Iranian Journal of Medical Microbiology

Brucellosis is the most common zoonotic disease that infects animals and humans through contaminated animals and their products. The disease is caused by an intracellular small gram- coccobacillus of the genus Brucella, which has no capsules, flagella and spores  cause destructive disease that leads to great economic losses to the livestock industry by reducing milk production Humans are usually infected through consumption of contaminated dairy products or close contact with infected animals (1). Human infection can lead to a chronic debilitating disease whose nonspecific symptoms affect various organs (2, 3). The genus Brucella has six classic species, including: . Brucella abortus, Brucella melitensis, Brucella ovis, Brucella suis, Brucella neotomae, Brucella canis. B. melitensis, B. abortus, B. canis, and B. suis can cause brucellosis in human (4). In addition, six non-classical species are known as Brucella species. Reports published in the last decade in Iran have introduced B. melitensis as the main pathogen responsible of brucellosis in human. Although. B. abortus was also involved to much lesser extent (6, 5). Rapid and accurate identification of Brucella species can indicate geographical and host origin. Currently, differentiation between species and biovars of Brucella are done using various analyze according to phage typing, phenotypic characteristics of lipopolysaccharide antigen, color sensitivity, need for carbon dioxide, the production of sulfide-hydrogen gas and metabolic properties (7). Diagnosis and detection of brucellosis based on biochemical tests is inefficient to differentiate Brucella species due to the presence of unknown behavioral strains in these experiments (8). Also, these experiments are potentially dangerous, complex and time consuming for laboratory technicians. Furthermore, Brucella identification complication can occurr due to similar differences between some species and inconstancy in reporting several phenotypic traits (9, 10). The use of genetic characteristics has been investigated using molecular DNA technology to address these shortcomings. Many PCR-based methods have been developed for rapid identification of Brucella species. For this purposes, molecular tests of the genome using the BCSP31 gene or 16SrRNA-23S operon are sufficient (11, 12), but for the other aims such as epidemiological tracking, more accurate methods are required. Due to the high DNA homology (above 90%) among Brucella species (14, 13), the significant genetic differences are mononucleotide polymorphisms, and regions with high genomic diversity between species are very rare (15). Several PCR-based methods have been used to determine the exact molecular biomarkers to determine Brucella molecular type (12). Various studies have reported that the use of the RNA polymerase (rpoB) β subunit gene, which is very suitable for phylogenetic analysis and identification Brucella strains, especially in highly homologous isolates (8, 15, 16 RpoB-based genotyping also allows the identification of new bacterial species and analysis  of the bacterial community (16). It can also describe rpoB gene mutations that play a very important role in rifampicin resistance (17). the present study evaluates the effectsof single nucleotide polymorphism (SNP) assays on the rpoB gene to show intra-species diversity occurring in different B. melitensis strains collected from various regions of Iran. (this part of manuscript is too long).

 

Sampling and bacteria isolation
In this study, a collection of 106 blood and 2 cerebrospinal fluid samples from patients with brucellosis symptoms from2017-2019 were cultured by the Brucellosis Department of Razi Vaccine and Serum Research Institute (Karaj, Iran). All samples were cultured in selective Brucella agar medium with Cycloheximide (50.0mg), Vancomycin (10.0mg), polymyxin B (2,500 IU), Bacitracin (12,500 IU), Nystatin (50,000 IU), and Nalidixic acid (2.5mg) (Oxoid, Basingstoke, UK) and 5% of inactivated horse serum. Bacterial cultures were incubated for 10 days at 37 ° C and 10% carbon dioxide. After this steps , the isolated bacteria were identified by classical typing methods (5, 18). Brucella isolated from patients and standard bacterium of B.melitensis 16M (ATCC 23456) were then used in this study to analyze rpoB gene by single nucleotide polymorphism.  Genomic DNA extraction was performed based on the manufacturing protocol by a high purity PCR template preparation kit (Roche, Germany) on isolated bacteria. The integrity of DNA was evaluated by 1% agarose gel and the concentration of DNA was assessed at 260/280 nm by a Nanodrop Spectrophotometer (Wilmington, DE, USA). The DNA of bacterial samples were then preserved at − 20 °C for analysis.

Molecular confirmation of isolated bacteria
Molecular identification of isolated bacteria in Brucella agar-specific medium was performed based on the IS711-based polymerase chain reaction (AMOS-PCR) and Bruce-ladder PCR were carried out on all extracted DNA to analyze the Brucella presence in samples. Amos-PCR amplification using 5 primers on the IS711 gene (Table 1) was done in a thermal program of 1 cycle of 95 ° C for 5 minutes, followed by 40 cycles of denaturation at 95 ° C for 30 seconds, annealing temperature at 55 ° C for 60 seconds, extension at 72 ° C for 3 minutes and final extension at 72 ° C for 10 minutes (19). Molecular typing also was performed by multiplex PCR (Bruce-ladder) with 16 primers on 8 different Brucella genes (Table 1) as follows: 95 ° C for 5 minutes, followed by 30 cycles at 95 ° C for 30 minutes,56 ° C for 90 seconds, 72 ° C for 3 minutes and at 72 ° C for 10 minutes (4). All reactions of PCR were conducted in a total volume of 25 μL including 0.2 mM deoxynucleotide triphosphate, 0.5 mM of each primer, 10 mM Tris–HCl (pH 8), 50 mM KCl, 1.5 mM MgCl₂, and 0.05 IU of Taq polymerase. The PCR products were run by electrophoresis on the 1.5 % agarose gel. All applied primers in this study are mentioned in the table 1.

PCR assay on the rpoB gene and its genotyping assignment
The PCR amplifications of the rpoB gene was done with specific primers using the B. melitensis 16M (accession number AE009516) as reference. Upstream primer, 1rB (5-ATGGCTCAGACCCATTCTTTC-3), and a downstream primer, 4134rB (5-TTATTCTGCCGCGTCCGGAA-3) were used to amplify the whole length of rpoB gene with 4,134-bp (17). PCR amplifications were performed with 25 μL of PCR mixture comprised of 10 mM Tris–HCl (pH 8), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM deoxynucleotide triphosphate, 0.05 IU of Taq polymerase, 0.5 mM of each primer and 100 ng of genomic DNA (evaluated by O.D. at 260 nm). To determine the rpoB types of different B. melitensis phenotypic biovars, the rpoB molecular targeting of specific residues on the codons 629, 985, 1249, and 1309 were performed using Mega 6 software (20).

Sequence analysis
The purification of PCR products were performed by PCR product purification kit (GeneAlI Company, South Korea) and sent to Fazapjooh Company for sequencing. The rpoB sequences were then aligned and assembled by the Mega 6 software program (21). In addition, the amino acids were deduced by the CLUSTAL W method of the Mega 6 program. After that all generated sequences with specific primers and length of 4134 bp, were compared by rpoB gene of B. melitensis 16M (accession number AE009516) for evaluation of nucleotide diversity. Sequencing was performed twice to confirm the results of reported mutations in this study.

Phylogenetic analysis
The consensuses data of all sequences were evaluated using the NCBI (Blast) alignment search tool to confirm rpoB genes matching. A phylogenetic tree was then drawn with Bootstrap 1000 (22). Bootstrap values from 1000 Bootstrap datasets were displayed at the end of the nodes.