گزارش گونه‏ های نوکاردیای جدا‏سازی شده ازخاک، توسط تعیین توالی ژن 16S rRNA، اصفهان، ایران

Introduction

The genus Nocardia belongs to the family Nocardiaceae, and members of the genus are all aerobic, Gram-positive, modified acid fast and non-motile actinomycete that form filamentous branched cells which fragment into pleomorphic rod-shaped or coccoid elements, non-spore-forming (1 & 2) catalase and urease positivebacteria that belong to Actinomycetes group (3). Soil is one of the best sources for maintaining Nocardia. However, these organisms are found in environments such as air, water, plants and rotten materials. Nocardia may be acquired in dry, dusty and often windy locations (2). Nocardia species are associated with opportunistic infections in human and animals that might become fatal. Anyway, some species can infect both immune-compromised and immune-competent individuals (4).

The genus is comprised of 105 acknowledged species according to the National Center for Biotechnology Information (NCBI) and German Collection of Microorganisms and Cell Cultures is called DSMZ. The process of using phenotypic and biochemical methods to identify Nocardia is difficult and tedious.

Moleculartechniques were developed in the 1990s, including a 16S rRNA gene PCR-based. Moleculartechniques were developed in the 1990s, including a
16S rRNA gene PCR-based technique for distinguishing the genus Nocardiaamong aerobic actinomycetes (5 & 6).

Members of these taxa organize a different phyletic line in the 16S rRNA tree that can be distinguished from one another by using a combination of morphological and biochemical features (7). rRNA genes are used to approximate evolutionary history and taxonomic designation of individual organisms in a wide range. The choice of rRNA genes as optimal tools for such purposes are based on both observations and presumptionsof ribosomal conservation.rRNA genes are necessarycomponents of the ribosome, which include 50 proteins and three classes of RNA molecules. In bacteria, three rRNA genes are categorized into a gene cluster which is expressed as a single operon and may be present in multiple copies in the genome (8). The 16S rRNA is nearly 1, 500-nucleotides sequence encoded by the 16S ribosomal RNA (rRNA). The sequence is an extremely conserved gene in which common areas covered in all living beings existed while nucleotide variations are concentrated on specific regions and also the hyper variable regions, which are defined by variability of specific species (9). To recognize various species of microorganismsthat use 16S rRNA hyper-variable regions called V1- V9, sequence analysis of the 16S rRNA gene was used to identify bacterial species and achieve taxonomic studies in a wide range (10 & 11).

Unfortunately, 16S rRNA hyper-variable regions perform different degrees of sequence diversity, and hyper-variable regions cannot distinguish all bacteria solitarily, and molecular diagnostic techniques, such as real-time PCR or melting temperature analysis, must be used (11). This paper was based on identification of Nocardia species by 16S rRNA gene sequencing, some of which were first reports from Iran soil at the time of isolation and detection, Such as
N. coubleae, N. cummidelens and
N. cyriacigeorgica. Then data regarding these isolates were entered inNCBI Database by Sequin software, and phylogenic tree and similarity matrix were drawn for isolates.

Materials and methods

A total of 70 soil samples were collected from several different locations in Isfahan’s suburb hospital areas, parks, agricultural lands, gardens and arid lands at different months of year. Fifty-gram of soil samples were collected from 3 cm to 5 cm depth. Soil samplestemperatureand the pH were measured. All samples were stored at low temperature (4°C) until they were tested (2).

Isolation methods: Slip-buried Method and Some characteristics of Phenotypic include the following:3-5 g of soil was added to 10 ml normal saline. Tubes were shaken for 3 min and the suspensions were incubated for 15 min in room temperature. 3-5 ml of the supernatant solution was transferred to another sterile tube by sterile pipette.

The streptomycin/ chloramphenicol solution(half of the total volume) was added to the supernatant. The mixture was incubated for ½ h. One drop (0.05 ml) of each sample was cultured on BHI agar with 5% human blood medium (12 & 13)and checked for hemolysis. Cycloheximide (0.5 g/l) and kanamycin (25 mg/l) was added to the tube or plate immediately. They were shaken and were incubated at 37°C for 2 weeks (2), Afterwards some of the Phenotypic characteristics were studied (7 & 14) such as: Modified acid fast, Growth in Lysosyme, Hydrolysis of Tyrosine, Xanthine, Hypoxanthine and Uric acid.

DNA extraction techniques: In this study, DNA extraction method was Microwave oven method (15) and as mentioned before, it was tested with Nocardiaasteroides DSM 43757-type strain as standard. In Microwave method, based on studies done by Salgado et al., the suspension was made with a single bacterial colony in 20 µl of deionized water subjected to 800 watt (W) microwave oven for 10, 15 and 20 seconds (S) at different potencies: 80, 160, 240, 320, 400, 560, 720 and 800 watts. An electrophoresis with agarose Gel 1% was applied to survey the presence and quality of the extracted DNA. In the present study, a suspension was made with single bacterial colony in 70 µl of deionized water. Then, suspensions were treated in a microwave oven for 10, 13, 20 seconds at 360 and 540 watts. Subsequently, the suspensions were put in -20°C.

