RESUMEN
Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K2). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Péptidos Cíclicos , Adenosina Trifosfato/metabolismo , Animales , Antituberculosos/química , Antituberculosos/farmacología , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Staphylococcus aureus/metabolismo , Estreptomicina/farmacología , Tuberculosis , Vitamina K 2/metabolismoRESUMEN
BACKGROUND: Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye. METHOD: A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR. RESULTS: The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5 ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria. CONCLUSION: A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.
Asunto(s)
Mycobacterium tuberculosis/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Compuestos Orgánicos/química , Tuberculosis/diagnóstico , Benzotiazoles , ADN Bacteriano/análisis , ADN Bacteriano/genética , Diaminas , Genes Bacterianos/genética , Humanos , Límite de Detección , Quinolinas , Recombinasas/metabolismo , Tuberculosis/microbiologíaRESUMEN
We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA(®) elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.
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Monitoreo del Ambiente/métodos , Complejo Mycobacterium avium/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Habitaciones de Pacientes , Microbiología del Agua , Humanos , Japón , Complejo Mycobacterium avium/genética , Complejo Mycobacterium avium/crecimiento & desarrollo , Infección por Mycobacterium avium-intracellulare/microbiología , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Sensibilidad y EspecificidadRESUMEN
Most people infected with Mycobacterium tuberculosis (Mtb) are believed to be in a state of latent tuberculosis (TB) infection (LTBI). Although LTBI is asymptomatic and not infectious, there is a risk of developing active disease even decades after infection. Here, to characterize mutations acquired during LTBI, we collected and analyzed Mtb genomes from seven Japanese patient pairs, each pair consisting of two active TB patients whose starting dates of developing active disease were >3 years apart; one had a high suspicion of LTBI before developing active disease, whereas the other did not. Thereafter, we compared these genomes with those of longitudinal sample pairs within a host of chronic active TB infections combined with public data. The bacterial populations in patients with LTBI were genetically more homogeneous and accumulated single nucleotide polymorphisms (SNPs) slower than those from active disease. Moreover, the lower proportion of nonsynonymous SNPs indicated weaker selective pressures during LTBI than active disease. Finally, the different mutation spectrums indicated different mutators between LTBI and active disease. These results suggest that the likelihood of the acquisition of mutations responsible for antibiotic resistance and increased virulence was lower in the Mtb population from LTBI than active disease.IMPORTANCEControlling latent tuberculosis (TB) infection (LTBI) activation is an effective strategy for TB elimination, where understanding Mycobacterium tuberculosis (Mtb) dynamics within the host plays an important role. Previous studies on chronic active disease reported that Mtb accumulated genomic mutations within the host, possibly resulting in acquired drug resistance and increased virulence. However, several reports suggest that fewer mutations accumulate during LTBI than during the active disease, but the associated risk is largely unknown. Here, we analyzed the genomic dynamics of Mtb within the host during LTBI. Our results statistically suggest that Mtb accumulates mutations during LTBI, but most mutations are under low selective pressures, which induce mutations responsible for drug resistance and virulence. Thus, we propose that LTBI acts as a source for new TB disease rather than as a period for in-host genome evolution.
