Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis.

BACKGROUND: Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. METHODS: In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. RESULTS: Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. CONCLUSIONS: The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting.

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare.

Alpha-2-Macroglobulin as a New Promising Biomarker Improving the Diagnostic Sensitivity of Bovine Paratuberculosis.

Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), which induces persistent diarrhea and cachexia. JD causes huge economic losses to the dairy industry due to reduced milk production and premature culling. Infected animals excrete MAP via feces during the prolonged subclinical stage without exhibiting any clinical signs. Therefore, accurate detection of subclinical stage animals is crucial for successful eradication of JD in the herd. In the current study, we analyzed serum samples of MAP-infected and non-infected cattle to identify potential biomarker candidates. First, we identified 12 differentially expressed serum proteins in subclinical and clinical shedder groups compared to the healthy control group. Second, we conducted ELISA for three selected biomarkers (alpha-2-macroglobulin (A2M), alpha-1-beta glycoprotein, and transthyretin) and compared their diagnostic performance with that of two commercial ELISA diagnostic kits. Serum A2M levels were significantly higher in the MAP-exposed, subclinical shedder, subclinical non-shedder, and clinical shedder groups than in the healthy control group, suggesting its possible use as a diagnostic biomarker for MAP infection. Furthermore, A2M demonstrated a sensitivity of 90.4%, and a specificity of 100% while the two commercial ELISA kits demonstrated a sensitivity of 67.83 and 73.04% and a specificity of 100%, respectively. In conclusion, our results suggest that measuring A2M by ELISA can be used as a diagnostic tool to detect MAP infection, considerably improving the detection rate of subclinical shedders and MAP-exposed animals that are undetectable using current diagnostic tools.

Increased Risk of Severe Gastric Symptoms by Virulence Factors vacAs1c, alpA, babA2, and hopZ in Helicobacter pylori Infection.

Two virulence factors of Helicobacter pylori, cagA and vacA, have been known to play a role in the development of severe gastric symptoms. However, they are not always associated with peptic ulcer or gastric cancer. To predict the disease outcome more accurately, it is necessary to understand the risk of severe symptoms linked to other virulence factors. Several other virulence factors of H. pylori have also been reported to be associated with disease outcomes, although there are many controversial descriptions. H. pylori isolates from Koreans may be useful in evaluating the relevance of other virulence factors to clinical symptoms of gastric diseases because the majority of Koreans are infected by toxigenic strains of H. pylori bearing cagA and vacA. In this study, a total of 116 H. pylori strains from Korean patients with chronic gastritis, peptic ulcers, and gastric cancers were genotyped. The presence of virulence factors vacAs1c, alpA, babA2, hopZ, and the extremely strong vacuolating toxin was found to contribute significantly to the development of severe gastric symptoms. The genotype combination vacAs1c/alpA/babA2 was the most predictable determinant for the development of severe symptoms, and the presence of babA2 was found to be the most critical factor. This study provides important information on the virulence factors that contribute to the development of severe gastric symptoms and will assist in predicting clinical disease outcomes due to H. pylori infection.

Comparative Evaluation of Band-Based Genotyping Methods for Mycobacterium intracellulare and Its Application for Epidemiological Analysis.

Mycobacterium intracellulare is a leading cause of nontuberculous mycobacterial pulmonary disease, with a rapidly increasing prevalence worldwide. This bacterium, commonly distributed in soil and water, is known to be transmitted through the environment rather than between people. Therefore, it is imperative to establish distinguishable genotyping methods to understand the clinical outcome, disease relapses, and epidemiology. Therefore, in this study, representative band-based genotyping methods were performed using M. intracellualre clinical isolates, and their Hunter-Gaston discriminatory index (HGDI) was 0.947, 0.994, and 1 for variable number tandem repetition (VNTR), VNTR-mycobacterial interspersed repetitive units, pulsed field gel electrophoresis, and repetitive sequence based-PCR, respectively. Although VNTR showed relatively low HGDI, co-infection with other M. intracellualre strains could be determined by loci showing allele diversity from 0 to 0.69. Additionally, genetic distance of clinical isolates from Gyeongnam/Korea, and other regions/countries were visualized by minimum spanning tree (MST) using the globally available VNTR profiles. The results of MST revealed that M. intracellulare isolated from patients in Gyeongnam/Korea had specific VNTR genotypes, which may be evidence of the geographic distribution of M. intracellulare specific genotypes. The comparative results of genotyping techniques and geographical characteristics in this study may provide fundamental information for the epidemiology of M. intracellulare.

Comparison of the Posterior Vertebral Column Resection With the Expandable Cage Versus the Nonexpandable Cage in Thoracolumbar Angular Kyphosis.

STUDY DESIGN: This was a retrospective study. OBJECTIVE: To compare the radiographic and clinical results of anterior support with nonexpandable cage with those of expandable cage. SUMMARY OF BACKGROUND DATA: The goals of surgical treatment in patients with thoracolumbar angular kyphosis are to improve the neurological deficit through neural decompression, to restore the normal alignment by correction of deformity, and to stabilize the spinal column by arthrodesis. Mesh cages have been traditionally used for reconstruction after corpectomy. However, expandable cages are gaining popularity due to some advantages over mesh cages, structural autograft or allograft, and poly-ether-ether-ketone/carbon fiber. MATERIALS AND METHODS: We performed a retrospective study in a consecutive series of 42 subjects with a male-to-female ratio of 15:27, a mean age of 65.3±10.9 years, and a mean follow-up period of 36.4±7.1 months, who underwent posterior vertebral column resection at our medical institutions between 2006 and 2011. They consist of 32 patients with posttraumatic kyphosis with a mean kyphotic angle of 33.1 degrees (range, 12.2-58.1 degrees) and 10 patients with tuberculous kyphosis with a mean kyphotic angle of 53.9 degrees (range, 22.6-126.0 degrees). The mean follow-up period was 36.4 months (range, 28-54 mo). We compared the clinical outcomes between the 2 groups based on radiographic findings, neurological status, and complications. Intervertebral heights and kyphosis at the level of vertebral column resection were used to measure the radiographic outcomes. Neurological outcome was evaluated using the modified Frankel grading system. RESULTS: Postoperatively, the mean kyphosis angle was 3.0±10.8 and 6.7±7.2 degrees (P=0.094). At a final follow-up, these values were 4.7±10.1 and 12.3±18.9 degrees in the corresponding order (P=0.099). There were 19 (80.0%) and 21 (86.4%) patients with solid arthrodesis of grade 1. On modified Frankel grading system, all the patients but one achieved improvement. But there was no significant difference in the grade between the 2 groups (P>0.05). Also in our series, there were 12 patients with cage subsidence and 6 with screw loosening. CONCLUSIONS: Nonexpandable cage and expandable cage are viable options for anterior support; both cages allow for correction of kyphosis with good fusion rate with similar neurological outcomes. Expandable cage is effective for the anterior support of posterior vertebral column resection.