Polymerase Chain Reaction (PCR) technique for 16SrRNA gene: PCR was implemented according to the previously explained (16 & 17), using universal Forward primer: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and Reverse primer: 1492R:5'-GGTTACCTTGTTACGACTT-3'.These 1500 bp PCR products were amplifiedby 27F and 1492R primers. PCR was carried out by using Prime Taq Premix (2X) kit (GENET BIO). In a final volume of 20 µl (10 µl 2XPrime Taq Premix, 1 µl F primer, 1 µl R primer, 6 µl sterilized deionized water), 2 µl of DNA was extracted. Amplification was done in thermal cycler (Eppendorf) programmed as the following: 5 min initial denaturation step at 94⁰C, pursued by 30 cycles (94⁰C for 30 Sec, 58⁰C for 30 Sec, 72⁰C for 60 Sec), and a 5 min final extension step at 72⁰C. The bands were evaluated in position of 1500 bp by 1% agarose gel electrophoresis. In the last step, 16S rRNA gene sequencing (11 & 18) and its analysis was done with proper software (11).

Molecular detection and identification of Nocardia isolates using sequencing of
16S rRNA: For preciseidentificationof Nocardia isolates, all of microorganisms wereidentified using PCR of a 1500-bp region of the 16S rRNA gene. Then, the samples were sequenced. 16S rRNA gene standard sequences of various species of Nocardia were received from international databases(NCBI) and the sequences of the isolates obtained in this study were aligned in the CLC Main Workbench 6 software.BLASTN was done onForward (F) sequences andReverse complement (RR) sequences in thedatabase of NCBI. After theBLAST, the Forward Reverse complement sequences and their related dendrogram, whichthecompanyhadsent, were comparedwith each other.

Positioningwas regardedmanually through positions of standard sequence (subject) close to the species obtained in this study which were obtained from NCBI databases. Forward sequences andReverse complement sequences were assembled manually in the Microsoft Word. After assembling these sequencesin the NCBI, BLASTN were blasted and in amoment, they could be examined in terms of percentage of similarity with thebacteriaspecies close to identified species in this study.Bacteria species, which had greater Max identity, Query coverage, less Gap and sequences with E-values close to zero are as our considered sequences in this research. The corrected sequences wereentered and then aligned in the CLC Main Workbench 6 software. Next, Fastaformat were made for entery of thegenes intheNCBIGenBank, phylogenetic tree and Matrix similarity.Nucleotide sequences accession numbers: N. cyriacigeorgica,
N. asteroides, N. cummidelens,
N. asteroides, N. asteroides, N. coubleae sequences in this study were submitted to the GenBank under the accession numbers KC577151, KC577152, KC577153, KC577154, KC577155, KC577156 for the 16S rRNA gene respectively.

Results

The initial isolation was identified by phenotypicisolation methods, such asModified acid fast, Growth in lysozyme, Hydrolysis of Tyrosine, Xanthine, Hypoxanthine and Uric acid (19).

To identify more precisely, PCR-Sequencingwas performedfor isolates of16S rRNA gene. A total of six isolates were obtained via the 16S rRNA gene sequencing. 16S rRNA gene sequencesrecorded in GenBank of NCBI and Accession numberswere received. Nucleotide sequences accession numbers as mentioned are in the following.
N. cyriacigeorgica, N. asteroides,
N. cummidelens, N. asteroides, N. asteroids and N. coubleae sequences in this study were submitted to the GenBank under the accession numbers KC577151, KC577152, KC577153, KC577154, KC577155, and KC577156 for the 16S rRNA gene respectively. In the following, isolated strains, N. cyriacigeorgica KC577151,
N. asteroides KC577152, N. cummidelens KC577153, N. asteroide KC577154,
N. asteroides KC577155, and N. coubleae KC577156 were compared with NCBI closely related type species, such as
N. cyriacigeorgicaDSM 44484T,
N. asteroidesDSM 43757T,
N. cummidelens DSM 44490T,
N. asteroids DSM 43757T,
N. asteroidesDSM 43757T, and
N. coubleae OFN N11 (data not shown), and these results were found 100, 100, 97, 100, 100 and 99% similarity with these strains and gaps in all of the isolated strains were 0%, respectively.The phylogenetic tree was constructed with the MEGA5software package with tree algorithms (Fig. 1), namely the neighbour-joining (20). The tree topology was determined by using 1000 bootstrap datasets. The evolutionary distances were computed using the Jukes-Cantor method (21).

Fig. 1: Neighbor-joining tree, inferred using MEGA 5 (20 & 22), based on 16S rRNA genesequences available from GenBank NCBI (accession numbers are shown in tree), as determined from 1000 bootstrap samples(1000 replicates), is indicated by percentages at each node The scale bar indicates 0.005 substitution per nucleotide position. The evolutionary distances were computed using the Jukes-Cantor method (21) and are in the units of the number of base substitutions per site. There were a total of 1501 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (23). Afterwards, Similarity values for 16SrRNA gene sequences of isolates of Nocardia with that of closely related type species was depicted by CLASTAL2.1 Multiple Sequence Alignments Server[1].

Table 1: Similarity values for 16S rRNA sequences of isolates of Nocardia with that of closely related type species by CLASTAL2.1 Multiple Sequence Alignments Server.