Asunto(s)
Genoma Bacteriano , Tuberculosis Latente , Mutación , Mycobacterium tuberculosis , Polimorfismo de Nucleótido Simple , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis Latente/microbiología , Virulencia/genética , Masculino , Femenino , Adulto , Tuberculosis/microbiología , Persona de Mediana Edad , Farmacorresistencia Bacteriana/genética , AncianoRESUMEN
The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Genotipo , Humanos , Tipificación Molecular/métodos , Proteínas Mutantes/genética , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Tuberculosis/diagnósticoRESUMEN
OBJECTIVES: To evaluate the usefulness of the JATA (12)-variable number of tandem repeats (VNTR) system for identifying the source of Mycobacterium tuberculosis outbreaks. DESIGN; JATA(12)-VNTR genotyping was performed on M. tuberculosis isolates from a total of 206 patients in whom group infection was confirmed by epidemiological studies ("group infection"), as well as from 64 patient clusters in whom group infection was suspected but not confirmed ("non-group infection"). The patients were diagnosed in Osaka Prefecture from April 1999 to December 2011. RESULTS: All isolates from the "non-group infection" patients showed a unique VNTR pattern, whereas isolates from 185 (89.9%) "group infection" patients showed a common and group-specific JATA (12)-VNTR pattern. However, single-locus variants were observed in 1 (1.6%) "non-group infection" case and in 21 (10.2%) "group infection" cases. CONCLUSION: Tuberculosis in 248 (91.9%) of the 270 study patients could be correctly identified based on the genotyping of the isolates by using the JATA (12)-VNTR. If proper attention is paid to the single-locus variant, the JATA (12)-VNTR system would be a useful tool for identification of sources of tuberculosis outbreaks.
Asunto(s)
Trazado de Contacto/métodos , Genotipo , Técnicas de Genotipaje/métodos , Repeticiones de Minisatélite , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , ADN Viral/genética , Brotes de Enfermedades/prevención & control , Humanos , Japón/epidemiología , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
OBJECTIVES: Japan Anti-Tuberculosis Association (JATA) (12)-variable numbers of tandem repeats (VNTR) is a standard method for genotyping of clinical isolates of Mycobacterium tuberculosis in Japan. As a model study for nationwide surveillance, this study aimed to describe the tendency and frequency of genotypes of M. tuberculosis in a large number of clinical samples. METHODS: Clinical isolates of M. tuberculosis (n = 1,778) were obtained from patients with tuberculosis in 3 areas, i.e., Osaka City, Osaka Prefecture, and Kobe City, during 2007 and 2008. The samples were analyzed using JATA (12)-VNTR. All genotypes were subjected to clustering analysis. RESULTS AND DISCUSSION: In total, 1,086 (61.1%) isolates showed clustering. The most common clusters were composed of 3 members. Such clusters were considered to reflect either actual transmission or low discriminatory power of JATA (12)-VNTR. Several prevalent JATA(12)-VNTR genotypes formed large clusters and were discussed in relation with epidemiological findings of other studies. The findings of this study will aid in the construction of an effective genotyping-based surveillance system of M. tuberculosis, through improvement of interpretation of VNTR types, observation of certain particular strains in an area, and efficient detection of unidentified outbreaks.
Asunto(s)
Análisis por Conglomerados , Técnicas de Genotipaje/métodos , Repeticiones de Minisatélite , Epidemiología Molecular/métodos , Mycobacterium tuberculosis/genética , Tuberculosis/epidemiología , Tuberculosis/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Trazado de Contacto/métodos , ADN Viral/genética , Brotes de Enfermedades/prevención & control , Femenino , Genotipo , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/transmisión , Adulto JovenRESUMEN
In spite of the importance of hyaluronan in host protection against infectious organisms in the alveolar spaces, its role in mycobacterial infection is unknown. In a previous study, we found that mycobacteria interact with hyaluronan on lung epithelial cells. Here, we have analyzed the role of hyaluronan after mycobacterial infection was established and found that pathogenic mycobacteria can grow by utilizing hyaluronan as a carbon source. Both mouse and human possess 3 kinds of hyaluronan synthases (HAS), designated HAS1, HAS2, and HAS3. Utilizing individual HAS-transfected cells, we show that HAS1 and HAS3 but not HAS2 support growth of mycobacteria. We found that the major hyaluronan synthase expressed in the lung is HAS1, and that its expression was increased after infection with Mycobacterium tuberculosis. Histochemical analysis demonstrated that hyaluronan profoundly accumulated in the granulomatous legion of the lungs in M. tuberculosis-infected mice and rhesus monkeys that died from tuberculosis. We detected hyaluronidase activity in the lysate of mycobacteria and showed that it was critical for hyaluronan-dependent extracellular growth. Finally, we showed that L-Ascorbic acid 6-hexadecanoate, a hyaluronidase inhibitor, suppressed growth of mycobacteria in vivo. Taken together, our data show that pathogenic mycobacteria exploit an intrinsic host-protective molecule, hyaluronan, to grow in the respiratory tract and demonstrate the potential usefulness of hyaluronidase inhibitors against mycobacterial diseases.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Ácido Hialurónico/metabolismo , Mycobacterium tuberculosis/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Recuento de Colonia Microbiana , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Glicosaminoglicanos/farmacología , Histocitoquímica , Humanos , Hialuronano Sintasas , Ácido Hialurónico/farmacología , Pulmón/química , Pulmón/metabolismo , Pulmón/microbiología , Macaca mulatta , Masculino , Ratones , Mycobacterium bovis/fisiología , Mycobacterium tuberculosis/metabolismo , RatasRESUMEN
