論文の公開元へMolecular characterization of Mycobacterium avium clinical isolates from Japan and development of diagnostic tools [an abstract of entire text]
概要
Mycobacterium avium (M. avium), an environmental bacterium, is the leading cause of nontuberculous mycobacterial (NTM) lung diseases worldwide. The increasing trend of M. avium infections in the recent decades poses a threat to the public health of not only the vulnerable populations such as the immunocompromised individuals, the elderly, and children but also in apparently healthy individuals. M. avium as well as other NTM infections represent some of the most difficult infections to eradicate due to their intrinsic resistance to antibiotics and common disinfectants. Hence, early detection is paramount for the timely management of infections because it allows for the institution of appropriate intervention measures since late interventions are associated with poor prognoses. There are several diagnostic tools currently available for the detection of M. avium, however, these tools are limited by low sensitivities, are time-consuming and associated with high costs. Hence, there is a sustained need for practical, accurate and rapid diagnostic tools to aid in the rapid diagnosis of M. avium infections.
On the other hand, the emergence of drug resistant M. avium strains has rendered the few available antimicrobials such as macrolides which form the cornerstone of M. avium therapy ineffective. Consequently, second-line drugs such as fluoroquinolones (FQ) have been used to treat drug resistant M. avium infections, unfortunately, FQ resistance has been increasing. In mycobacteria, resistance to FQ has been largely attributed to alterations in DNA gyrase, which is the target for FQ. FQ resistance mechanisms have been extensively studied in Mycobacterium tuberculosis, and reports have shown that over 90% of FQ resistant isolates have mutations in the two DNA gyrase subunits; GyrA and GyrB encoded by gyrA and gyrB. However, in M. avium, there is still very limited information on FQ resistance mechanisms.
Considering the above, in Chapter I, an IS1245 LAMP assay for the rapid detection of M. avium was developed. Secondly, due to the high geographic and host-related genetic diversity known to exist among M. avium strains, the influence of this diversity on the applicability of the
LAMP assay was assessed using human and pig samples from Japan. In Chapter II, the role of MfpA in levofloxacin resistance among M. avium clinical isolates was investigated by minimum inhibitory concentration (MIC) determination and sequencing FQ resistance associating genes.
In Chapter I, Loop-mediated isothermal amplification (LAMP) technology was utilized to develop a rapid diagnostic assay targeting a widely used species-specific marker, IS1245. The applicability of this assay in Japan were further assessed using extracted DNA from human (n = 137) and pig (n = 91) M. avium isolates. LAMP reactions were performed in 25uL reaction volumes and results were obtained by observing rising curves on the LoopAmp turbidimeter. The sensitivity of the assay was evaluated by 10-fold serial dilutions of genomic DNA of M. avium HP 22 strain and to confirm the specificity, the assay was assessed against 22 Nontuberculous mycobacterial type strains, 2 MTBC reference strains, 14 MTBC clinical isolates, and an additional 5 closely related non-mycobacterial species that cause respiratory infections.
The sensitivity analysis showed that the assay was able to detect up to 6 fg (equivalent to 1 genome copy) of M. avium DNA within 30 minutes of the reaction. Furthermore, the assay was specific to M. avium and did not amplify any other bacterial species. When evaluated with Japanese isolates, all 91 (100%) M. avium isolates from pigs were detected positive, interestingly, among the 137 clinical M. avium isolates, 41 (30%) were undetectable with this LAMP assay. The negative isolates all lacked the IS1245, the absence of which was revealed by PCR and whole- genome sequencing.
The discrepancy in the IS1245 carriage rates between M. avium isolates from humans and pigs underscores one of the possibly many molecular aspects whereby Japanese M. avium clinical strains may be genetically distinct from the pig strains. Moreover, M. avium clinical isolates in Japan have been previously reported to have high relatedness with the genotypic profiles of environmental strains, whereas pig-derived M. avium strains in Japan were similar genetically to strains isolated from the European human population.
IS1245 marker is known as a gold standard for the detection of M. avium, however, the evaluation showed that M. avium genetic diversity has a major influence on the applicability of the diagnostic tool across different geographical areas. Due to low IS1245 carriage rates in the circulating M. avium strains, the newly developed IS1245 LAMP system may not be very ideal for the Japanese human population. Nevertheless, the assay will be highly suitable for use in Euro- American countries that have a high IS1245 prevalence and routinely use IS1245 as a marker of M. avium for both detection and differentiation. The findings of this study are a classic example of how genetic diversity in M. avium can directly affect the application of a diagnostic tool globally especially those tools targeting non-conserved genes like IS1245. This subsequently limits rapid diagnosis and ultimately prevents timely management of M. avium infections.
In conclusion, the incidences of M. avium infections have been increasing especially in industrialized countries causing higher burdens than M. tuberculosis. Unlike, M. tuberculosis, M. avium is especially harder to treat due to the lack of effective treatment regimens and high drug resistance rates. Therefore, early detection before full disease progression is paramount for the timely control of M. avium infections. Hence, in Chapter I, an accurate and cost-effective LAMP assay targeting IS1245 for the rapid detection of M. avium was established, and the effects of genotypic diversity on the applicability of this tool in Japan was also explored.
