mail: reil@veinst.hr), Silvio \u0160PI\u010cI\u0106<\/strong>, DVM, PhD, Croatian Veterinary Institute, Zagreb, Croatia<\/div>\n![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/03\/pdf.png\")
<\/a>https:\/\/doi.org\/10.46419\/vs.53.1.9<\/a><\/div>\n<\/p>\nAbstract<\/a>Introduction<\/a>Materials and methods<\/a>Results<\/a>Discussion<\/a>Acknowledgements<\/a>References<\/a>Sa\u017eetak<\/a><\/span><\/div>\n\n
Abstract<\/h2>\n
\n
\nThis study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis<\/em> 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.
\nMolecular 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<\/em> infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results.
\nOxford 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.
\nMLST 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.
\nMLST 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<\/em> 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.
\nMinION sequencing proved to be a strong, accessible solution for species determination.
\nFuture study is required to determine how detailed genome information it can give, considering the error rate.<\/p>\nIntroduction<\/h2>\n
\n
\nUnfortunately, this is not the case (Taleski et al<\/em>., 2002; Pappas, 2010).<\/p>\n
\nClassical bacteriological methods are the gold standard for identification and classification. Since these methods are time-consuming, highly subjective and dangerous, molecular methods have been in use for over two decades.
\nIn the case of Brucellae<\/em>, classical multiplex PCR and genotyping technics like MLVA and MLST are the most frequently used (Spicic et al<\/em>., 2010; Duvnjak et al<\/em>., 2015).<\/p>\n
\nThe quality of its reads is still unmatching, library preparation takes hours and PCR is necessary, and the sequencing is not possible in the field. Further, the reads are usually small (max 300 bp) and the first results are expected after approximately 50-60 hours.<\/p>\n
\nThis enables the sequencing of very long individual DNA molecules and reading in real-time. The device itself is pocket- sized and library preparation can take just 10 minutes, making it a powerful tool for on-site real-time sequencing. The error rate is higher than for Illumina, though it enables denovo assembly of whole and complex genomes because of the long stretches it produces (Goodwin et al<\/em>., 2016). Its low price and constant improvements enable this device to become a better research tool that is being used to build and explore model (Tyson et al<\/em>., 2017) and non-model organisms (Quick et al<\/em>., 2017).<\/p>\n
\nHowever, the error rate is higher than Illumina sequencing. Therefore, the objective of this study was to determine its applicability for species and biovar typing of Brucellae<\/em>.<\/p>\nMaterials and methods<\/h2>\n
\nB. suis<\/em> strains<\/h3>\n
![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/table01-efficacy-of-ngs.png\")
Methods<\/h3>\n
\nBacterial cells were collected in nuclease- free water and resuspensions made with an absorbance of approximately 0.55 at 600 nm (Densimat, Biomerieux).<\/p>\n
\nThe cycling regime differed from test to test but was done according to references. Amplifications were performed on the ProFlex thermocycler (Applied Biosystems, USA). For RFLP enzyme restriction, 20 \u00b5L reaction mixture contained 5 \u00b5L amplified DNA, 5U restriction enzyme, 2 \u00b5L associated buffer (Fermentas, Burlington, Canada) and 12.5 \u00b5L distilled water (Dnase\/Rnase Free Distilled Water, GIBCO, Invitrogen, Paisley, UK). Digestion was done at 37\u00baC for 3 hours. Restriction products were analysed using capillary electrophoresis on the QIAxcel system (QIAGEN, Hilden, Germany) using High-Resolution DNA cartridge with size markers 100-2500 bp.<\/p>\n
\nWe prepared the sequencing library using the Rapid Barcoding Kit (ONT) with modifications to the RBK_9054_v2_revC_23Jan2018 protocol.<\/p>\n
\nThe remainder of the protocol was conducted on 10 \u00b5L obtained DNA suspension. The samples were barcoded and sequenced.<\/p>\nIn-silico analysis<\/h3>\n
Results<\/h2>\n
\n![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/figure01-efficacy-of-ngs.jpg\")
<\/a>![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/figure02-efficacy-of-ngs-864x1024.jpg\")
<\/a>![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/figure03-efficacy-of-ngs.jpg\")
![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/figure04-efficacy-of-ngs.jpg\")
\nWe used approximately 1 \u00b5g DNA for sequencing on the flowcell according to Tapestations calculations.<\/p>\nIn silico analysis<\/h3>\n
![\"\"](\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/08\/table02-efficacy-of-ngs.png\")
Discussion<\/h2>\n
\n
\nThe results from this study show that biotyping is not the most suitable method for biovar assignation.<\/p>\n
\nAccording to Tapestation 2200 (Agilent), DNA was of good concentration and length, enabling good sequencing output and resulting in good in silico analysis.<\/p>\n
\nThese findings are similar to those observed by other authors (Jumas-Bilak et al<\/em>., 1998).<\/p>\n
\nUnlike MLVA typing (Duvnjak et al<\/em>., 2015), MLST 21 seems to be sensitive enough for biovar typing of Brucella suis<\/em> strains. Separation of CVI_60 might be explained through Nanopore error rate though this assumption requires further investigation. Biovar 1 strains 58, 59 and 60 clustered with biovar 1 strains from the USA, Argentina, Mexico, Colombia, and Switzerland. Biovar 2 strains 50, 76, 105, 196 and 213 clustered with biovar 2 strains from Croatia and also with international strains, mostly from Europe. The referent strains of biovar 3, 4 and 5 (origin Slovenia) clustered with other strains of a certain biovar (Figure 4). It is also clear that the differences between biovars are minor, which might explain the reason why biovars are so difficult to identify, especially using non-molecular methods.<\/p>\nAcknowledgements<\/h2>\n
\n
\nWe are grateful for the excellent technical support of Marijana Novosel from the Croatian Veterinary Institute, Zagreb, Croatia.<\/p>\n
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\n24.\tMEDAKA: https:\/\/github.com\/nanoporetech\/ medaka
\n25.\tOIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2012): Chapter 2.8.5. Porcine brucellosis http:\/\/www.oie.int\/fileadmin\/Home\/ eng\/Health_standards\/tahm\/2.08.05_PORCINE_ BRUC.pdf (cited 26.4.2013.)