Multidrug-resistant tuberculosis (MDR-TB) poses a serious threat to TB control. Early diagnosis and proper treatment are essential factors to limit the spread of the disease. The existing molecular tests for MDR-TB usually require specific instruments, steady power supply, and routine maintenance, which might be obstacles for low-resource settings. This study aimed to develop allele-specific isothermal recombinase polymerase amplification (allele-specific RPA) to simultaneously detect the most common mutations in the rpoB gene at codons 516, 526, and 531, which are associated with rifampicin resistance, and in the katG gene at codon 315, which is related to isoniazid resistance. Allele-specific primers targeting four major mutations, rpoB516, rpoB526, rpoB531, and katG315, were constructed and used in individual RPA reactions. The RPA amplicons were endpoints detected by the naked eye immediately after applying SYBR Green I. The optimised RPA assay was evaluated with the Mycobacterium tuberculosis wild-type strain H37Rv and 141 clinical M. tuberculosis isolates. The results revealed that allele-specific RPA combined with SYBR Green I detection (AS-RPA/SYBR) detected these four major mutations with 100% sensitivity and specificity relative to DNA sequencing. The limits of detection for these particular mutations with AS-RPA/SYBR were 5 ng. As a result of the outstanding performance of AS-RPA/SYBR, including its easy setup, speed, lack of a specific instrument requirement, and lack of cross-reaction with other bacteria, this technique may be integrated for the molecular diagnosis of MDR-TB, especially in low-resource settings.
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Colorimetría/métodos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Alelos , Sustitución de Aminoácidos/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Genes Bacterianos/genética , Humanos , Mutación/genética , Mycobacterium tuberculosis/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Mycobacterium bovis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Bovinos , Humanos , Mycobacterium bovis/genética , Sensibilidad y EspecificidadRESUMEN
Early detection and treatment are paramount for the timely control of Mycobacterium avium infections. Herein, we designed a LAMP assay targeting a widely used species-specific marker IS1245 for the rapid detection of M. avium and evaluated its applicability using human (n = 137) and pig (n = 91) M. avium isolates from Japan. The developed assay could detect as low as 1 genome copy of M. avium DNA within 30 minutes. All 91 (100%) M. avium isolates from pigs were detected positive while all other tested bacterial species were negative. Interestingly, among the 137 clinical M. avium isolates, 41 (30%) were undetectable with this LAMP assay as they lacked IS1245, the absence of which was revealed by PCR and whole-genome sequencing. These findings highlighted genotypic differences in M. avium strains from humans and pigs in Japan and how this diversity can influence the applicability of a detection tool across different geographic areas and hosts.
Asunto(s)
Elementos Transponibles de ADN/genética , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium avium/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Animales , Variación Genética , Genoma Bacteriano/genética , Humanos , Japón , Infecciones por Mycobacterium/microbiología , Infecciones por Mycobacterium/veterinaria , Mycobacterium avium/clasificación , Mycobacterium avium/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Especificidad de la Especie , Porcinos , Enfermedades de los Porcinos/microbiologíaRESUMEN
BACKGROUND: Species identification of isolates belonging to the Mycobacterium tuberculosis complex (MTC) seems to be important for the appropriate treatment of patients, since M. bovis is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of M. bovis among people or cattle has not been investigated. METHODS: Genetic regions cfp32, RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR. RESULTS: All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the cfp32, RD9 and RD12 and determined as M. tuberculosis. Two isolates lacked cfp32 PCR product and one lacked RD12, however, those three samples were further examined and identified as M. tuberculosis by the sequence analyses of hsp65 and gyrB. CONCLUSIONS: The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as M. tuberculosis and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.
