The International Journal of Mycobacteriology

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 11  |  Issue : 2  |  Page : 145--149

Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis


Serihan Kübra Emikoglu Cerit1, Gülnur Tarhan2, Ismail Ceyhan3,  
1 Department of Medical Microbiology, Health Science Institute, Adiyaman University, Adiyaman, Turkey
2 Department of Medical Microbiology, Faculty of Medicine, Adiyaman University, Adiyaman, Turkey
3 Department of Public Health Nursing, Health Science Faculty, Ankara Yildirim Beyazit University, Ankara, Turkey

Correspondence Address:
Gülnur Tarhan
Adiyaman Üniversitesi, Tip Fakültesi, Tibbi Mikrobiyoloji Anabilim Dali, Altinsehir Mah. 3005, Sok. No: 13, 02040, Merkez, Adiyaman
Turkey

Abstract

Aims: Rapid and accurate identification of mycobacteria is important for the species-specific treatment of the disease. The aim of this study was the identification at the species level of 34 nontuberculous mycobacteria strains isolated from respiratory tract samples and 14 reference strains as by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Materials and Methods: Isolates derived from clinical specimens were subcultured in the Lowenstein–Jensen medium. Deoxyribonucleic acid isolation was carried out using the boiling method. PCR amplification was performed using primers specific to the hsp65 gene region. The PCR products were digested BstEII and HaEIII enzymes. All samples were studied comparatively by two different centers. Results: In our study, the most common species were found to be Mycobacterium intracellulare in 23.52% (8/34). The performance of the PCR-RFLP method in detecting mycobacteria was found to be 82.35%. Conclusions: The PCR-RFLP method is a rapid, cheap, and practical method for the identification of mycobacteria.



How to cite this article:
Emikoglu Cerit SK, Tarhan G, Ceyhan I. Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis.Int J Mycobacteriol 2022;11:145-149


How to cite this URL:
Emikoglu Cerit SK, Tarhan G, Ceyhan I. Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis. Int J Mycobacteriol [serial online] 2022 [cited 2022 Jul 6 ];11:145-149
Available from: https://www.ijmyco.org/text.asp?2022/11/2/145/347528


Full Text



 Introduction



The Mycobacteriaceae family differs greatly from other bacteria in terms of their pathogenicity, reservoirs, reproduction times, and morphology in humans and animals To date, there are more than 180 mycobacteria species described in nature.[1],[2] Within these species, mycobacteria other than Mycobacterium tuberculosis complex are called nontuberculous mycobacteria (NTM). Most therapeutic drug monitoring (TDM) is widespread in nature and is known as opportunistic pathogens.[3] NTM infections originate from the environment and are not transmitted from person to person. There has been an increase in NTM infections in recent years due to cancer, diabetes mellitus, HIV, and various immune system suppressing causes.[4],[5] NTMs have been isolated from clinical cases more frequently in recent years. NTMs mostly cause disease in the lungs. Treatment of NTM lung infections is time-consuming, difficult, and costly. It has been reported that the NTM species that are frequently isolated in lung infections are slow-growing TDMs such as Mycobacterium avium complex (MAC) (M. avium, Mycobacterium intracellulare), Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium abscessus, and Mycobacterium xenopi.[6]

Identification of the agent at the species level in mycobacterial infections is important in establishing an appropriate treatment protocol. Conventional methods used in the diagnosis of NTM are based on the growth rate, growth temperature, colony appearance, pigment production, and biochemical properties of bacteria. Traditional biochemical methods and phenotypic tests are laborious and time-consuming, but their costs are low.[7] However, the application of biochemical tests for species identification of NTMs may not always give accurate results when used alone. Moreover, they are also insufficient in the identification of new species. Molecular methods were employed because they more accurately identified species in a shorter time frame.[8] Numerous standardized commercial methods exist for typing mycobacteria, including Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFF), Innogenetics Line Probe Assay (INNO-LIPA), deoxyribonucleic acid (DNA) sequence analysis and more. However, these methods have their limitations, such as insufficient species identification and being too costly for routine use.[9],[10]

Consequently, these limitations have led researchers to employ polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in mycobacteria type identification as it not only has a rapid turnaround time but also for its simplicity as it is based on the PCR technique. Thus, it has been found that PCR-RFLP reduces the dependence on commercial products and is a method with results in a short time.[1],[4],[6],[11]

The aim of this study was to identify NTM isolated from respiratory specimens at the species level using the PCR-RFLP method.

