Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples

Kim, Ki-Jeong; Hong, Seok Hyun; Kim, Byoung Jun; Kim, Bo Ram; Lee, So Young; Kim, Ga Na; Shim, Tae Sun; Kook, Yoon Hoh; Kim, Bum Joon

doi:10.1186/s12879-015-1076-8

Cited by 9 publications

(7 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Typically, these methods use sequencing of several housekeeping genes, including hsp65, rpoB, secA, either individually [19][20][21][22] , or in a combined manner 23 . Moreover, there are several PCR-based methods, i.e., repetitive sequence-based PCR (rep-PCR) 22 , deletion-mapping PCR 24 , and peptide nucleic acid multi-probe-real-time PCR 25 . Most commonly, the detection of deletion is used to differentiate M. abscessus subsp.…”

mentioning

confidence: 99%

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

Minias

Żukowska

Lach

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Mycobacterium abscessus complex (MABC) is a taxonomic group of rapidly growing, nontuberculous mycobacteria that are found as etiologic agents of various types of infections. They are considered as emerging human pathogens. MABC consists of 3 subspecies—M. abscessus subsp. bolletti, M. abscessus subsp. massiliense and M. abscessus subsp. abscessus. Here we present a novel method for subspecies differentiation of M. abscessus named Subspecies-Specific Sequence Detection (SSSD). This method is based on the presence of signature sequences present within the genomes of each subspecies of MABC. We tested this method against a virtual database of 1505 genome sequences of MABC. Further, we detected signature sequences of MABC in 45 microbiological samples through DNA hybridization. SSSD showed high levels of sensitivity and specificity for differentiation of subspecies of MABC, comparable to those obtained by rpoB sequence typing.

show abstract

mentioning

confidence: 99%

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

Minias

Żukowska

Lach

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…abscessus and M. abscessus subsp. bolletii by identifying the fragmented erm(41) gene, it still requires sequence analysis of several housekeeping genes (20,21). In addition, PCR methods that depend upon gene sequencing are costly.…”

mentioning

confidence: 99%

Evaluation of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Mycobacterium abscessus Subspecies According to Whole-Genome Sequencing

Luo

Liu

et al. 2016

J Clin Microbiol

View full text Add to dashboard Cite

cThis study was undertaken to evaluate the utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry with the Vitek MS Plus system for identifying Mycobacterium abscessus subspecies in order to facilitate more rapid and appropriate therapy. A total of 175 clinical M. abscessus strains were identified by whole-genome sequencing analysis: 139 Mycobacterium abscessus subsp. abscessus and 36 Mycobacterium abscessus subsp. massiliense. The research-use-only (RUO) Saramis Knowledge Base database v.4.12 was modified accordingly by adding 40 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massiliense reference spectra to construct subspecies SuperSpectra. A blind test, used to validate the remaining 116 isolates, yielded 99.1% (n ‫؍‬ 115) reliability and only 0.9% (n ‫؍‬ 1) error for subspecies identification. Among the two subspecies SuperSpectra, two specific peaks were found for M. abscessus subsp. abscessus and four specific peaks were found for M. abscessus subsp. massiliense. Our study is the first to report differential peaks 3,354.4 m/z and 6,711.1 m/z, which were specific for M. abscessus subsp. massiliense. Our research demonstrates the capacity of the Vitek MS RUO Saramis Knowledge Base database to identify M. abscessus at the subspecies level. Moreover, it validates the potential ease and accuracy with which it can be incorporated into the IVD system for the identification of M. abscessus subspecies. Mycobacterium abscessus is one of the most common pathogens isolated from patients with cystic fibrosis and the second most prevalent, rapidly growing nontuberculous mycobacteria (NTM) species causing NTM pulmonary diseases (1, 2). Although it might not be the most virulent NTM pathogen, inherent antibiotic resistance makes it notoriously difficult to treat (3-7). Among multiple resistance mechanisms, resistance to macrolides is noteworthy (8). In this regard, acquired resistance to clarithromycin is always associated with a mutation in the 23S rRNA gene; intrinsic resistance is conferred mainly by the erythromycin methylase (erm) gene (3). The erm gene plays a significant role in the drug susceptibility and antibiotic resistance of M. abscessus. Mycobacterium abscessus is divided into three subspecies: Mycobacterium abscessus subsp. massiliense, Mycobacterium abscessus subsp. bolletii, and Mycobacterium. abscessus subsp. abscessus (9). The most meaningful genomic difference between the three subspecies is that M. abscessus subsp. massiliense possesses an incomplete and inactive erm gene, which is 276 bp in length and harbors deletion mutations at two positions (10-13). M. abscessus subsp. massiliense can acquire clarithromycin resistance as a consequence of a 23S rRNA mutation at position A2058 (12,14). Due to an inactive erm gene, M. abscessus subsp. massiliense does not exhibit inducible resistance after exposure to macrolides. M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, on the other hand, exhibit inducible resistance (9, 15). As a result, lung diseas...