Asunto(s)
Técnicas Bacteriológicas/métodos , Mycobacterium bovis/clasificación , Mycobacterium tuberculosis/clasificación , Reacción en Cadena de la Polimerasa/métodos , Tuberculosis/microbiología , Proteínas Bacterianas/genética , Bangladesh , ADN Bacteriano/genética , Humanos , Mycobacterium bovis/genética , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (Mtb), caused by amino acid substitutions in DNA gyrase, has been increasingly reported worldwide. WQ-3810 is a newly developed FQ that is highly active against FQ-resistant pathogens; however, its activity against Mtb has not been evaluated. Herein we examined the efficacy of WQ-3810 against Mtb through the use of recombinant Mtb DNA gyrases. In addition, in vitro antimycobacterial activity of WQ-3810 was evaluated against recombinant Mtb var. bovis Bacille Calmette-Guérin strains in which gyrase-coding genes were replaced with Mtb variants containing resistance-conferring mutations. WQ-3810 showed a higher inhibitory activity than levofloxacin against most recombinant DNA gyrases with FQ-resistance mutations. Furthermore, WQ-3810 showed inhibition even against a DNA gyrase variant harboring a G88C mutation which is thought to confer the highest resistance against FQs in clinical Mtb isolates. In contrast, the FQ susceptibility test showed that WQ-3810 had relatively weak mycobactericidal activity compared with moxifloxacin. However, the combination of WQ-3810 and ethambutol showed the greatest degree of synergistic activity against recombinant strains. Since FQs and ethambutol have been used in multi-drug therapy for tuberculosis, WQ-3810 might represent a new, potent anti-tuberculosis drug that can be effective even against FQ-resistant Mtb strains.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Azetidinas/farmacología , Proteínas Bacterianas/metabolismo , Girasa de ADN/metabolismo , Farmacorresistencia Bacteriana , Fluoroquinolonas/farmacología , Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Proteínas Bacterianas/genética , Girasa de ADN/genética , Farmacorresistencia Bacteriana/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Etambutol/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium bovis/enzimología , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genéticaRESUMEN
Mycobacterium tuberculosis (Mtb) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original Mtb isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent Mtb are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of Mtb Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.
Asunto(s)
ADN Bacteriano/genética , Farmacorresistencia Bacteriana/genética , Evolución Molecular , Genoma Bacteriano , Mutación , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Beijing , ADN Bacteriano/análisis , Humanos , Japón/epidemiología , Tasa de Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/epidemiología , Tuberculosis/genéticaRESUMEN
Mycobacterium triplex (M. triplex) is a bacterial species that can cause severe pulmonary diseases. Despite its clinical importance, only a few cases of M. triplex infection have been reported. Here, we present a rare case of pulmonary disease due to M. triplex in an immunocompetent patient who showed abnormal findings on chest X-ray and computed tomography scans. In this patient, the bacterium was identified by DNA sequencing analysis of the 16S rRNA and hsp65 genes. The patient was successfully treated with the appropriate antimicrobial agents. To put this case into the context of the current literature, we also reviewed other case reports of M. triplex infection.