\n26.\tOSTERMAN, B. and I. MORIYON (2003): International committee on systematics of prokaryotes; subcommittee on the taxonomy of Brucella: minutes of the meeting, 17 September 2003, Pamplona, Spain. Int. J. Syst. Evol. Microbiol. 56, 1173-1175. Documentation of the Brucella nomenclature committee to return to species designations in an effort to document their usefulness in avoiding potential harm.
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\n<\/em><\/p>\n<\/div>\n\n
U\u010dinkovitost sljede\u0107e generacije sekvenciranja u dijagnostici bakterijskih zoonoza<\/h2>\n
\nDr. sc. Sanja DUVNJAK<\/strong>, dipl. ing., dr. sc. \u017deljko PAVLINEC<\/strong>, Hrvatski veterinarski institut, Zagreb, Hrvatska; dr. sc. Robert VASER<\/strong>, dipl. ing., dr. sc. Kre\u0161imir KRI\u017dANOVI\u0106<\/strong>, dipl. ing., docent, Fakultet elektrotehnike i ra\u010dunarstva, Zagreb, Hrvatska; dr. sc. Mile \u0160IKI\u0106<\/strong>, dipl. ing. redoviti professor, Institut za genom Singapur, A*STAR, 60 Biopolis St, Singapur; dr. sc. Maja ZDELAR-TUK<\/strong>, dr. med. vet., dr. sc. Irena REIL<\/strong>, dr. med. vet., dr. sc. Silvio \u0160PI\u010cI\u0106<\/strong>, Hrvatski veterinarski institut, Zagreb, Hrvatska<\/div>\n
\n
\nPravilno prepoznavanje i razumijevanje patogena klju\u010dan je korak u epidemiologiji i epizootiologiji bilo koje bakterijske vrste, \u010dija identifikacija mo\u017ee biti izazovna, osobito u slu\u010daju zoonotskih vrsta. Cilj je ovog rada bio implementirati sekvenciranje \u010detvrte generacije u tipizaciji 11 sojeva Brucella suis<\/em> koje se \u010duvaju u na\u0161oj arhivi te ovu metodu usporediti s klasi\u010dnim i molekularnim metodama koje se trenuta\u010dno primjenjuju, a ne zasnivaju se na sekvenciranju.
\nKlasi\u010dno je biotipiziranje vrlo subjektivno i dalo je podvojene rezultate za 3 soja.
\nOd molekularnih metoda koristili smo vi\u0161estruku lan\u010danu reakciju polimerazom (engl. Polymerase Chain Reaction<\/em>, PCR) i polimorfizam duljine restrikcijskih fragmenata (engl. Restriction Fragment Lenght Polymorphism<\/em>, RFLP) budu\u0107i da niti jedna od metoda ne mo\u017ee zasebno identificirati i vrstu i biovar, a \u0161to je va\u017eno u slu\u010daju Brucella suis<\/em> infekcije. Vrsta i biovar svih sojeva uspje\u0161no su potvr\u0111ene i u skladu s rezultatima biotipizacije.
\nSekvenciranje sljede\u0107e generacije (engl. Next Generation Sequencing<\/em>, NGS) provodili smo na Oxford Nanopore MinION ure\u0111aju koji sekvencira duge lance DNK. Za sastavljanje genoma rabljeni su razli\u010diti algoritmi, a za identifikaciju i analizu rezultata MLST-a kori\u0161ten je softver BioNumerics 8.0. MLST 21 je kori\u0161ten za identifikaciju biovara i epidemiolo\u0161ku usporedbu ispitivanih sojeva.
\nGenomi su bili veli\u010dine 3,2 Mb i sastavljeni u dva kromosoma. Analiza MLST 21 smjestila je na\u0161e sojeve u vrsne i biovarne skupine u skladu s drugim kori\u0161tenim molekularnim testovima. Koliko je nama poznato, ovo je prva dokumentirana uporaba sekvenciranja dugih lanaca DNK u identifikaciji Brucella suis<\/em> u jugoisto\u010dnoj Europi. Zaklju\u010dujemo da je bakteriolo\u0161ka biotipizacija zastarjela i da je identifikacija biovara u ovom rodu, ovisno o doma\u0107inu, neto\u010dna te da je molekularna karakterizacija uvijek sigurnija, br\u017ea i prikladnija opcija. MinION sekvenciranje pokazalo se kao vrlo pristupa\u010dno rje\u0161enje za odre\u0111ivanje vrste i biovara Brucella suis<\/em>.
\nDaljnja su istra\u017eivanja potrebna da bi se ustvrdilo koliko detaljne informacije o genomu ono mo\u017ee dati, imaju\u0107i u vidu zna\u010dajniji postotak pogre\u0161ke prilikom sekvenciranja.<\/p>\n