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

نوع مقاله: پژوهشی- انگلیسی

نویسندگان

1 دانشجوی دکترای تخصصی میکروب‏شناسی، دانشگاه‌علوم‌ پزشکی اصفهان، ایران

2 دانشیار میکروب‏شناسی، دانشگاه‌علوم‌ پزشکی اصفهان، ایران

چکیده

مقدمه: جنس نوکاردیا جزو اکتینومیست‏های هوازی هستند که یک گروه بزرگ از باکتری‏های ساکن خاک را شامل می‏شوند، که در جهان پخش شده‏اند. در این مطالعه، با استفاده از آزمون‏های تشخیصی رایج، مرسوم و مولکولی گوناگون، چندین ایزوله‏های نوکاردیا جداسازی و تشخیص داده شد.

مواد و روش ‏‏ها: در این پژوهش، برای جداسازی از خاک، روش Slip-buried و برای استخراج DNA، روش Microwave oven استفاده شد و در این روش‏ها، سویه استاندارد DSM 43757 Nocardia asteroides استفاده شد. در ادامه، روش PCR با استفاده از پرایمرهای عمومی انجام شد.

نتایج: با تحلیل اطلاعات فنوتیپی و مولکولی، به‏ویژه تعیین توالی ژن 16S rRNA، ژن‏های نوکاردیای‏های جداسازی شده از خاک اصفهان، که در ادامه به آن‏ها اشاره شده است، درپایگاه داده NCBI باAccession number مربوط به هر ژن ثبت شدند. نوکاردیاهای ثبت شده در NCBI:

Nocardia cyriacigeorgica KC577151, Nocardia asteroides KC577152, Nocardia cummidelens KC577153, Nocardia asteroides KC577155, Nocardia asteroides KC577155, Nocardia coubleae KC577156 ..

بحث و نتیجه ‏گیری: در این پژوهش 6 گونه نوکاردیا جداسازی و تشخیص داده شد، که برخی از این گونه‏های جداسازی شده از خاک ایران، اصفهان، در زمان جداسازی و تشخیص،گونه‏های جدیدی بودند. برای تشخیص بیشترگونه‏های مختلف نوکاردیا می‏توان در مطالعات بعدی از تکثیر و تعیین توالی ژن‏های دیگر این باکتری از جمله ITS,hsp65 secA, rpoB  و sod بهره گرفت. 

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Report of Nocardia species isolated from soil by 16S rRNA gene sequencing method in Isfahan, Iran

نویسندگان [English]

  • Samane Bourbour 1
  • Jamshid Faghri 2
  • Sharare Moghim 2
1 M.Sc. of Microbiology, Isfahan university of Medical Sciences, Iran
2 Associate professor of Microbiology, Isfahan University of Medical Sciences, Iran
چکیده [English]

Introduction: The genus Nocardia belongs to aerobic Actinomycetes. They are a large group of soil-dwelling bacteria that are distributed worldwide. Using various key conventional and molecular diagnostic tests, several Nocardia isolates were identified, characterized and distinguished.
Materials and methods: In our study, soil isolation method was Slip-buried typeand DNA extraction was obtained through Microwave oven method and Nocardia asteroides DSM 43757-type strain as standard was tested to approve the above method. Following this study, Polymerase Chain Reactionwas carried out using universal primers.
Results: Phenotypic and molecular data analysis, particularly 16S rRNAgene sequencing, provided evidence on Nocardia cyriacigeorgica KC577151, Nocardia asteroides KC577152, Nocardia cummidelens KC577153, Nocardia asteroides KC577154, Nocardia asteroides KC577155, Nocardia coubleae KC577156 involvement in Iran soil in Isfahan and these isolates were eventually registered in NCBI gene bank.
Discussion and conclusion: In this research, six Nocardiaspecies were isolated and identified some of which isolated species were novel in Iran soil in Isfahan, at the time of isolation and detection. To identify more species of Nocardia in subsequent studies, proliferation and sequencing of other genes of the bacteria such as hsp65, ITS, secA, rpoB and sod can be applied.

کلیدواژه‌ها [English]

  • Novel Nocardia species
  • 16S rRNA sequencing
  • Soil samples
  • Isfahan
  • Iran

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.

% Similarity to

Taxon

1

2

3

4

5

6

7

8

9

10

11

1. N. asteroides

DSM 43757

-

 

 

 

 

 

 

 

 

 

 

2. N. cyriacigeorgica DSM 44484

98.53

 

 

 

 

 

 

 

 

 

 

3. N. cummidelens DSM 44490

97.45

96.72

 

 

 

 

 

 

 

 

 

4. N. soli

DSM 44488

97.45

96.72

100.00

 

 

 

 

 

 

 

 

5. N. coubleae

OFN N11

96.75

96.14

98.41

98.41

 

 

 

 

 

 

 

6. N. cyriacigeorgica

(KC577151)

98.27

100.00

96.54

96.54

95.08

 

 

 

 

 

 

7. N. cummidelens

(KC577153)

93.44

91.90

96.51

96.51

96.23

91.90

 

 

 

 

 

8. N. asteroides

(KC577155)

100.00

98.39

97.29

97.29

95.84

98.24

93.44

 

 

 

 

9. N. coubleae

(KC577156)

95.22

94.56

97.89

97.89

98.56

94.56

82.26

95.22

 

 

 

10. N. asteroides

(KC577152)

100.00

98.36

97.24

97.24

95.69

98.36

93.44

100.00

95.22

 

 

11. N. asteroides

(KC577154)

100.00

98.32

97.17

97.17

92.36

98.32

89.80

100.00

95.22

98.24

-

 