The newly developed IS1245 LAMP assay exhibited high sensitivity and specificity, thereby making it a fast, cost-friendly alternative method of M. avium detection especially in the Euro Americas which have reported very high IS1245 carriage rates. Despite IS1245 marker been known as a gold standard for the detection of M. avium, the application of the IS1245-based LAMP assay was limited in Japan due to the absence of IS in about a third of the Japanese M. avium clinical isolates. This demonstrated how genetic diversity among M. avium strains limits the applicability of diagnostic tools across different geographical areas. Thus, placing an emphasis of the importance of understanding the local genetic make-up of a pathogen population before adopting internationally set standards.
Chapter II was focused on elucidating FQ resistance mechanisms in M. avium. FQ are broad-spectrum antimicrobials that are used for the treatment of macrolide-resistant cases. FQ target DNA gyrase, an essential enzyme that is involved in DNA replication, transcription, and stress responses. DNA gyrase is a type II topoisomerase composed of two GyrA and two GyrB subunits, GyrA binds DNA, while GyrB is an ATPase. Many structural studies have demonstrated that mutations in gyrA and gyrB conferring quinolone resistance, play important roles in drug- protein interactions, however, the correlation between antibiotic resistance and particular mutation sites in M. avium clinical isolates is not strong. This phenomenon suggests that there are other yet unknown mechanisms that may also be important contributing factors to resistance and that the DNA gyrase mechanisms may not be the only factor determining drug resistance.
The most recently described mechanism of FQ resistance is DNA mimicry by pentapeptide repeat proteins and the most notable examples are Mycobacterium fluoroquinolone resistance protein A (MfpA) and plasmid mediated quinolone resistance protein (Qnr) in mycobacteria and Gram-negative bacteria respectively. So far, MfpA has been associated with inducing intrinsic FQ resistance in Mycobacterium smegmatis and Mycobacterium tuberculosis exclusively. Particularly, in M. smegmatis, mfpA reportedly played a role in determining the innate MICs of FQ in the mfpA mutant strain which had a two to four-fold decrease in the level of FQ resistance. MfpA has been shown to limit the efficacy of FQ by decreasing the FQ-induced DNA cleavage, and thereby protecting DNA gyrase from the inhibitory activity of FQ. Consequently, mutations that alter or increase the expression of MfpA could lead to the development of FQ resistance. However, the role of MfpA in FQ resistance in M. avium clinical strains has not yet been investigated.
Hence the objective of Chapter II was to investigate the role of MfpA in levofloxacin (a representative FQ) resistance among M. avium clinical isolates. This was done by first determining the minimum inhibitory concentrations (MIC) of a total of 88 M. avium isolates from Japan by using the broth microdilution method. Thereafter, the FQ resistance associating genes (gyrA, gyrB, mfpA, and mfpB) of these isolates were sequenced and analyzed in BioEdit software.
Among the isolates, 21.6 % (19/88) were susceptible to LVX, whereas 78.4 % (69/88), were resistant. Only four of the resistant isolates (4/69, 5.8 %) harbored resistance-conferring mutations (D94Y or D94G) within the QRDR of gyrA, and no resistance-associating mutations were found in gyrB as well as mfpB. Sequencing of the mfpA revealed two distinct mfpA genotypes among the M. avium isolates. Specifically, some isolates harbored mfpA with a C170frameshift deletion hereby referred to as “C170deleted mfpA” while other isolates had an “Intact-mfpA”, where the frameshift mutation was absent. Owing to this finding, a multiplex PCR for rapid differentiation of the above described mfpA genotypes in M. avium was developed to simplify genotype differentiation of all isolates. The multiplex had good diagnostic accuracy because all the results agreed with Sanger sequencing results.
From the total 88 isolates analyzed in this study, 58 had the Intact-mfpA genotype while 30 had C170deleted mfpA type. Among the resistant isolates, 53/69 (76.8%) had an intact mfpA, whilst 16/69 (23.2%) had a C170deleted mfpA. On the other hand, 73.7% (14/19) of the susceptible isolates had C170deleted mfpA and the remaining 26.3% (5/19) were of the Intact-mfpA genotype. A high proportion (53/58, 91.4%) of isolates with the Intact-mfpA were also LVX resistant. Furthermore, MIC determination revealed a significant association (p < 0.001) between isolates with Intact-mfpA genotype and decreased LVX susceptibility in comparison with isolates bearing the C170deleted mfpA. This observation although preliminary, suggests that mfpA has some contributory role in either inducing and/or enhancing LVX resistance in M. avium thereby warranting more attention in future studies.
In conclusion, fluoroquinolones are considered important alternative therapy for M. avium pulmonary infections when the recommended regimen is insufficient. However, the resistance to fluoroquinolones is increasing and this has impacted the effective management of M. avium infections. In this chapter, the basis for levofloxacin resistance in 88 M. avium isolates from Japan was investigated through MIC determination and sequencing of gyrA, gyrB, mfpA, and mfpB. Moreover, there was little correlation of mutations in gyrA and gyrB with LVX resistance and instead a statistically significant (p-value <0.001) association was observed between mfpA (Intact- mfpA genotype) and resistance to LVX. The results of this study will form the basis on which further studies can be conducted to further elucidate MfpA’s mechanism of resistance in M. avium and the resultant knowledge should be helpful in the effective management of M. avium infections.
Overall, the findings of this research work are a new addition to the existing scientific knowledge which will continue to deepen our understanding of M. avium in order to institute effective control measures and put a stop to the rising M. avium- related pulmonary infections worldwide.