 Materials and Methods



Bacterial strains

The study evaluated 34 NTM strains that were volunteered from the Atatürk Chest Diseases and Thoracic Surgery Training and Research Hospital Microbiology Laboratory and the Adiyaman University Educational and Research Hospital. The 34 strains were isolated from respiratory tract samples of patients with suspected tuberculosis and were defined by conventional methods and by using 14 reference strains (Mycobacterium smegmatis ATCC 14468, M. kansasii ATCC 12478, Mycobacterium fortuitum ATCC 6841, M. abscessus ATCC19977, Mycobacterium scrofulaceum ATCC 19981, Mycobacterium szulgai ATCC 35799, Mycobacterium gordonae ATCC 14470, M. intracellulare ATCC 13950, H37Ra ATCC 25177, M. xenopi ATCC 19250, M. avium ATCC 2529 Mycobacterium triviale ATCC 23292, Mycobacterium diernhoferi ATCC 19340, Mycobacterium marinum ATCC 927). Consecutive sputum samples from patients were used. According to the Centers for Disease Control and WHO criteria, NTM isolated in at least two consecutive cultures were accepted as disease agents.[1],[2],[4],[5],[6],[7],[8],[9],[10],[11],[12] Decontamination, homogenization, and concentration processes were performed on sputum samples according to the 4% NaOH-NALC method. Acid-fast bacilli were detected by Ehrlich Ziehl–Neelsen staining, and cultures on Löwenstein–Jensen medium were incubated at 37°C for 2–8 weeks. Growing cultures were evaluated for typical/atypical mycobacteria by ARB staining, niacin, nitrate, and para-nitrobenzoic acid test. During the incubation period, growth controls were made twice a week, and they were examined for their growth time, pigment formation, and colony morphology.[11]

Deoxyribonucleic acid extraction

DNA isolation from the cultured samples was carried out using the boiling method.[12] A microcentrifuge tube containing 1 ml of sterile distilled water was suspended by taking an extract from the bacterial colonies produced in Löwenstein–Jensen media. The tube was vortexed and centrifuged. The pellet was washed with 500 μl of TE buffer (10 mM Tris, 1 mM Ethylenediaminetetraacetic acid, pH 8.0). Then, 300 μl of TE buffer was added, and the supernatant was discarded. The caps of the microcentrifuge tubes were pierced at several places with a lancet to allow air to enter. By incubating the tubes in a boiling water bath for 20 min, the bacterial cells were lysed, and their DNA was released. The tubes were then centrifuged at 2000 × g for 20 min, allowing bacterial residues to precipitate. The supernatant containing the DNA was separated and transferred into two sterile tubes in a volume of 125 μL. The samples were kept at −20°C before use.

Deoxyribonucleic acid amplification

A 50 μl PCR reaction mixtures were prepared by using 5 μl 10X buffer, 5 μl 25 mM MgCl₂ 1 μl dNTPs (Fermantase), 0.5 μl primer Tb11 (5-ACCAACGATGGTGTGTCCAT), 0.5 μl primer Tb12 (5-CTTGTCGAACCGCATACCCT), 0.2 μl Taq polymerase (Fermantase) and dH₂O for the PCR amplification of the hsp65 gene using the NTM DNA.[12]

A volume of 40 μl of the PCR mixture was distributed into the tubes, and 10 μl of the extracted sample DNA was added to each tube. The reaction tubes were then placed in the thermal cycler and amplification was performed over denatured at 94°C for 3 min, 44 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension for 72°C at 90 s and finally an additional extension at 72°C for 4 min reaction. Following completion of the PCR, amplification products were separated out by agarose gel electrophoresis, and a band length of 441 bp was confirmed.

Restriction digestion and analysis

The restriction digest mixture was prepared using BstEII (Fermantase) and HaEIII (Fermantase) enzymes for cleavage of the DNA at the restriction sites. Each enzyme mixture was prepared separately and contained 2 μl of enzyme buffer and 1 μl of enzyme per sample. The prepared mixture was distributed in sample amplification tubes, and 10 μl of the PCR product was added to the tubes. The mixtures were incubated overnight at 37°C.

Evaluation of enzyme cleavage patterns

Following enzymatic digestion of the PCR product, a TIBO gel (TrendsinInnovativeBiotechnologyOrganization, Istanbul) mixture was prepared. The 10X TAE buffer was used as a loading buffer and 3 μl of the SYBR gold enzyme staining product was added to the mixture. The gel was then loaded onto an ORTE (Observable Real-Time Electrophoresis, TIBO, Istanbul) device. The electrophoresis was run at 100 volts for 45 min with continuous monitoring and image recording every 5 min. M. tuberculosis H37Ra was used as a control in every run. The results were evaluated using a reference algorithm created from various sources.[13],[14],[15],[16],[17]

All samples were studied comparatively by two centers. Incompatible and unidentified test results were verified by performing the GenoType Mycobacterium AS/CM test (Hain, Germany) and the DNA sequence PRISM 3100 Genetic analyzer (Applied Biosystems) and the sequences were compared with the nucleotide database in GenBank at NCBI (www.ncbi.nlm.nih.gov/blast/).

 Results



This study was carried out by two centers, Adıyaman University Faculty of Medicine Medical Microbiology Research Laboratory and Atatürk Chest Diseases and Thoracic Surgery Training and Research Hospital Microbiology Laboratory. The same samples were studied independently with the PCR-RFLP method in Adıyaman University Faculty of Medicine Medical Microbiology Research Laboratory and GenoType Mycobacterium AS/CM test (Hain, Germany) in Atatürk Chest Diseases and Thoracic Surgery Training and Research Hospital Microbiology Laboratory. The results of the studies in both centers were evaluated comparatively.