show abstract

“…massiliense (type I, type II-1 and type II-2). This method was subsequently tested using 228 cultures of clinical MABSC isolates and later applied to the sputum of clinical patients [35]. Syrmis et al .…”

Section: Discussionmentioning

confidence: 99%

“…Kim et al [34] developed a peptide nucleic acid (PNA) real-time PCR method targeting the hsp65 gene to detect M. abscessus and three types of M. massiliense (type I, type II-1 and type II-2). This method was subsequently tested using 228 cultures of clinical MABSC isolates and later applied to the sputum of clinical patients [35]. Syrmis et al [36] designed two individual qPCR assays for distinguishing the MABSC on the species level and MAM.…”

Section: Table 2 Continuedmentioning

confidence: 99%

“…A few problematic points can be found in all the above studies. Assays based on culturing predominate; testing on sputum from clinical patients was carried out only in the study by [35]. Determination of the LOD was performed only in the study by Marras et al [37] and was based on determination of different quantities of DNA added as a template in the qPCR reaction.…”

Section: Table 2 Continuedmentioning

confidence: 99%

See 1 more Smart Citation

Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR

Dziedzinská

Okunkova

Králík

et al. 2022

Journal of Medical Microbiology

View full text Add to dashboard Cite

Introduction. Cystic fibrosis (CF) is a serious disease with multisystemic clinical signs that is easily and frequently complicated by bacterial infection. Recently, the prevalence of nontuberculous mycobacteria as secondary contaminants of CF has increased, with the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) being the most frequently identified. The MABSC includes subspecies of significant clinical importance, mainly due to their resistance to antibiotics. Gap statement. Sensitive method for early detection and differentiation of MABSC members and MAC complex for use in routine clinical laboratories is lacking. A method based on direct DNA isolation from sputum, using standard equipment in clinical laboratories and allowing uncovering of possible sample inhibition (false negative results) would be required. The availability of such a method would allow accurate and accelerated time detection of MABSC members and their timely and targeted treatment. Aim. To develop a real time multiplex assay for rapid and sensitive identification and discrimination of MABSC members and MAC complex. Methodology. The method of DNA isolation directly from the sputum of patients followed by quadruplex real-time quantitative PCR (qPCR) detection was developed and optimised. The sensitivity and limit of detection (LOD) of the qPCR was determined using human sputum samples artificially spiked with a known amount of M. abscessus subsp. massiliense (MAM). Results. The method can distinguish between MAC and MABSC members and, at the same time, to differentiate between M. abscessus subsp. abscessus /subsp. bolletii (MAAb/MAB) and MAM. The system was verified using 61 culture isolates and sputum samples from CF and non-CF patients showing 29.5 % MAAb/MAB, 14.7 % MAM and 26.2 % MAC. The LOD was determined to be 1 490 MAM cells in the sputum sample with the efficiency of DNA isolation being 95.4 %. Verification of the qPCR results with sequencing showed 100 % homology. Conclusions. The developed quadruplex qPCR assay, which is preceded by DNA extraction directly from patients’ sputum without the need for culturing, significantly improves and speeds up the entire process of diagnosing CF patients and is therefore particularly suitable for use in routine laboratories.

show abstract

Separation of Mycobacterium abscessus into subspecies or genotype level by direct application of peptide nucleic acid multi-probe- real-time PCR method into sputa samples

Cited by 9 publications

References 35 publications

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

Subspecies-specific sequence detection for differentiation of Mycobacterium abscessus complex

Evaluation of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Mycobacterium abscessus Subspecies According to Whole-Genome Sequencing

Identification of and discrimination between the Mycobacterium abscessus complex and Mycobacterium avium complex directly from sputum using quadruplex real-time PCR

Contact Info

Product

Resources

About