Discussion and conclusion

The aim of this study was isolation, phenotypic and molecular identification of various Nocardia species from soil samples of Isfahan suburb hospitals.Since these organisms are widely distributed in environment and fragmented nocardial cells may be scattered and increased their acquisition through the respiratory path and favor traumatic inoculation in both immune-compromised and immune-competent hosts, and as they may sometimes be mistaken with tuberculosis, even these bacteria may be fatal. Thus, we decided to isolate these bacteria from surrounding soils of Isfahan hospitals to find a connection between environmental isolates from clinical isolates of these bacteria in future, and then keep patients under more intensive care (2 & 24). At the time of the study, other researches which had been done with regard to this bacteria in Iran were mostly on clinical isolates or those merely isolated by phenotypic tests, which are mentioned in the following. In the present study, as mentioned, Slip-burid-method was surveyed for isolation of Nocardia from soil. Indeed, in our study
N. cyriacigeorgica KC577151,
N. asteroides KC577152, N. cummidelens KC577153, N. asteroide KC577154,
N. asteroides KC577155, N. coubleae KC577156 species were isolated from Isfahan, Iran soil samples. Herein, at the time of the study, N. cyriacigeorgica KC577151, N. cummidelens KC577153, and N. coubleae KC577156 were reported for the first time from Isfahan, Iran soil. Due to high similarity in 16S rRNA gene variable regions, Nocardia cummidelens were not discriminated from Nocardia soli. The following several studies refer to this field. In a study done by Trivisan et al. in 1889, Nocardia asteroides were isolated from clinical specimens. In 2001, a study done by Corring Yassin presented Nocardia ciriacigeorgica from bronchial secretions in Germany (25) as well as in 2011, Shojaei reported this species from clinical specimens in Iran. In Shojaei, full sequence of 16S rDNA and partial sequence of hsp65 were used. 16S rDNA showed 100% similarity with
N. cyriacigeorgica DSM 44484T and
N. asteroides typeVI and hsp65 gene sequence showed 100% similarity with
N. cyriacigeorgica DSM 44484T and 99.55% with N. asteroides typeVI. (26). In 2001, Maldonado spotted Nocardia cummidelens from soil and water in England. In Maldona findings, According to chemical and morphological features, Twenty-eight isolates were nocardiae. These organisms formed a monophyletic clade in the 16S rDNA tree with Nocardia salmonicida. Due to genotype data, three of the strains, isolates S1, W30 and R89 were distinguished from one another. These organisms were called Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. And Nocardia soli sp. nov. respectively (27). In 2007, Rodriguez Nava reported Nocardia coubleae from oil-contaminated soil in Kuwait, in which they detected two bacterial isolates (OFN N11 and OFN N12T) based on a multi-genic method that included 16S rRNA, hsp65 and sod gene sequencing. The closest species was Nocardia ignorata (with 99.4, 99.5 and 98.6% gene sequencelikeness to
16S rRNA, hsp65 and sod genes, respectively). In their study, novel isolates were recognized phenotypically from the type strains of the genus Nocardia. The novel isolates had 26% relatedness to type strain of N. ignorata in DNA–DNA hybridization experiments. On the basis of genotypic and phenotypic methods, two novel species were named Nocardia coubleae sp. nov. with type strain OFN N12T (14). In a 2012 study by Kachuei, Nocardia was presented in Isfahan, Iran. They studied on Nocardia with conventional biochemical tests and physiological Characteristics (14). In Kachuei’s study, from 153 (19.1%) Nocardia isolates identified, Nocardia asteroids complex (45.5%) and Nocardia brasiliensis (24.7%) were the most frequently isolated species, followed by Nocardia otitidiscaviarum (2.2%), Nocardiopsis dassonvillei, Actinomadura actinomadura (each 1.7%) and Nocardia transvalensis (1.1%) and also unknown spp. (23.0%) (2). In the present study, Nocardia cyriacigeorgica, Nocardia asteroides, Nocardia cummidelens, Nocardia coubleae were isolated from Isfahan soil. Among these species, Nocardia cyriacigeorgica, Nocardia cummidelens, and Nocardia coubleae were isolated from Isfahan soil, Iran for the first time and their 16s rRNA genes were submitted to the Gene Bank of NCBI database and were recorded there. Afterwards, relevant phylogeny or evolutionary tree was depicted using Molecular Evolutionary Genetics Analysis version 5 (MEGA5) Software (20) based on 16S rRNA gene sequences available from NCBI Gene Bank. K Tamura (23) and Patrycja Golinska (28) also did analysis using MEGA5 Software. Then, similarity values were plotted for 16S rRNA sequences of isolates of Nocardia with that of closely related type species by CLASTAL2.1 Multiple Sequence Alignments Server. While Nocardia species genetic diversity was demonstrated using the NJ phylogenetic tree generated from the 16srRNA sequences, as determined from 1000 bootstrap samples (1000 replicates), and indicated by percentages at each node, the scale bar indicates 0.005 substitution per nucleotide position. To summarize in this research, we isolated three Nocardia novel species for the first time from Isfahan soil by PCR-Sequencing. Moreover, their 16s rRNA genes were submitted to the Gene Bank of NCBI database and were recorded there.

Consequently, we suggest for better identification of different species of this bacteria, Appling the sequencing
16S rRNA, hsp65, ITS, secA, rpoB and sod genes; moreover, analyzing the bacteria cell wall by using relevant technique, such as HPLC, but it cost you a lot of money.

Acknowledgment

This study was supported by Grant No.390510 from Isfahan University of Medical Science, Isfahan, Iran.

 

 



[1]- https://www.ebi.ac.uk/tools/msa/clustalw2/ (Table 1).

 

 

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