All steps were applied as indicated in this study. PCR standardization resulted in a 441 bp PCR product of the desired hsp65 gene region, as illustrated in [Figure 1].{Figure 1}

After the enzyme cutting, the evaluation of the band patterns and the identification of the species was made using the reference algorithm[13],[14],[15],[16],[17] [Figure 2].{Figure 2}

As a result of this evaluation, the species distribution of the samples examined in the study is shown in [Table 1].{Table 1}

In this study, when the results defined by the PCR-RFLP method were compared with the results of other centers; Of the 34 isolates, 82.35% (28/34) were found to be compatible and 5.88% (2/34) were found to be incompatible. Four (11.76%) isolates could not be identified by both centers. In addition, 14 reference strains used for control purposes were also correctly typed.

 Discussion



Mycobacterial species identification methods are cumbersome. The need for rapid methods for the isolation and identification of mycobacteria from patients has increased.[4],[5],[6] Moreover, recent data have indicated an increase in NTM-related infections. Mycobacteria are a rapidly growing species that have long been considered nonpathogenic. Nonetheless, over the past few year's studies have indicated NTM as the causative agent in various diseases of the bones and joints, the soft tissue, and the lungs.[18] The interaction between a microorganism's pathogenicity and the host's immune system plays an important role in disease sensitivity. Old age, immunodeficiency, and lung diseases are all factors that increase the risk of NTM infections.[19]

Numerous standardized commercial methods exist for typing mycobacteria, including MALDI-TOFF, INNO-LİPA, DNA sequence analysis, and more. However, these methods have their limitations, such as insufficient species identification and being too costly for routine use. Consequently, these limitations have led researchers to employ PCR-RFLP in mycobacteria type identification as it not only has a rapid turnaround time but also for its simplicity as it is based on the PCR technique. Thus, it has been found that PCR-RFLP reduces the dependence on commercial products and is a method with results in a short time.[9],[10]

Of the mycobacteria isolates used in our study, the most commonly identified species were M. intracellulare, M. abscessus, and M. simiae. A Korean study undertaken by Kim and Rheem in 2013 also employed the PCR-RFLP method on respiratory samples.[20] This study reported that MAC and M. kansasii as the most common mycobacteria species. In 2017, Nour-Neamatollahie et al. They used the PCR RFLP method to examine 59 NTM isolates (56 respiratory samples, three non-respiratory samples). In this study, M. kansasii and M. simiae were identified as the most common species. These results were in keeping with the results of this study which found M. simiae to be one of the most common species.[21] In 2018, Appak et al. compared PCR-RFLP and DNA sequence analysis methods. They found the highest rate of M. abscessus, M. xenopi, M. fortuitum and M. peregrinum species in common in the two methods.[22] Nasiri et al. reported that among the samples isolated as NTM in 2018, species-level identification was made using the hsp65 PCR-restriction fragment length polymorphism analysis (hsp65-PRA) method and identified the highest prevalence in M. simiae, M. kansasii, and M. fortuitum.[23] A further Iranian study was conducted by Mortazavi et al. in 2019 and this also employed NTMs isolated from respiratory samples. The most common species identified in this study were M. fortuitum, M. simiae, and M. kansasii.[24]

According to the findings of the aforementioned studies, it was evident that the most common mycobacteria species in sputum samples were similar, but that the variation encountered may be caused by differences in geographical regions and environmental conditions.[25]

In some studies evaluating the effectiveness of the PCR-RFLP method in the identification of mycobacterial species, PCR-RFLP has been reported to be simple, inexpensive, fast, and with high discrimination power.[23],[24],[25],[26],[27],[28],[29]

The findings of this study established that the PCR-RFLP method is a useful method for species-level assessment of mycobacteria. Nonetheless, the results also indicated the need for further standardization of the method and particularly of the imaging processes. Such standardization was deemed crucial for correct species identification. Furthermore, this study noted some disadvantages of this method, including difficulties in distinguishing and therefore evaluating mycobacteria with similarly sized patterns and the small band patterns that result from DNA cleavage. Critical points in the implementation of the test are the evaluation of the enzyme with postcut imaging, appropriate marker use, and algorithms.

 Conclusions



We believe that if this method is well standardized, it is a fast, simple, and inexpensive method that can be used routinely for the differentiation of mycobacterial species. We believe that if this method is well standardized, it is a fast, simple, and inexpensive method that can be used routinely for the differentiation of mycobacterial species.

Limitation of the study

The number of samples in our study was limited. There is a need for extensive studies with more examples.

Ethical clearance

Our study was derived from MDR M. tuberculosis strains isolated in our laboratory and does not require an ethics committee decision.

Acknowledgments

This study was supported by Adıyaman University Scientific Research Projects Unit as project number TIPFYL/2018-0002. We would like to thank the Scientific Research Projects Commission of Adıyaman University for financial support. We would also like to thank Acıbadem University Faculty of Medicine, Head of Medical Microchiology Department, Prof. Dr. Tanıl Kocagöz and his team for their technical support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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