RESUMEN
Japan reportedly has high incidence rate of nontuberculous mycobacterial lung disease (14.7 cases per 100,000 person in 2014). In Japan, the most common etiology is Mycobacterium avium subsp. hominissuis (MAH). MAH is a typical inhabitant of the environment, especially bathrooms, which are considered as a potential source of infection. To corroborate this hypothesis, we determined the detection rate of MAH in bathrooms of healthy volunteers by an ordinary culture method and we analyzed the genetic relatedness of these isolates with those from patients and other sources. We collected swabs of bathtub inlets, showerheads, bathroom drains, and shower water from 180 residences throughout Japan. The overall MAH detection rate was 16.1%, but the rate varied among regions: it was high in Kanto (9/34, 26.5%) and Kinki (9/33, 27.3%), but low in Kyushu (0/11, 0%), Tohoku (1/23, 4.3%), and Hokkaido (2/23, 8.7%). MAH was detected primarily in bathtub inlet samples (25 out of 170 residences). Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of 57 MAH isolates from bathrooms of the healthy volunteers with human clinical isolates. A minimum spanning tree generated on the basis of the VNTR data indicated that isolates from the bathrooms of the healthy volunteers had a high degree of genetic relatedness with those from Japanese patients, bathrooms of patients, and river water, but not with those from Russian patients and Japanese pigs. These results showed that bathtub inlets in Japan provide an environmental niche for MAH and suggest that bathrooms are one of the important infection sources of MAH in Japan. Understanding country-specific lifestyle habits, such as bathing in Japan, as well as the genetic diversity of MAH, will help in elucidating the sources of this pathogen.
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Enfermedades Pulmonares/microbiología , Repeticiones de Minisatélite , Mycobacterium avium/clasificación , Ríos/microbiología , ADN Bacteriano/genética , Voluntarios Sanos , Humanos , Japón , Mycobacterium avium/genética , Mycobacterium avium/aislamiento & purificación , Filogeografía , Federación de Rusia , Microbiología del Suelo , Cuartos de Baño , Microbiología del AguaRESUMEN
A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.
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Mycobacterium tuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Esputo/microbiología , Tuberculosis Pulmonar/diagnóstico , Cartilla de ADN , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Humanos , Mycobacterium tuberculosis/genética , Nepal , Valor Predictivo de las Pruebas , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Tuberculosis Pulmonar/microbiologíaRESUMEN
The distribution of Mycobacterium avium-intracellulare complex (MAC) in residences was examined. MAC was only recovered from bathrooms but not from other sites of residences. The appearance ratio in the bathrooms of patients with pulmonary MAC was significantly higher than that in healthy volunteers' bathrooms (P=.01). For 2 patients, the genotypes of environmental isolates were identical to their respective clinical isolates.
Asunto(s)
Complejo Mycobacterium avium/aislamiento & purificación , Infección por Mycobacterium avium-intracellulare/microbiología , Mycobacterium avium/aislamiento & purificación , Características de la Residencia , Infecciones del Sistema Respiratorio/microbiología , Humanos , Higiene , Japón , Mycobacterium avium/clasificación , Complejo Mycobacterium avium/clasificación , Infecciones Oportunistas/diagnóstico , Infecciones Oportunistas/microbiología , Valores de Referencia , Microbiología del AguaRESUMEN
The molecular mechanism of high level tetracycline resistance in T serotypes 4 and 11 group A streptococcal (GAS) isolates was examined in 61 tetracycline-resistant isolates in Japan. PCR and sequencing analyses revealed that the T serotype/emm genotype, T4/4 isolates carried tet(O) genes, which were genetically homogenous. The T11/11 and T11/89 isolates carried different subtypes of tet(M) genes, which were present on transposons Tn916 and Tn1545, respectively. In addition, these T11 isolates may have obtained the tet(M) gene after the 1990s, because resistance to tetracycline in T11 isolates was rarely found before then. These results strongly suggested that the T4 and T11 GAS isolates acquired tetracycline-resistance via different molecular mechanisms.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Streptococcus pyogenes/efectos de los fármacos , Resistencia a la Tetraciclina/genética , Elementos Transponibles de ADN , Electroforesis en Gel de Campo Pulsado , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Serotipificación , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/genética , Streptococcus pyogenes/aislamiento & purificaciónRESUMEN
Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as 'M-strain' has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB.